Anatol Kontush

Functionally Defective High-Density Lipoprotein: A New Therapeutic Target at the Crossroads of Dyslipidemia, Inflammation, and Atherosclerosis

High-density lipoproteins (HDL) possess key atheroprotective biological properties, including cellular cholesterol efflux capacity, and anti-oxidative and anti-inflammatory activities. Plasma HDL particles are highly heterogeneous in physicochemical properties, metabolism, and biological activity. Within the circulating HDL particle population, small, dense HDL particles display elevated cellular cholesterol efflux capacity, afford potent protection of atherogenic low-density lipoprotein against oxidative stress and attenuate inflammation. The antiatherogenic properties of HDL can, however be compromised in metabolic diseases associated with accelerated atherosclerosis. Indeed, metabolic syndrome and type 2 diabetes are characterized not only by elevated cardiovascular risk and by low HDL-cholesterol (HDL-C) levels but also by defective HDL function. Functional HDL deficiency is intimately associated with alterations in intravascular HDL metabolism and structure. Indeed, formation of HDL particles with attenuated antiatherogenic activity is mechanistically related to core lipid enrichment in triglycerides and cholesteryl ester depletion, altered apolipoprotein A-I (apoA-I) conformation, replacement of apoA-I by serum amyloid A, and covalent modification of HDL protein components by oxidation and glycation. Deficient HDL function and subnormal HDL-C levels may act synergistically to accelerate atherosclerosis in metabolic disease. Therapeutic normalization of attenuated antiatherogenic HDL function in terms of both particle number and quality of HDL particles is the target of innovative pharmacological approaches to HDL raising, including inhibition of cholesteryl ester transfer protein, enhanced lipidation of apoA-I with nicotinic acid and infusion of reconstituted HDL or apoA-I mimetics. A preferential increase in circulating concentrations of HDL particles possessing normalized antiatherogenic activity is therefore a promising therapeutic strategy for the treatment of common metabolic diseases featuring dyslipidemia, inflammation, and premature atherosclerosis.

Small, Dense HDL Particles Exert Potent Protection of Atherogenic LDL Against Oxidative Stress

Objectives—The relationship of the structural and functional heterogeneity of HDL particles to protection of LDL against oxidative stress is indeterminate. Methods and Results—HDL subfractions of defined physicochemical properties were isolated by density gradient ultracentrifugation from normolipidemic human serum (n=8), and their capacity to protect LDL from oxidation was evaluated. Under mild oxidative stress induced by AAPH or Cu(II), HDL subfractions (at equal cholesterol or protein concentration or equal particle number) significantly decreased LDL oxidation rate (−20% to −85%) in the propagation phase (234 nm), which was prolonged by up to 82% with decreased maximal diene formation. Antioxidative activity of HDL subfractions increased with increment in density, as follows: HDL2b<HDL2a<HDL3a<HDL3b<HDL3c (confirmed by thiobarbituric acid–reactive substance content and LDL electrophoretic mobility). Concordantly, antioxidative activity of small HDL prepared by FPLC was significantly higher (+56%) than that of large HDL. Antioxidative action of HDL subfractions was primarily associated with inactivation of LDL lipid hydroperoxides. The potent protective activity of small HDL could not be accounted for exclusively by enzymatic activities (PON1, platelet-activating factor acetylhydrolase, and lecithin-cholesterol acyltransferase). Conclusions—Small, dense HDL exhibit potent antioxidant activity, which may arise from synergy in inactivation of oxidized LDL lipids by enzymatic and nonenzymatic mechanisms, in part reflecting distinct intrinsic physicochemical properties.

HDL Measures, Particle Heterogeneity, Proposed Nomenclature, and Relation to Atherosclerotic Cardiovascular Events

BACKGROUND A growing body of evidence from epidemiological data, animal studies, and clinical trials supports HDL as the next target to reduce residual cardiovascular risk in statin-treated, high-risk patients. For more than 3 decades, HDL cholesterol has been employed as the principal clinical measure of HDL and cardiovascular risk associated with low HDL-cholesterol concentrations. The physicochemical and functional heterogeneity of HDL present important challenges to investigators in the cardiovascular field who are seeking to identify more effective laboratory and clinical methods to develop a measurement method to quantify HDL that has predictive value in assessing cardiovascular risk. CONTENT In this report, we critically evaluate the diverse physical and chemical methods that have been employed to characterize plasma HDL. To facilitate future characterization of HDL subfractions, we propose the development of a new nomenclature based on physical properties for the subfractions of HDL that includes very large HDL particles (VL-HDL), large HDL particles (L-HDL), medium HDL particles (M-HDL), small HDL particles (S-HDL), and very-small HDL particles (VS-HDL). This nomenclature also includes an entry for the pre-β-1 HDL subclass that participates in macrophage cholesterol efflux. SUMMARY We anticipate that adoption of a uniform nomenclature system for HDL subfractions that integrates terminology from several methods will enhance our ability not only to compare findings with different approaches for HDL fractionation, but also to assess the clinical effects of different agents that modulate HDL particle structure, metabolism, and function, and in turn, cardiovascular risk prediction within these HDL subfractions.

