The physiology of lipoproteins

TOPICS IN MOLECULAR BIOLOGY The physiology of lipoproteins Thomas N. Tulenko, PhD,a and Anne E. Sumner, MDb The seminal studies of Brown and Goldstein (Science 1986;232:34-47) coupled with the findings of the Framingham study revolutionized our understanding of the metabolic basis for vascular disease. These studies led to the widespread use of the coronary risk lipid profile, which uses the total cholesterol/high-density lipoprotein (HDL) ratio (or low-density lipoprotein [LDL]/HDL ratio) in predicting risk for vascular disease and as a tool for therapeutic management of patients at risk for vascular disease. However, although these methods are predictive of coronary artery disease (CAD) in general, it is also well known that the extent of occlusive disease and CAD varies greatly between individuals with similar cholesterol and HDL lipid profiles. For this reason, the National Cholesterol Education Program Expert Panel revised these guidelines and now recommends monitoring LDL and HDL cholesterol in the context of coronary heart disease risk factors and “risk equivalents.” In addition, more recent findings indicate that specific alterations in individual lipoprotein subclasses may account for the variations in CAD in subjects with similar lipid profiles. For example, a preponderance of small, dense LDL particles correlates with a marked increase in risk for myocardial infarction independent of LDL levels. In particular, the association of small, dense LDL with elevated triglycerides (large, less dense VLDL) and reduced HDL has been defined as the atherogenic lipoprotein profile, and the key metabolic defect driving this profile may be elevated levels of triglycerides, specifically large, less dense VLDL. In an attempt to explain the physiologic basis for lipoprotein variations, this review describes the basic metabolic scheme underlying the traditional view of lipoprotein metabolism and physiology. It then examines the identity and role of the various lipoprotein subfractions in an attempt to distill a working model of how lipoprotein abnormalities might account for vascular disease in general and the metabolic syndrome in particular. (J Nucl Cardiol 2002;9:638-49.) Lipoprotein metabolism is a complicated process, and our understanding of it is still somewhat fragmentary and challenging. Since the seminal studies by Brown and Goldstein,1 coupled with the findings of the Framingham study, a schematic overview has evolved that has proved effective in guiding our understanding and management of dyslipidemias. It reflects the fundamental concept that elevated low-density lipoprotein (LDL) levels, reduced high-density lipoprotein (HDL) levels, or their combination characterizes the dyslipidemias and their underlying vascular disease. However, although this view is predictive of coronary artery disease (CAD),2 the extent of From the Department of Surgery, Thomas Jefferson University College of Medicine, Philadelphia, Paa; and National Institutes of Health, Diabetes Branch, Bethesda, Md.b Support for this project was provided in part by National Institutes of Health grant HL-66273 and a grant from Pfizer Pharmaceutical Company. Reprint requests: Thomas N. Tulenko, PhD, Department of Surgery, Thomas Jefferson University College of Medicine, 1025 Walnut Street, Suite 605, Philadelphia, PA 19107; [email protected]. Copyright © 2002 by the American Society of Nuclear Cardiology. 1071-3581/2002/$35.00 ⫹ 0 43/1/128959 doi:10.1067/mnc.2002.128959 638

occlusive disease3 and CAD4 varies greatly between individuals with similar lipid profiles. More recent findings indicate that specific alterations in individual lipoprotein subclasses may account for these variations.5-7 Nuclear magnetic resonance lipoprotein profiling, for example, is confirming and extending the concept that not all LDL particles or HDL particles are the same and that these variations may result from differences in particle size and number.8 An overriding emergent concept in vascular disease is that atherosclerosis is a systemic disease, not limited to CAD. It includes cerebral vascular disease (ie, stroke) and the peripheral vascular targets involving claudication, aneurysm, and renovascular disease. Patients with any one of these vascular disorders at presentation are, by definition, at significant risk for the other disorders as a result of the systemic manifestations of dyslipidemias. In this context, further refinement of the understanding of lipoprotein particle “physiology” will improve our ability to reduce the burden of vascular disease, the mortality of which, contrary to popular belief, has not decreased over the last 60 years.9 The objective of this review is to revisit lipoprotein physiology, first from the traditional view of

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Figure 1. Lipoprotein map showing the origin of lipoproteins from dietary fats (chylomicron [CM]) to their processing and resynthesis into VLDL, IDL, and HDL. The major apoproteins associated with each particle are indicated and their characteristics and functions are listed in Table 2. LpL, Lipoprotein lipase; HL, hepatic lipase; CE, cholesteryl ester; A, B48, C, and E, apolipoproteins; B, apo B-100; TG, triglyceride; PL, phospholipid; FFA, free fatty acid; Rem, remnant. (Modified and used with permission from Olson R. Disorders of lipoprotein metabolism. In: Gilbert-Barnes E, Barnes L, editors. Metabolic diseases: foundations of clinical management, genetics and pathology. Volume I. Natick [MA]: Eaton Publishing; 2000. p. 283-322.)

