The HDL Handbook: Biological Functions and Clinical Implications

1

Introduction of HDL Molecules, Past and Brief Future

Mari Tabuchi∗ and Tsugikazu Komoda∗∗,    ∗Department of Chemistry, Faculty of Science, Rikkyo University, Tokyo, Japan,    ∗∗Department of Urology, Toho University School of Medicine, Tokyo, Japan

Abstract

Our understanding of high-density lipoprotein cholesterol (HDL-C) and its relationship to coronary heart disease (CHD) has changed dramatically over the past half century. The initial discovery of the protective role of HDL-C was made by Gofman in the mid-1950s. In the mid-1970s, Glomset’s pioneering studies on reverse cholesterol transport (RCT) and Gordon’s Framingham study supported the concept that HDL-C was a good lipoprotein, since there was a strong inverse correlation between the levels of serum HDL-C and the subsequent risk of atherosclerosis. Furthermore, in the late-1980s, a decreased concentration of serum HDL-C emerged as one of the major risk factors for coronary artery disease. However, recent studies in the early 2000s have shown that high HDL-C levels do not necessarily have anti-atherogenic effects. In patients with cholesterol ester transfer protein (CETP) deficiency, high HDL-C levels have no anti-atherogenic effect. Furthermore, drugs that inhibit CETP elevate HDL-C levels but do not decrease the risk of cardiac events. Thus, the traditional anti-atherogenic role of HDL-C has been questioned. There is evidence that HDL-C molecules have additional atheroprotective roles beyond bulk cholesterol removal from cells through RCT. Other studies suggest that the widely used diagnostic measurement of HDL-C may have a limitation and that the qualitative measurement of HDL-C molecules may be important in the near future.

Keywords

Atherosclerosis; Cholesterol ester transfer protein (CETP); Coronary heart disease (CHD); Cardiovascular disease (CVD); High-density lipoprotein (HDL); Low-density lipoprotein (LDL); Reverse cholesterol transfer (RCT)

Contents

1. Introduction

1.1. HDL-C as Good Cholesterol

2. HDL May be an Independent Predictor of CHD

3. High Levels of HDL Sometimes Cause Atherosclerosis: the Example of CETP Deficiency

4. Is HDL-C Really Good Cholesterol?

5. Quantitative and Qualitative Measurement of HDL-C Molecules

6. Conclusion

References

1 Introduction

The high-density lipoprotein (HDL) molecule consists of particles that are heterogeneous in size, density, and composition. Recently, views on the relevance and utility of HDL have been changing dramatically. For over a half century, plasma HDL-cholesterol (HDL-C) was believed to play an important role in the protection against atherosclerosis. HDL-C was termed good cholesterol and thought to be a negative risk factor for atherosclerosis. However, an important discovery has been made recently. Although a deficiency or low-level of HDL-C causes premature atherosclerosis, some conditions where there is elevated HDL-C have been found to increase the risk of coronary atherosclerosis. Thus, HDL-C can sometimes become bad cholesterol and increase the risk of atherosclerosis. This chapter gives a brief introduction on the history of HDL-C molecules and suggests future directions for investigation.

1.1 HDL-C as Good Cholesterol

HDL was first isolated from horse serum in 1929 [1] and from human serum in 1949 [2]. By 1950 total cholesterol and LDL-C took center stage, although the role of HDL-C was unclear [3]. Several novel techniques for lipoprotein separation were developed, including ultracentrifugation [4,5] and electrophoresis [6], that allowed evaluation of the role of HDL-C in atherosclerosis.

The initial discovery of the protective role of HDL-C was made by Gofman at the Donner Laboratory of the University of California at Berkeley in 1956 [7]. He separated HDL-C subclasses using an ultracentrifuge and demonstrated an inverse association between HDL-C levels and coronary heart disease (CHD). However, his work was not widely accepted, because other researchers were unable to replicate his results, and total cholesterol and low-density lipoprotein cholesterol (LDL-C) were the primary focus at that time. Subsequently, many researchers have investigated the role of HDL-C in CHD.

In the 1960s, Glomset [8] investigated the mechanism of the plasma cholesterol esterification reaction and hypothesized that HDL-C plays a role in the transport of cholesterol from peripheral tissues to the liver that was termed reverse cholesterol transport (RCT).

In 1975, Miller found that plasma HDL-C was unrelated to the plasma concentration of total cholesterol and other lipoproteins [9]. This finding supported the RCT hypothesis. He also showed that a reduction of plasma HDL-C concentration accelerated the development of atherosclerosis, which supported the protective role of HDL-C against CHD. The anti-atherogenic role of HDL-C was proposed to be a result of its role in RCT—removal of excess cholesterol from peripheral tissues to the liver for re-use.

