Low-density lipoproteins (LDL) and their oxidized derivatives initiate and promote the atherosclerotic process, leading to the development of coronary artery disease.¹ Although the metabolism of HDL is not completely understood, there is increasing clinical evidence and fundamental research to support the significant antiatherogenic role of HDL.¹ HDL has multiple functions within the body, including reverse cholesterol transport, providing the cholesterol molecule substrate for bile acid synthesis, transport of clusterin, transport of paraoxanase, prevention of lipoprotein oxidation and selective uptake of cholesterol by adrenal cells.²

To better understand how HDL is antiatherogenic, a brief explanation of the atherosclerotic process is necessary. The atherosclerotic process begins when LDL becomes trapped within the vascular wall. Oxidation of this LDL results in the binding of monocytes to the endothelial cells lining the vessel wall. These monocytes are activated and migrate into the endothelial space where they are transformed into macrophages, leading to further oxidation of the LDL. The oxidized LDL is taken up through the scavenger receptor on the macrophage, leading to the formation of foam cells. A fibrous cap is generated through the proliferation and migration of arterial smooth muscle cells, thus creating an atherosclerotic plaque.¹

High Density Lipoproteins and Friends

Lipoproteins are complexes of lipids and proteins held together by non-covalent bonds. Each type of lipoprotein has a characteristic mass, chemical composition, density and physiological role. Irrespective of density or particle size, circulating lipids consist of a core of cholesteryl esters and triglycerides, an envelope of phospholipids and free cholesterol and apolipoproteins. The apolipoproteins are involved in the assembly and secretion of the lipoprotein, provide structural integrity, activate lipoprotein-modifying enzymes, and are the ligand for a large assortment of receptors and membrane proteins.² Each type of lipoprotein has a characteristic apolipoprotein composition or ratio. The most prominent apolipoprotein in HDL is apolipoprotein-AI (apo-AI), which accounts for approximately 70% of the protein mass, with apo-AII accounting for another 20%.² The ratio of apoA-I to apoA-II determines HDL functional and antiatherogenic properties.³ Circulating HDL particles consist of a hetergeneous mixture of discoidal and spherical particles with a mass of 200 to 400 kilo-daltons and a diameter of 70 to 100 angstroms.²

Apo-AI

Apolipoprotein-AI is synthesized in the liver and intestine.² It is a 243-residue protein containing a globular amino-terminal domain and a lipid-binding carboxyl-terminal domain. Apo-AI is an integral component of both spherical circulating HDL particles and of the simpler, discoidal, nascent HDL particles.⁴ Apo-AI is more effective than apo-AII in promoting cellular cholesterol efflux, the first step in reverse cholesterol transport.⁵ Apo-AI is a primary structural protein for HDL and serves as a cofactor for the enzyme lecithin cholesterol acyltransferase. Elevated levels of apo-AI are associated with decreased risk of coronary artery disease.³

Apo-AII

HDL Subclasses

Differences in the quantitative and qualitative content of lipids, apolipoproteins, enzymes and lipid transfer proteins result in the presence of various HDL subclasses, which are characterized by differences in shape, density, size, charge and antigenicty.⁶ On the basis of flotation rate in the preparative ultracentrifuge, there are two major subclasses of HDL: HDL₂ and HDL₃.⁷ Due to the higher PAFAH activity associated with HDL₂, it has a greater oxidative protective effect than HDL₃. The difference in the distribution of antioxidants between HDL₂ and HDL₃ also helps to explain the difference in their anti-oxidative protective effect against LDL oxidation.⁸ HDL₂ particles are depleted in cholesteryl ester and enriched in triglycerides. Hepatic lipase acts on the large, triglyceride-rich HDL₂ to hydrolyze the triglycerides, converting HDL₂ to HDL₃. HDL₃ then serves as an acceptor for free cholesterol, perpetuating the HDL₂-HDL₃ cycle.⁹

Inhibition of Oxidation

Reverse Cholesterol Transport

HDL is essential for the transport of cholesterol from extra-hepatic tissues to the liver, where it is excreted into bile as free cholesterol or as bile acids that are formed from cholesterol.¹ The process requires several steps. The first is the formation of nascent or pre-beta HDL particles in the liver and intestine.^2,13 Excess cholesterol moves across cell membranes into the nascent HDL where lecithin cholesterol acyl transferase (LCAT) converts it to cholesteryl ester and ultimately to mature HDL. Esterified cholesteryl is then transferred by cholesteryl ester transfer protein (CETP) from HDL to apolipoprotein-B containing lipoproteins, which are taken up by numerous receptors in the liver.¹ Nascent HDL is regenerated via hepatic triglyceride lipase and phospholipid transfer protein and the cycle continues.² In addition to the cholesterol removed from peripheral cells, HDL accepts cholesterol from LDL and erythrocyte membranes.¹⁴ Another somewhat less complicated mechanism of reverse cholesterol transport is passive diffusion of cholesterol between cholesterol-poor membranes and HDL or other acceptor molecules.⁴