the role of basic lipoprotein levels underlying atherosclerotic syndromes, as this view provides the basis for our current approach to clinical management. We then build on this scheme to identify new and emerging concepts of lipoprotein particle subclasses and their physiology. FORWARD CHOLESTEROL TRANSPORT Figure 1 illustrates the fundamental flow scheme for synthesis, interactions, and degradation of lipoproteins, and Table 1 identifies the broad classes of lipoproteins and their unique characteristics. Table 2 lists the apoproteins resident on the different particles and what we understand their functions to be. A

convenient starting point for the evolution of the LDLs begins with the lowest-density class, the chylomicron (Figure 1). These particles are synthesized in the intestinal mucosal cells directly from dietary fats, namely triglycerides, cholesterol and phospholipids, and apoprotein (apo) B48, which is synthesized in these cells. Their density is low because their size is large (ⱖ100 nm) and they contain large amounts of lipid (especially buoyant triglycerides). Their large size precludes penetration of the capillary membrane, so they are secreted by intestinal mucosa into the lymphatics and then enter the circulation by way of the thoracic duct. As a side note, injury of the thoracic duct during cardiothoracic surgery can be determined

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Table 1. Differential lipoprotein characteristics

Lipoprotein class Chylomicron VLDL IDL LDL HDL Lp(a)

Density (g/mL)

Size (nm)

Major lipids

Major apoproteins

⬍0.93 0.93-1.006 1.006-1.019 1.019-1.063 1.063-1.210 1.040-1.090

100-500 30-80 25-50 18-28 5-15 25-30

Dietary TGs Endogenous TGs CEs and TGs CEs CEs CEs

B-48, C-II, E B-100, C-II, E B-100, E B-100 A, C-II, E B-100 and glycoprotein

Reprinted from reference 63. TG, Triglyceride; CE, cholesteryl ester; LP(a), lipoprotein little “a”.

Table 2. Apolipoprotein characteristics and functions

Apolipoprotein A-I A-II B-100 B-48 C-I C-II C-III E

Molecular weight

Function

29,000 17,500 549,000 264,000 7,000 9,000 9,000 34,000

LCAT activator HDL structure LDL receptor ligand Chylomicrons LCAT activator LpL activator HDL structure LDL receptor ligand

Modified from reference 63. LpL, Lipoprotein lipase.

by the presence of triglycerides (ie, chylomicrons) in pleural aspirant (chylous effusion). Once in the blood, chylomicrons acquire apo E and apo C-II and are progressively reduced in size by the action of lipoprotein lipase, which is bound to the capillary endothelium and catalyzes the removal of free fatty acids from the chylomicron triglyceride pool. Interestingly, lipoprotein lipase is activated by apo C-II on the chylomicron, so lipoprotein lipase can then hydrolyze most of the chylomicron triglycerides to free fatty acids, which are then released from the particle, bind to albumin, and are ultimately deposited in adipose tissue. The continued action of lipoprotein lipase leaves the chylomicron nearly depleted of triglyceride within an hour after a meal. The depleted chylomicron remnant particle exits this pathway by its uptake into the hepatocyte through a receptor-mediated process, with the chylomicron apo E serving as the ligand for the hepatic LDL receptor. In the hepatocyte the chylomicron remnant releases its contents (ie, the remaining triglycerides, cholesteryl esters, phospholipids, and apoproteins). The hepatocyte reassembles these chylomicron remnant– de-

rived products, along with endogenous triglycerides and cholesteryl esters, into very low-density lipoproteins (VLDLs) and secretes them into the circulation for the next phase of delivery of lipids to the periphery. Note that like chylomicrons, VLDLs are also triglyceride-rich and contain apo C-II and apo E. However, unlike chylomicrons, they contain fewer triglycerides, are smaller, and now carry apo B-100 instead of apo B-48. The emergence of apo B-100 here is important, in that it is a physiologic ligand for the LDL receptor. Over the next half hour, lipoprotein lipase (activated again by apo C-II on the VLDL particle) reduces VLDL triglyceride content further, leaving the particles progressively smaller, more dense, and more cholesterol-enriched as it moves down this well-known cascade to intermediatedensity lipoprotein (IDL). Hydrolysis of IDL triglycerides to free fatty acids is mediated by yet another lipase, hepatic lipase, and LDL soon appears as the terminal particle in this pathway. Actually, about one third of the VLDL is cleared by the hepatic LDL receptor, whereas two thirds pass down this cascade, terminating as LDL. The transition of VLDL to IDL is accompanied by the transfer of apo C-II (to HDL) and is also removed (again to HDL) during the transition of IDL to LDL apo E. Hence, LDL, whose half-life in the circulation is on the order of several days, contains only apo B-100 and essentially only one copy per particle. Note that the upstream precursor particles (IDL and VLDL) also contain apo B-100. This is important because apo B levels correlate with mortality from myocardial infarction to a greater degree than do LDL levels,10 suggesting that the postprandial lipoproteins are also atherogenic.11,12 REVERSE CHOLESTEROL TRANSPORT The forward movement of lipids to the peripheral cells is the predominant pathway for delivery of free fatty acids, the body’s primary deep metabolic fuel. It is