Berg also investigated the relationship between serum HDL-C levels and atherosclerotic heart disease [10]. The mean HDL-C concentration was significantly lower in men with CHD in comparison with those without CHD. These results were in accordance with the hypothesis that high levels of HDL-C protected against CHD to some extent. Gordon also confirmed that HDL-C played a protective role against CHD in the Framingham study in 1977 [11]. In that study, there was an inverse association between HDL-C levels and the incidence of CHD.

2 HDL May be an Independent Predictor of CHD

The anti-atherogenic property of HDL-C was gradually accepted in the mid-1970s, since there was a strong inverse correlation between the levels of serum HDL-C and the subsequent risk of atherosclerosis. Furthermore, in the late-1980s, a decreased concentration of serum HDL-C emerged as one of the major risk factors for CHD.

Several studies including the Framingham heart study found that low HDL-C levels, widely prevalent in countries with Western diets, was an independent predictor for cardiovascular disease (CVD) risk [11–14]. This risk was true even in the presence of low LDL-C levels [15]. These findings increased the attention paid to HDL-C as a secondary prevention target for CVD risk [16,17]. Moreover, some researchers were convinced that HDL-C might be an even stronger risk factor for CHD than LDL-C. Conventionally, cardiovascular prevention strategies emphasize therapeutic reductions in LDL-C [18,19]. However, increasing attention had been focused on HDL-C as a secondary prevention target to address residual CVD risk [16,17].

3 High Levels of HDL Sometimes Cause Atherosclerosis: the Example of CETP Deficiency

Some recent studies have shown that high HDL-C levels do not necessarily have anti-atherogenic effects, although the risk for atherogenesis is lower in the many patients with high HDL-C. Plasma Cholesterol Ester Transfer Protein (CETP) deficiency was originally reported in Japanese siblings with hyperalphalipoproteinemia [20]. Increased HDL-C is sometimes clustered in families. The CETP deficiencies were associated with high HDL-C levels and relatively low LDL-C levels (mean levels of 164 mg/dL and 77 mg/dL, respectively) [21]. Many studies showed that a low CETP mass and increased HDL-C was associated with a decreased risk of CHD [22–24]. In a Kochi Prefectural Institute of Public Health’s cross-sectional survey, no cases of CHD were found in 300 subjects with HDL-C > 100 mg/dL [25]. A meta-analysis of multiple clinical studies also supported the concept that lower CETP levels might have an anti-atherogenic effect [26].

However, epidemiological studies in Japanese Americans living in Hawaii and Japanese in the Omagari area have shown a relatively increased incidence of CHD in CETP deficiency [27], and homozygous CETP deficiency in some cases was thought to be pro-atherogenic [28]. Thus, high-HDL levels in some patients do not appear to have an anti-atherogenic effect.

4 Is HDL-C Really Good Cholesterol?

Some clinical treatments, such as niacin [29], torcetrapib [30], and dalcetrapib [31], do not reduce cardiac risk, although HDL-C and/or LDL-C levels are successfully regulated. Statins effectively lower LDL-cholesterol levels, but residual cardiovascular risk remains [29]. Inhibition of CETP with torcetrapib successfully elevated HDL-C levels, but unexpectedly increased cardiovascular morbidity and mortality [30]. Dalcetrapib, another inhibitor of CETP, raised HDL-C levels in patients hospitalized with acute coronary syndrome, but this change failed to translate into a reduction in cardiovascular events [31]. There was no association between gene variants exclusively related to HDL-C concentration and myocardial infarction [32]. These results indicate that HDL-C might not have a causal role in preventing CHD. Although many early clinical studies showed evidence for a beneficial effect of HDL-C on CHD, the cardioprotective effect of increasing levels of HDL-C has been questioned.

5 Quantitative and Qualitative Measurement of HDL-C Molecules

It is thought that HDL-C molecules have additional atheroprotective roles beyond bulk cholesterol removal from cells through RCT. The anti-inflammatory and anti-oxidant activities of HDL-C molecules have recently attracted considerable attention [33,34]. These activities of HDL-C are associated with protection from cardiovascular disease. One of the promising mechanisms was reported as response to injury by Ross in 1993 [35].

There are currently seven different direct homogenous HDL-C assays [36] that use chemical precipitation methods with reagents such as dextran sulfate instead of physical ultracentrifugation. In a recent report, the error between the seven direct assays was higher than 12%, resulting in inaccurate HDL-C measurement and subsequent CVD risk estimated by LDL-C [37]. Recently, results of long-term follow-up using an analytical ultracentrifuge have demonstrated that the subfractions HDL2 and HDL3 are independently related to CHD risk [38]. CETP-deficient patients have very large HDL-C particles [39], and CHD patients often have small discoidal HDL-C particles [40]. Large HDL-C particles increase with weight loss, niacin, certain statins, and CETP inhibitors [41–46]. These findings suggest that the widely used diagnostic measurement of HDL-C may have a limitation, and that the measurement of size and shape of HDL-C molecules could be more important than previously thought [47,48]. Thus, the definition should be changed from quantitative measurement of HDL-C to qualitative evaluation of HDL-C subfractions [35,49–51]. However, since HDL-C consists of polydisperse particles that are heterogeneous in size, density, shape, and composition, separations require complicated methods, such as electrophoresis [50] or chromatography [52], and are not available for routine clinical use.