LCAT, a protein with 416 amino acids and four N-linked glycosylation sites, is secreted by the liver and released into the plasma in association with HDL.¹⁵ LCAT is vital in the conversion of discoidal to spheroidal HDL. It is responsible for esterification of the cholesterol molecules of HDL to cholesteryl ester using lecithin as the fatty-acyl donor.⁴ LCAT mediates the transfer of linoleate from lecithin to free cholesterol on HDL to form cholesterol esters that are transferred to very low density lipoprotein (VLDL) and LDL.¹⁶ LCAT is activated by apolipoprotein-AI. The eight 22-amino acid and two 11-amino acid tandem repeats of the Apo-AI molecule are responsible for lipid binding and activation of LCAT.¹⁶ LCAT does not affect the rate of cholesterol efflux from cells, but rather decreases the back transfer, thus promoting net efflux from cells.¹⁴ LDL oxidation products are potent inhibitors of LCAT. This adversely effects the metabolism of HDL via two mechanisms. The first, a direct inhibitory effect on LCAT activity and the second, a cross-linking of apo-AI, thereby limiting the ability of HDL to function efficiently in the reverse cholesterol transport pathway.¹⁶ LCAT, together with CETP and phospholipid transfer protein, are key determinants of the structure and composition of HDL.¹⁴

CETP is a plasma lipid transfer protein secreted by the liver, which mediates the transfer and exchange of neutral lipids and phospholipids.¹⁶ CETP exchanges cholesteryl esters of HDL with triglycerides of VLDL, IDL, and LDL.⁶ It also mediates catabolism of HDL cholesteryl esters, decreasing both HDL size and protein content. CETP also plays a central role in reverse cholesterol transport by moving cholesterol from the periphery back to the liver.¹⁶ Once considered a proatherogenic modulator of HDL metabolism, CETP maintains its antiatherogenic properties, through the generation of lipid-free Apo-AI, the enhancement of reverse cholesterol transport and the transfer of oxidized lipids.⁶

The HDL-associated enzymes PAFAH, LCAT and paraoxonase (PON) are all involved in the inhibition of LDL peroxidation.⁸ Paraoxonase is a component of HDL, which lowers risk from coronary heart disease by destroying pro-inflammatory molecules that may promote atherosclerosis through oxidative damage.¹ Paraoxonase is a component of a spectrum of circulating high-density lipoprotein particles and can hydrolyse oxidized phospholipids and cholesteryl ester hydroperoxides.¹⁸ PON is tightly bound to Apo-AI, as a result of the hydrophobic N-terminal domain of the enzyme.¹⁸ The enzyme activity of PON is calcium dependent. Calcium has two roles in the enzymatic reaction. First, it maintains the active site, either by participating directly in the catalytic reaction or by maintaining the appropriate conformation of the active site. Secondly, calcium facilitates the removal of diethyl phosphate from the active site, rendering the phosphorus susceptible to nucleophilic attack.¹⁹

Paroxonase has a genetic polymorphism that results in a single amino acid substitution at position 192.¹⁹ The A, Q, or low activity isoenzyme has glutamine at position 192 and the B, R, or high activity isoenzyme has arginine at position 192.¹¹ The polymorphisms affect the hydrolytic activity of the PON isoenzymes with respect to certain substrates, such as paraoxon and lipid peroxides.²⁰ Both the serum concentration of paraoxanase and an individual’s genotype are related to plasma lipid and lipoprotein concentrations and thereby coronary heart disease.¹⁹ Plasma levels of HDL are a major correlate of paraoxonase protein levels, while paraoxonase genotype was the major predictor of plasma paraoxonase activity.¹⁶ RR homozygotes while most active in hydrolysis of paraoxon, are less effective at protecting LDL against lipid peroxidation that either QQ homozygotes or QR heterozygotes.²⁰ Another polymorphism at amino acid 55, a leucine to methionine substitution, effects PON concentration through modulation of activity in non-insulin dependent diabetes mellitus (NIDDM).^21,22 Paraoxonase is inactivated by oxidized LDL. Oxidized phospholipids, oxidized cholesteryl esters or lysophosphatidylcholines, formed during LDL oxidation, interact with the enzyme –free sulfhydryl group, leading to paraoxonase inactivation.⁸

Adhesion Molecules/VCAM

Endothelial cells interact and communicate with macrophages, platelets, smooth muscle cells and T-lymphocytes; they are also a potential site of oxidation of LDL.²³ Endothelial dysfunction, one of early steps of atherogenesis, is caused by a variety of injurious actions, including elevated and modified LDL, free radicals and inflammatory reactions.²⁴ Localized up-regulation of adhesive endothelial adhesion molecules, a prerequisite for monocyte adherence and migration, is a dynamic process sensitive to inflammatory cytokines, sheer stress and oxidative insults.²³

Adhesion of monocytes to the vascular endothelium, mediated by adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 and E-selectin, is one of the earliest events in atherogenesis. The expression of VCAM-1 coincides with the development of early foam cell lesions. VCAM-1 is also found in the arterial endothelium over existing atheromatous plaques.²⁵ Additional functions of HDL, unrelated to it’s role in plasma lipid transport, include the inhibition of transmigration of monocytes induced by oxidized LDL and the inhibition of the adhesion of monocytes to endothelial cells.²⁶ The ability of HDL to inhibit cytokine induced cell surface expression of adhesion molecules indicates that HDL may inhibit atherogenesis at an early stage by preventing monocyte adhesion to the endothelium. The inhibitory effects of HDL also influence the progression of atherogenesis by altering the expression of adhesion molecules which attract T-lymphocytes to these lesions, thus contributing to the protective effects of an acute inflammatory response.²⁷ The reduction of VCAM-1 expression reflects the antioxidant properties of HDL, as this diminishes both oxidative stress and production of pro-inflammatory lipoproteins.²³ The ability of HDL to inhibit adhesion molecule expression may be secondary to its known capacity to remove lipid oxidation products.²⁵ HDL₃ has substantially greater inhibitory activity than HDL₂. The superiority of HDL₃ as an inhibitor of VCAM-1 may be a consequence of differences in either particle size or composition or it may be simply that there are more HDL₃ particles than HDL₂ particles.²⁵