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also the only pathway for delivering cholesterol. Note that cholesterol is obligate to every cell for the synthesis and maintenance of cell membranes. Whereas the forward movement of triglycerides is balanced by the metabolic consumption of free fatty acids as a primary metabolic fuel, there is essentially no analogous (ie, catabolic) mechanism for the metabolic disposal of cholesterol. This is problematic because there is accumulating evidence that cholesterol may be atherogenic in vascular wall cells at lower levels of exposure13-16 and outright cytotoxic at higher levels.17,18 The metabolic balance of cholesterol, which is both essential and dangerous, is accomplished by reverse cholesterol transport by HDLs. HDL is synthesized in the intestinal mucosal cells and liver and secreted as a disk-shaped nascent HDL particle containing only a small amount of phospholipid and apo A-I (pre–␤-HDL). It avidly absorbs unesterified (free) cholesterol from peripheral cells by an aqueous diffusion pathway from the cell membrane to the particle. The efflux of cholesterol is facilitated by the binding of the nascent HDL particle to cell-surface receptors (eg, scavenger receptor B1 [SRB1]). Free cholesterol, being amphipathic, is absorbed onto the surface of the HDL from a “donor” cell membrane. Lecithin-cholesterol acyltransferase (LCAT), activated by apo A-I, catalyzes cholesterol’s esterification, causing the lipophilic cholesteryl ester molecule to enter the core of the HDL particle, thereby freeing up space on the surface of the particle for further cholesterol adsorption. This coordinated action of apo A-I, HDL, and LCAT promotes the accumulation of cholesteryl esters in HDL and is instrumental in maintaining the unidirectionality (ie, cholesterol efflux) of reverse cholesterol transport. With continuation of this process, HDL gradually matures to its spherical shape (␣-HDL). Along with cholesterol absorption, HDL accumulates apo C-II and apo E from the VLDL and IDL. Interestingly, one of the functions of HDL is to serve as a reservoir of apoproteins, especially apo C-II, which is essential for the activation of lipoprotein lipase. Removal of lipids from the mature ␣-HDL particle occurs by two pathways, a direct pathway and an indirect pathway. The direct pathway involves two branches, a selective lipid uptake pathway mediated by SR-B119 and by a “holoparticle” uptake pathway by apo E receptors (eg, cubilin [renal] and probably other receptors).20 The indirect pathway involves transfer of cholesteryl esters from ␣-HDL to apo B– containing lipoproteins (VLDL, IDL, and LDL) mediated by cholesteryl ester transfer protein (CETP).21 Because the bulk of cholesterol removal by HDL is accomplished by shuttling to these particles, their uptake by the liver is essential for the disposal of HDL cholesterol. At the liver, the hepatocytes express LDL receptors on their cell surface that

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recognize apo B as well as apo E as physiologic ligands. Thus the hepatic LDL receptor cleverly mediates the uptake of chylomicron remnant, VLDL, IDL, and LDL. A common misconception is that in reverse cholesterol transport, HDL delivers its cholesterol load to the liver. Indeed this occurs, but the CETP-mediated shuttling of cholesteryl esters to LDL for delivery to the liver is by far the more quantitative pathway.22 In this sense, “bad” cholesterol also serves a vital good role! ATHEROGENESIS Oddly, only in human beings does the balance between forward and reverse cholesterol transport become a significant health problem. When this occurs, LDL levels in the blood rise, causing LDL accumulation within the wall of large arteries, which increases in proportion to their level in the blood. In addition, the retention of LDL increases independently of plasma LDL levels in subjects with atherosclerotic disease as well as in various animal models.23 In either case, excessive accumulation of LDL in the arterial wall appears to initiate the early genesis of atherosclerotic lesions.23 Numerous reviews are available that detail the current level of understanding of this process.23,24 In brief, however, vesicular transport of LDL through the endothelial cell (EC) monolayer is accompanied by modest oxidative modification of the LDL particle. Perhaps as a result of this, damage to the endothelium by LDL (both native and oxidized LDL) activates the cellular inflammatory pathway mediated by activation of redox-sensitive transcription factors (eg, nuclear factor kappa b, NF␬B) and subsequent upregulation of intercellular adhesion molecule and vascular cell adhesion molecule synthesis. On the EC luminal membrane, intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 serve as docking molecules and recruit circulating monocytes into the vessel wall. On binding to the endothelium, extravasation of monocytes occurs and they undergo phenotypic modulation to macrophages. Oxidative modifications of LDL render the particle susceptible to uptake into macrophages (and ECs) by the unregulated macrophage scavenger receptor. Because cell cholesterol levels exquisitely regulate (negatively) the synthesis of LDL receptor in most cells, cholesterol overload by native LDL is unlikely. However, because the scavenger receptor pathway is unregulated, this pathway easily overwhelms the macrophage with cholesterol. Because of the pathogenic capacity of excess free cholesterol,17,18 most cells respond to cholesterol overload by esterifying cholesterol to cholesteryl esters, primarily cholesteryl oleate, an inert storage form of cholesterol. In macrophages, this cholesterol overload results in the accumulation of cholesteryl esters, which