Proteomic Analysis of Defined HDL Subpopulations Reveals Particle-Specific Protein Clusters Relevance to Antioxidative Function

Objective—Recent proteomic studies have identified multiple proteins that coisolate with human HDL. We hypothesized that distinct clusters of protein components may distinguish between physicochemically-defined subpopulations of HDL particles, and that such clusters may exert specific biological function(s). Methods and Results—We investigated the distribution of proteins across 5 physicochemically-defined particle subpopulations of normolipidemic human HDL (HDL2b, 2a, 3a, 3b, 3c) fractionated by isopycnic density gradient ultracentrifugation. Liquid chromatography/electrospray mass spectrometry identified a total of 28 distinct HDL-associated proteins. Using an abundance pattern analysis of peptide counts across the HDL subfractions, these proteins could be grouped into 5 distinct classes. A more in-depth correlational network analysis suggested the existence of distinct protein clusters, particularly in the dense HDL3 particles. Levels of specific HDL proteins, primarily apoL-I, PON1, and PON3, correlated with the potent capacity of HDL3 to protect LDL from oxidation. Conclusions—These findings suggest that HDL is composed of distinct particles containing unique (apolipo)protein complements. Such subspeciation forms a potential basis for understanding the numerous observed functions of HDL. Further work using additional separation techniques will be required to define these species in more detail.

Antiatherogenic small, dense HDL—guardian angel of the arterial wall?

Our understanding of the relationship between the atheroprotective activities of HDL and heterogeneity of HDL particles has advanced greatly. HDL particles are highly heterogeneous in structure, intravascular metabolism and antiatherogenic activity. In this review, we discuss new findings on the antiatherogenic properties of HDL particles. Small, dense HDL possesses potent antioxidative activity but this is compromised under conditions of atherogenic dyslipidemia. HDL functional deficiency frequently coincides with reductions in HDL-cholesterol concentration and alterations in HDL metabolism and structure. Formation of small, dense HDL particles with attenuated antiatherogenic activity can be mechanistically related to HDL enrichment in triglycerides and in serum amyloid A, depletion of cholesteryl esters, covalent modification of HDL apolipoproteins and attenuated antiatherogenic function of apolipoprotein AI. Low circulating levels of HDL cholesterol might, therefore, be associated with the defective functionality of small HDL particles of abnormal structure and composition. In common metabolic diseases, such as type 2 diabetes and metabolic syndrome, deficiency of HDL particle number and function favor accelerated atherosclerosis. Therapeutic normalization of the quantity, quality and biological activities of HDL particles thus represents a novel approach to attenuating atherosclerosis in dyslipidemic individuals with metabolic disease. Cholesteryl ester transfer protein inhibitors, nicotinic acid, reconstituted HDL and other HDL-raising agents are being investigated. Induction of selective increase in the circulating concentrations of small, dense HDL3 particles with increased antiatherogenic activity seems especially promising, particularly for therapy of atherogenic dyslipidemia.

Cholesteryl ester transfer protein: at the heart of the action of lipid-modulating therapy with statins, fibrates, niacin, and cholesteryl ester transfer protein inhibitors

Subnormal plasma levels of high-density lipoprotein cholesterol (HDL-C) constitute a major cardiovascular risk factor; raising low HDL-C levels may therefore reduce the residual cardiovascular risk that frequently presents in dyslipidaemic subjects despite statin therapy. Cholesteryl ester transfer protein (CETP), a key modulator not only of the intravascular metabolism of HDL and apolipoprotein (apo) A-I but also of triglyceride (TG)-rich particles and low-density lipoprotein (LDL), mediates the transfer of cholesteryl esters from HDL to pro-atherogenic apoB-lipoproteins, with heterotransfer of TG mainly from very low-density lipoprotein to HDL. Cholesteryl ester transfer protein activity is elevated in the dyslipidaemias of metabolic disease involving insulin resistance and moderate to marked hypertriglyceridaemia, and is intimately associated with premature atherosclerosis and high cardiovascular risk. Cholesteryl ester transfer protein inhibition therefore presents a preferential target for elevation of HDL-C and reduction in atherosclerosis. This review appraises recent evidence for a central role of CETP in the action of current lipid-modulating agents with HDL-raising potential, i.e. statins, fibrates, and niacin, and compares their mechanisms of action with those of pharmacological agents under development which directly inhibit CETP. New CETP inhibitors, such as dalcetrapib and anacetrapib, are targeted to normalize HDL/apoA-I levels and anti-atherogenic activities of HDL particles. Further studies of these CETP inhibitors, in particular in long-term, large-scale outcome trials, will provide essential information on their safety and efficacy in reducing residual cardiovascular risk.