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Table 3. Apoproteins and plasma enzymes essential to normal metabolism of lipoproteins

Type

Disorder

I

Hypertriglyceridemia

II-a II-a II-a II-a II-b III

Hypercholesterolemia Familial hypercholesterolemia Familial hypercholesterolemia Hypercholesterolemia Familial combined hypercholesterolemia Dysbetalipoproteinemia

IV V

Hypertriglyceridemia Hypertriglyceridemia

Phenotype Chylomicrons (increased TGs) LDL Increased LDL Increased LDL Increased LDL Increased LDL and VLDL Increased chylomicron remnants and ␤-VLDL Increased VLDL Increased chylomicron remnants and VLDL

Defect Impaired LpL – LDL-R (homozygous) LDL-R (heterozygous) Polygenic Increased apo-B synthesis Altered apo-E

Pathology

Frequency

Pancreatitis/ gastrointestinal Atherosclerosis Atherosclerosis Atherosclerosis Atherosclerosis Atherosclerosis

Uncommon

Atherosclerosis

1:10,000

Increased TG synthesis Atherosclerosis Increased TG synthesis/ Pancreatitis/ clearance gastrointestinal

Common 1:106 1:500 1:20 1:100

Common 1:500

Modified from reference 63. LpL, Lipoprotein lipase; TG, triglyceride.

form lipid droplets and give the cells the appearance of the classical foam cell. Unfortunately, both LDL and oxidized LDL activate macrophages still further, causing their release of a variety of proinflammatory cytokines. In addition, LDL and oxidized LDL induce phenotypic modulation of smooth muscle cell (SMC) from the normal growth-stable, quiescent phenotype to the proliferative (and synthetic) “atherosclerotic” phenotype. By mechanisms that are not clear, SMCs also acquire excess cholesteryl esters and develop an appearance nearly indistinguishable from that of macrophage-derived foam cells. Thus, as a result of SMC proliferation, matrix synthesis (ie, collagen and glycosaminoglycans), and accumulation of macrophage and smooth muscle– derived foam cells (and their lipids), the lesion expands, taking on the characteristics of a space-occupying (ie, vessel lumen) lesion. Ironically, the majority of lesions have little to no clinical significance, but the small minority of lesions that become unstable and rupture account for most of the acute atherosclerotic syndromes. It is important to point out that popular consensus holds that atherosclerosis is fundamentally an inflammatory disease.24 However, this paradigm fails to address the root cause of this important disease.25 Clearly, inflammatory processes contribute importantly to mediating many of the cellular events during atherogenesis; however, they are not likely the underlying disturbance. Getting back to basics, the root cause is more likely at the level of increased retention of atherogenic lipoproteins in the arterial wall,23,26 which results in an imbalance between forward and reverse cholesterol transport locally. It needs to be stressed that focusing attention on

the inflammatory aspect of atherosclerosis27-30 should not divert attention away from the true fundamental problem: restoring balance between forward and reverse cholesterol transport (ie, normalizing blood lipid levels). CLINICAL CLASSIFICATION OF DYSLIPIDEMIAS With use of the scheme outlined in Figure 1, the basic dyslipidemias that appear in clinical practice have been defined31 and are listed in Table 3. Although these phenotypes are straightforward, they are not inclusive of lipoprotein alterations underlying vessel disease, and the underlying etiology is not always evident. Type I Type I hyperlipidemia is an uncommon lipid disorder caused by impaired lipoprotein lipase activity so that triglyceride removal is retarded and the circulating halflife of chylomicrons is greatly extended, raising triglyceride levels. Impaired lipoprotein lipase can result from genetic defects in its synthesis and/or expression on the EC-surface membrane. Patients with type I dyslipidemia do not have accelerated atherosclerotic disease, but the increased chylomicron levels complicate pancreatic function and they usually have pancreatic/digestive disorders. Type II Type II hyperlipidemia is the most common and includes type II-a and type II-b, both of which are characterized by elevated blood cholesterol levels. Type

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II-a includes the rare monogenic form, familial hypercholesterolemia, which is caused by a variety of defects in the LDL receptor. Type II-a also includes the polygenic form (by far the most common dyslipidemia), which is much more complicated and appears to require environmental factors as well as genetic defects that impact negatively at a variety of loci in the LDL metabolic pathway. Type II-b dyslipidemia, familial combined hyperlipidemia, is less common and is characterized by an increase in both cholesterol and triglyceride levels. It is caused by an increase in the hepatic synthesis of apo B-100. Because apo B is important in VLDL synthesis, VLDL levels are elevated, an effect that drives the increase in the downstream lower-density lipoproteins IDL and LDL, thus giving rise to increased cholesterol levels as well as triglyceride levels (hence, “combined hyperlipidemia”). Type III Type III hyperlipidemia, dysbetalipoproteinemia, is a rare disorder characterized by elevated levels of chylomicrons and ␤-VLDLs. It is caused by the synthesis of a dysfunctional apo E, resulting from a mutation in the ApoE gene on chromosome 19. The normal catabolism of remnant lipoproteins, which is directed by apo E, is altered in this dyslipidemic state. The altered apo E is not recognized by the LDL receptor, and hence, clearance of apo E– containing lipoproteins (chylomicrons and VLDLs) is impaired, thereby resulting in elevation of chylomicron and VLDL remnants (␤-VLDL) in fasting plasma. Because they remain in the circulation longer, the action of lipoprotein lipase depletes triglyceride levels further, leaving these particles cholesterol-enriched. Thus the VLDL remnant is a terminal particle that migrates more slowly on gel electrophoresis (hence, “␤-VLDL”) and is particularly atherogenic. These particles are readily taken up by the unregulated scavenger receptor on macrophages, leading to the deposition of cholesteryl esters and formation of foam cells in peripheral tissues. As a result, coronary artery atherosclerosis is usually severe. Interestingly, atherosclerotic plaques also appear in the endocardium of the left atrium and ventricle, as well as on the mitral leaflets and in the pulmonary artery. As a side note, when triglycerides are presented to the circulation at a rate too great for the body to dispose of them (eg, in the postprandial period), hypertriglyceridemia results in the first hours after a meal. In the common laboratory rabbit, increasing the cholesterol content in the diet produces fasting hypercholesterolemia, but the primary cholesterolcarrying particle is ␤-VLDL, not LDL as in human type II hyperlipidemia. This results from an inability

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of the rabbits to clear apo B– and apo E– containing lipoproteins efficiently, as the LDL receptor is downregulated by excess cholesterol. This extends the circulating half-life of VLDL, and coupled with the delipidating action of lipoprotein lipase, cholesterolrich ␤-VLDL is generated and becomes the primary cholesterol-carrying lipoprotein. Hence, cholesterol feeding to rabbits produces fatty streak lesions in the short term (2-4 months) and mature humanlike lesions in the long term (years). Thus the hypercholesterolemia in rabbits is fundamentally different from the common hypercholesterolemia in human beings, in that increased ␤-VLDL instead of LDL is the most abundant cholesterol-carrying lipoprotein, rendering this model more similar to human type III dyslipidemia. In addition, a popular animal model of human atherosclerosis is the apo E knockout mouse. These animals cannot make apo E, and thus, apo E– containing lipoprotein levels elevate (elevated chylomicron remnant, apo B-48). Thus, although they develop lesions similar to those in mature human atherosclerosis, they do so by a dyslipidemia fundamentally different than that seen in human beings. Type IV Type IV hyperlipidemia is a common disorder characterized by elevations in triglyceride levels due to elevations in VLDL levels but with maintenance of normal levels of cholesterol. In these patients, increased synthesis of triglycerides is the metabolic disorder underlying the elevated triglyceride levels. It is inheritable in nature and is thus referred to as familial hypertriglyceridemia. Type V Type V hyperlipidemia is rare but biochemically similar to the more common type IV hyperlipidemia, in that it is characterized by elevated triglyceride levels. However, it is genetically distinct from type IV. In this disorder, increased synthesis of triglycerides occurs, leading to elevated triglyceride levels, but there is also a decrease in clearance of chylomicrons and VLDLs. In this way, it is clinically similar to lipoprotein lipase deficiencies, in that pancreatic/digestive disorders are more likely than atherosclerotic disease. LIPOPROTEIN SUBCLASSES The paradigm that VLDL, IDL, and LDL are linked in a continuous metabolic cascade in which triglycerides are lost in a series of small delipidation steps is an overly simplistic view of the generation of the downstream

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remnants of VLDL (ie, IDL and LDL). It is now clear that within each class of the LDLs, distinct subclasses exist that can differ in their physical properties, clinical significance, and even origin.32 The most notable example comes from studies that demonstrated that within the LDL class, particles of the smallest discrete size range are associated with CAD to a greater degree than are larger, less dense LDL particles. Separation by both size and density reveals that the smaller atherogenic LDL particles are also more dense (ie, less lipid/unit volume). Density differences between lipoproteins result mainly from differences in triglyceride content of the particle core, as the phospholipid and apoprotein coat is of constant thickness.33,34

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study.40 IDL1 particles are larger and less dense, and IDL2 particles are smaller and more dense (a third yet smaller class, IDL3, can also be identified). IDL1 particles appear to delipidate preferentially to intermediatesized LDL2 particles; IDL2 particles, on the other hand, give rise to the largest-sized LDL1 particles. Thus distinctly different pathways appear to exist for IDL metabolism, raising the question of whether clinical significance can be differentially attributed to the different IDL subclasses. This is an important and unanswered question, as elevated IDL levels have been linked to increased risk of CAD, suggesting that IDL may have atherogenic potential.41 LDL