Biological activities of HDL subpopulations and their relevance to cardiovascular disease

The concept of raising high-density lipoprotein (HDL) has been the focus of increasing attention as a strategy to reduce cardiovascular disease. HDL particles are, however, highly heterogeneous in structure, intravascular metabolism and biological activity. In this review, we describe major HDL subpopulations and discuss new findings on the antiatherogenic properties of HDL particles. Across the HDL subpopulation spectrum, small, dense, protein-rich HDLs display potent atheroprotective properties, which can be attributed to specific clusters of proteins and lipids; such activities can be compromised under conditions of atherogenic dyslipidemia. Comprehensive structural and compositional analyses of HDL may provide key information to identify subpopulations displaying specific biological functions and acquiring deficient functionality, with the potential to reveal novel biomarkers of cardiovascular risk and new pharmacological targets.

Antiatherogenic function of HDL particle subpopulations: focus on antioxidative activities

Oxidative stress, an emerging risk factor for premature atherosclerosis and cardiovascular disease, mediates the formation of proinflammatory, pro-atherogenic oxidized low-density lipoprotein (oxLDL) in the arterial intima. Circulating HDL particles, and particularly small, dense, protein-rich HDL3, may provide potent protection of LDL in vivo from oxidative damage by free radicals in the arterial intima, resulting in the inhibition of the generation of proinflammatory oxidized lipids, primarily lipid hydroperoxides (LOOH) but also short-chain oxidized phospholipids (oxPL). HDL-mediated inactivation of LOOH involves initial transfer of phospholipid hydroperoxides (PLOOH) from LDL to HDL3, which is governed by the rigidity of the surface monolayer of HDL, and subsequent reduction of PLOOH by redox-active Met residues of apolipoprotein A-I (apoA-I) with the formation of phospholipid hydroxides (PLOH) and methionine sulphoxides. HDL-associated enzymes may in turn contribute to the hydrolytic inactivation of short-chain oxPL. Mounting evidence suggests that the integrated antioxidative activity of HDL appear to be defective in atherogenic dyslipidaemias involving low HDL-cholesterol levels; anomalies in the proteome and lipidome of HDL particles in dyslipidaemic patients may underlie such functional deficiency. Pharmacological normalization of HDL metabolism concomitantly with correction of circulating levels, composition and biological activities of HDL particles, with enrichment in apoA-I and reduction in HDL surface rigidity, may constitute an efficacious therapeutic approach to attenuate atherosclerosis in dyslipidaemic patients at high cardiovascular risk.

HDL-targeted therapies: progress, failures and future

Since the discovery in the 1970s that plasma levels of high-density lipoprotein cholesterol (HDL-C) are inversely associated with cardiovascular outcome, it has been postulated that HDL is anti-atherogenic and that increasing HDL-C levels is a promising therapeutic strategy. However, the recent failure of three orally active, HDL-C-raising agents has introduced considerable controversy, prompting the question of whether increasing the cholesterol cargo of HDL in a non-selective manner is an effective pharmacological approach for the translation of its atheroprotective and vasculoprotective activities. The interrelationships between HDL-C concentration, HDL particle number and levels of diverse HDL particle subpopulations of defined composition are complex, as are their relationships with reverse cholesterol transport and other anti-atherogenic functions. Such complexity highlights the incompleteness of our understanding of the biology of HDL particles. This article examines the HDL hypothesis in molecular and mechanistic terms, focusing on features that have been addressed, those that remain to be tested, and potential new targets for future pharmacological interventions.

Apolipoprotein A-I structural organization in high-density lipoproteins isolated from human plasma

High-density lipoproteins (HDLs) mediate cholesterol transport and protection from cardiovascular disease. Although synthetic HDLs have been studied for 30 years, the structures of human plasma–derived HDL and its major protein apolipoprotein apoA-I are unknown. We separated normal human HDL into five density subfractions and then further isolated those containing predominantly apoA-I (LpA-I). Using cross-linking chemistry and mass spectrometry, we found that apoA-I adopts a structural framework in these particles that closely mirrors that in synthetic HDL. We adapted established structures for synthetic HDL to generate the first detailed models of authentic human plasma HDL in which apoA-I adopts a symmetrical cage-like structure. The models suggest that HDL particle size is modulated by means of a twisting motion of the resident apoA-I molecules. This understanding offers insights into how apoA-I structure modulates HDL function and its interactions with other apolipoproteins.