VLDL VLDL subclasses have been divided into three groups ranging from large, less dense particles (VLDL1) to small, more dense particles (VLDL3). Delipidation by lipases underlies, in part, the generation of the intermediate density VLDL (VLDL2) and VLDL3 from VLDL1, but the process is complicated further by the independent synthesis of both VLDL1 and VLDL2 by the liver. The delipidation of VLDL1 to VLDL2 is critically dependent on lipoprotein lipase activity but independent of hepatic lipase activity. The significance of VLDL subclasses was revealed by the observation that variation in plasma triglyceride levels is principally a function of changing VLDL1 levels,35 and it is this subclass that is lowered by fibrate therapy.36 Patients with hypertriglyceridemia produce an excess of VLDL1 apo B synthesis (type IV hyperlipidemia),32 whereas those with raised cholesterol levels overproduce VLDL2 apo B (type II-b hyperlipidemia).37 In addition, lipoprotein lipase activity defects lead to elevation of the large, less dense VLDL1 particles. IDL IDL subclasses have also been identified. IDL has long been recognized as an intermediate lipoprotein in the delipidation cascade. However, it was later shown that not all IDLs were derived from VLDLs and that not all IDLs were delipidated to LDLs; a significant fraction of the IDL class was taken up by the LDL receptor directly by the liver, as reflected by the finding that IDL clearance was greatly reduced in familial hypercholesterolemia homozygotes.38 Moreover, hepatic lipase was shown to be essential for the IDL to LDL conversion, as subjects with hepatic lipase deficiency had elevated IDL levels.39 At least two subclasses of IDL particles have been identified, which, unfortunately, overlap in size and density, making them difficult to easily isolate and

LDL subclasses have recently attracted attention as a result of observations that small, dense LDL is the most atherogenic subclass of LDL and that this class is elevated in diabetes mellitus and metabolic syndrome. Originally, LDL was considered a population of particles of continuously variable size and density. Krauss and Burke42 were the first to demonstrate that this paradigm was not accurate, by providing evidence that in virtually all subjects, LDL is made up of a small number of discrete size and density subclasses. Ranging in size and density, LDL1 is the largest and least dense, whereas LDL2 is intermediate and LDL3 is the smallest and most dense.32 Working with Krauss, Austin et al43 later showed that patients with a preponderance of small, dense LDL had a 3-fold increase in risk of myocardial infarction independent of the total LDL level. In addition, it was noted that subjects with a preponderance of small, dense LDL had higher levels of triglycerides.44 Austin et al43 also identified the pattern B phenotype: elevated small, dense LDL coupled with elevated triglyceride, elevated IDL, and decreased HDL levels; pattern A (large, less dense LDL) was always present at low plasma triglyceride levels.44 The observation that pattern A (large, less dense LDL) was always present at low plasma triglyceride levels (⬍0.05 mmol/L) and pattern B was found in most subjects with high triglyceride levels (⬎2.0 mmol/L) has led to the suggestion that a plasma triglyceride level of 1.5 mmol/L is the threshold value, above which either LDL2 is increasingly converted to LDL3 in the circulation or LDL3 rather than LDL2 is the preferred product of VLDL delipidation.32 In 1990 Austin et al44 coined the term atherogenic lipoprotein profile, which describes the syndrome of small, dense LDL, elevated VLDL, and low HDL. This syndrome is emerging as a significant lipid risk profile for CAD. Moreover, elevated VLDL1 levels are likely the key metabolic disturbance in this syndrome,32 and its eleva-

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tion with obesity (ie, metabolic syndrome) and diabetes supports this concept. The logic behind this hypothesis is driven by the notion that small, dense LDL results from the removal of “excess” triglyceride from LDL. However, the cholesteryl ester–rich core of LDL normally limits excessive delipidation of LDL to the smaller, denser particles. It has been proposed that through the action of CETP, which mediates the exchange of cholesteryl esters for triglycerides between lipoprotein particles, small, dense LDL is generated when VLDL levels are high (as, for example, in hyperinsulinemic conditions).32 In this scenario, with high chylomicron/VLDL levels (ie, hypertriglyceridemia), a portion of the core triglycerides are transferred to LDL (and HDL) and in exchange for cholesteryl esters removed from LDL. In this way, triglyceride-rich LDL is formed and acted on by the lipases, leading to the generation of the small, dense LDL species. This unifying hypothesis highlights a role for hypertriglyceridemia in the initiation of the atherogenic lipid profile associated with the pathogenesis of vascular disease. HDL An inverse and independent relationship between HDL levels and risk of CAD has been firmly established.45 More than 40% of patients with acute myocardial infarction have low HDL levels.46 Low HDL and apo A-I levels have been identified as the most important lipid risk factor for coronary events in patients with confirmed CAD.47 In addition, low HDL levels were the most frequent familial dyslipidemia in patients with premature myocardial infarction,48 and the Helsinki Heart Study49 and the Veterans Affairs High-Density Lipoprotein Cholesterol Intervention Trial Study50 demonstrated that increases in HDL on treatment with gemfibrozil correlated with a reduction in coronary events. Thus the preponderance of evidence supports a role for HDL in the physiologic protection against atherosclerotic vascular disease, and HDL cholesterol levels have become an important factor in the assessment of global risk for peripheral vascular disease. As with LDLs, HDLs have been shown to comprise at least 3 distinctly different subclasses based on size. Interestingly, identification of subclasses of HDL preceded the discovery of subclasses of LDL. Figure 2 illustrates the physiology of HDL subclasses and the relative role each plays in the reverse cholesterol transport scheme. The more common subclasses of HDL particles are nascent HDL (ie, pre–␤-HDL), HDL3, and HDL2 (smaller, more dense to larger, less dense, respectively) (Table 4). Nascent HDL is the progenitor particle of the HDL class. Although the identity and physiology

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of this particle are not clear, consensus holds that it is synthesized by the liver and intestine, initially as only the apo A-I protein, a modest 29-kd protein that acquires phospholipid in a manner that is not yet fully understood. Once associated with a small amount of phospholipid, a disklike particle forms that avidly adsorbs free cholesterol from cell membranes. Relative to concentrations of lipid-rich ␣-HDL (␣-HDL ⫽ HDL2 ⫹ HDL3), the concentration of lipid-poor pre–␤-HDL is increased in the extravascular compartment including the lymph, in which reverse cholesterol transport is initiated in vivo.51 In support of a role for pre–␤-HDL in reverse cholesterol transport, this particle is especially efficient at removing cholesterol from peripheral cells.21 Free cholesterol accumulates on the surface of the particle with the 3-hydroxyl group oriented to the aqueous phase and the ring structure oriented toward the hydrophobic core (as in membranes). LCAT catalyzes the esterification of cholesterol by adding a long-chain fatty acid, usually oleic acid, to the 3-hydroxyl group. In this step, cholesterol loses its amphipathic property and is now fully hydrophobic, so it moves into the internal core of the particle where it can exist in a thermodynamically more stable lipophilic environment. In this way the disk structure inflates with cholesteryl esters. Apoproteins A-II, E, and C are acquired from the lower-density lipoproteins as it circulates in the blood, and the particle matures into HDL3, the next larger particle of this subclass. HDL3 continues to adsorb cholesterol from the surface membranes of peripheral cells and expands to HDL2, the largest of the HDL particles. Current evidence supports a role for all three HDL particles in reverse cholesterol transport,51,52 but interestingly, clinical studies suggest that CAD is most strongly correlated (negatively) with levels of the HDL2 subclass.53-55 The mechanism by which HDLs acquire cholesterol is intriguing. It was originally thought that cholesterol was acquired solely by aqueous diffusion from the cell-surface membrane to the particle’s surface without a need for specific binding of the particle to the cell.56 However, more recent studies have identified distinct HDL receptors, namely SR-B1, that bind HDL and anchor it near the cell surface, and while in this configuration, cholesterol efflux from the cell to the HDL particle, by aqueous diffusion, is facilitated. Another important cholesterol efflux pathway involves the adenosine triphosphate– binding cassette transporter 1 (ABCA-1). This cell-surface transporter hands off cholesterol and membrane phospholipid (“microsolubilization”57) to apo A-I, thereby providing another source for nascent HDL (pre–␤-HDL) particles. This explains why, in Tangier disease, in which mutations occur in the ABCA-1 transport protein, there is a defect in cell lipid efflux that leads to the absence of lipid-rich ␣-HDL.

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Figure 2. HDL map showing the origin of HDL as pre–␤-HDL and its evolution to the mature HDL3 and HDL2 particles. Efflux of free cholesterol from peripheral cells occurs by aqueous diffusion but is facilitated by HDL binding to the HDL receptor SR-B1 (not shown; see text). Note free (unesterified) cholesterol (FC) initially associates with the particle surface, but by the action of LCAT, it is esterified, allowing it to enter the core of the particle. In addition, the bulk of HDL cholesterol (ie, now cholesteryl ester [CE]) is removed to apo B– containing lipoproteins by CETP for eventual disposal by the hepatic LDL receptor. (Modified from von Eckardstein et al. Arterioscler Thromb Vasc Biol 2001;21:13-27.)

Table 4. Lipoprotein subclasses by size and density

Density Low Intermediate High Low Intermediate High Low Intermediate High Low Intermediate High

Lipoprotein VLDL1 VLDL2 VLDL3 IDL1 IDL2 IDL3 LDL LDL LDL HDL2 HDL3 pre–␤-HDL

Size (nm) 60-100 40-60 30-40 28-30 27-28 — 23-30 20.5-23 18-20 8-13 7-8 ⬍7

Compiled from data in references 8 and 32. Attempts to account for size and density between laboratories are difficult, probably because of the differences in techniques used by laboratories in the emerging new field of lipoprotein biology.

Lastly, although a clear role for all of the HDLs in reverse cholesterol transport and atheroprotection is indisputable, interesting functions for HDL beyond reverse cholesterol transport have also come to light in recent

years.58 For example, HDL also inhibits the chemotaxis of monocytes, leukocyte adhesion to EC, EC dysfunction, LDL oxidation, and complement activation. In addition, HDL stimulates the proliferation of ECs. Hence, beyond reverse cholesterol transport, HDL appears to have antioxidant, antiinflammatory, antiadhesive, antiaggregatory, and profibrinolytic effects as well. ASSESSING CARDIOVASCULAR RISK Since the advent of statins, the first of the truly efficient LDL cholesterol–lowering agents, numerous interventional trials have clearly shown that decreases in LDL levels reduce vascular events as either primary or secondary interventional therapies. Their ability to reduce LDL cholesterol levels with modest elevation in HDL levels improves the coronary risk profile, supporting the notion that LDL and HDL cholesterol are general predictors of vascular disease risk. However, we are painfully aware that the extent of occlusive disease3 and CAD4 varies greatly between individuals with similar lipid profiles. Interestingly, the new Adult Treatment Panel III guidelines do not use the LDL/HDL ratio to assess CAD risk.59 Instead, the new recommendation relies on the presence of elevated LDL in concert with

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traditional CAD risk factors or “risk equivalents” (cigarette smoking, hypertension, family history, and diabetes) to predict CAD risk by means of the Framingham Risk Score. Thus the new guidelines now focus on LDL cholesterol levels but in the context of the well-documented CAD risk factors. The emergence of lipoprotein subclasses, as well as their metabolic and clinical significance, has shed new light on lipoprotein physiology that may extend our understanding of the relationship between dyslipidemias and CAD and provide even greater accuracy in predicting CAD risk. This is particularly evident from the studies giving rise to the concept of the atherogenic lipoprotein profile that introduces the role of the triglyceride-rich large VLDL1 in the genesis of small, dense LDL3 and large HDL2 subfractions. For this reason, accounting for these lipoprotein subfractions in the algorithm for predicting global cardiovascular risk, along with the new Adult Treatment Panel III guidelines, should improve risk assessment. Unfortunately, ready access to services for providing lipoprotein subfraction profile screening is not yet available for routine screening. As these services become more available, expansion of our database from the general hospital population will more clearly define these associations and likely provide new targets for lipidlowering strategies. CHOLESTEROL-LOWERING DRUGS The preferential inhibition of the formation of small, dense LDL would seem to be a beneficial atheroprotective strategy. Studies aimed at determining whether statins have the capability to normalize lipid particle distribution are currently under way. Statins are inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase, the rate-limiting step in cholesterol synthesis. Thus all of the statins upregulate hepatic LDL receptors, as the synthesis of these receptors is under the control of cell cholesterol levels.60 They are highly effective in lowering both total cholesterol levels (about 35%) and LDL cholesterol levels (about 45%) in primary hypercholesterolemia, but they have limited capacity in reducing triglyceride levels (about 10%). They also increase HDL levels, but only by about 5%, and this effect does not appear to contribute to the reduction in the mortality rate (approximately 30%) seen with statin therapy. Fibrates, on the other hand, inhibit VLDL secretion, increase lipoprotein lipase activity, and have the opposite action, decreasing triglyceride levels (up to 50%) with little effect on LDL levels (about 10%). With regard to statins, the general body of evidence suggests that in most patients, statins uniformly lower all LDL subclasses without preferential actions on any one class.

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For example, pravastatin-induced alterations in lipid subclasses were not different in patients with large or small LDLs at baseline.60 Instead, the levels of the most abundant LDL subclass at baseline were preferentially lowered regardless of LDL subclass. Accordingly, analyses in this study indicated that the mean LDL size increased in patients with a preponderance of small LDL particles, leading to the conclusion that high-risk patients with small, dense LDL particles gained as much therapeutic benefit as those patients with large LDL particles. However, in another study, patients with familial combined hyperlipidemia (type II-b) were treated with atorvastatin (10 mg/d for 6 weeks), which also equally reduced all of the LDL subclasses. Interestingly, in this study atorvastatin also reduced VLDL1 levels, and in terms of absolute lipoprotein mass, the reduction in small, dense LDL was greatest, and a shift in the LDL profile toward the more buoyant larger particles was seen.61 In a more recent study by the same investigators, again in subjects with type II-b dyslipidemia, atorvastatin dose-dependently (10 mg/d vs 40 mg/d for 6 and 12 weeks) reduced the small, dense LDL subfraction. Interestingly, an increased ability of the patient’s plasma to extract cholesterol out of cells in an vitro assay was also observed.62 These findings are consistent with the notion that at least some of the statins can normalize dyslipidemias associated with an abundance of atherogenic small, dense LDL. It needs to be emphasized, however, that we are now only in the early phase of therapeutic investigations directed at modulating lipoprotein subclasses, and the next several years should provide a broader view of the ability of statins, and possibly other drugs, to normalize lipoprotein subclasses and CAD risk. Beyond statins, the pharmaceutical industry is aggressively pursuing agents directed toward elevating HDL levels. In particular, CETP inhibitors are currently in the industry pipeline and show promise of raising HDL cholesterol levels by 50% or more. However, the clinical impact of these agents and their overall safety have yet to be confirmed. In summary, there is little doubt that dyslipidemias constitute the largest single factor underlying causality in atherosclerotic syndromes. Tremendous progress has been made in understanding the molecular nature of dyslipidemias and how they can be modified with lifestyle changes and/or pharmacotherapy. Adding to this is the potential for still more exciting new breakthroughs to further modulate dyslipidemias toward the normal lipid profile and achieve still greater improvements in morbidity and mortality rates associated with atherosclerotic disease. Acknowledgment The authors have indicated they have no financial conflicts of interest.

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