One of the promising methods for the measurement of HDL-C molecules is a technique that uses bioformulated-fiber matrix electrophoresis [53]. High- and low-density fractions of HDL-C were well separated by this method. Although a higher density is generally believed to correlate with a smaller particle size, this separation contradicted this belief. Direct microscopic observation of fractioned HDL-C confirmed the lack of a relationship between density and size. This technique may be useful for the diagnosis of dyslipidemia in the future.

6 Conclusion

In conclusion, according to the many studies including clinical and epidemiological investigations over more than half a century, the effect of HDL-C on CVD may be more complicated than previously thought. These important new findings may be useful in estimating the risk of CHD in the near future.

References

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Chapter 2

Apolipoprotein A-I Mutations and Clinical Evaluation

Akira Matsunaga,    Department of Laboratory Medicine, Faculty of Medicine, Fukuoka University, Fukuoka, Japan

Abstract

Apolipoprotein (apo) A-I accounts for 70% of the total protein in high-density lipoprotein (HDL) and plays a key role in HDL biogenesis and function. Analyses of the apoA-I amino acid sequence have revealed that most of its 243 amino acid residues are grouped into amphipathic α-helices of 11 or 22 amino acids in length. Since the hydrophobic C-terminal domain (residues 190–243) of the human apoA-I molecule is critical for lipid binding, deletion of this segment reduces the ability of the protein to solubilize lipid. Sixteen different types of homozygous, compound heterozygous, and heterozygous apoA-I deficiencies, including large deficiency, inversion, frameshift, and nonsense mutations, have been reported. Most of the missense mutations causing low HDL cholesterolemia are associated with alterations to amino acids 143–187, forming α-helices 6–7 of apoA-I. Mutations in this region are accompanied by activation failure of lecithin:cholesterol acyltransferase (LCAT). Other point mutations leading to low HDL cholesterolemia include ApoA-I(S36A), (K107del), (R173C)Milano, (L178P), and (E235del)Nichinan. Twenty-one mutations that cause amyloidosis have been reported. The hereditary amyloidogenic mutations are clustered within amino acids 26−107 and 154−178 of apoA-I. ApoA-I (R151C)Paris and ApoA-I (R173C)Milano are rare cysteine variants that can form dimers. ApoA-IMilano, in particular, has been proven to exert anti-atherogenic effects in animal studies and small clinical trials. Although there are difficulties associated with its formulation, clinical applications are expected.

Keywords

apolipoprotein A-I; mutation; HDL deficiency; apolipoprotein A-I deficiency; ABCA1; apolipoprotein A-I Milano (apoA-IMilano); amyloidosis; apolipoprotein mimetic peptide

Contents

1. Introduction

2. Physiological Functions of apoA-I

3. Genomic and Protein Structure of apoA-I

4. ApoA-I Deficiencies with Large Deficiency, Inversion, Nonsense, or Frameshift Mutations

5. ApoA-I Missense Mutations with Low HDL-C

6. ApoA-I Mutations Related to Amyloidosis

7. Cysteine Mutants of apoA-I

8. Anti-Atherogenic apoA-I and apoA-IMilano

9. Conclusion

References

1 Introduction

Plasma HDL (hydrated density, 1.063–1.21 g/mL) comprises a heterogeneous group of small discoid and spherical particles (7–12 nm in diameter) that differ in density, size, and electrophoretic mobility. The HDL apolipoproteins include apoA-I and apoA-II, as well as apoA-IV, apoA-V, apoC-I, apoC-II, apoC-III, apoD, apoE, apoJ, apoL, and apoM. The main protein component of HDL is apoA-I, a 28-kDa protein that contains 10 amphipathic α-helical domains of 11 or 22 amino acids each. Approximately 70% of total plasma HDL protein is apoA-I (present in normolipidemic human plasma at ∼130 mg/dL), and it is located in essentially all HDL particles. The second most abundant protein is apoA-II (two 77-amino acid molecules forming a homodimer in humans with a disulfide-link at Cys6), which comprises 15–20% of total plasma HDL protein. However, this component is not present in all HDL particles. In human plasma, about 25% of apoA-I is present in HDL particles containing only apoA-I (LpA-I), and the remaining HDL particles contain both apoA-I and apoA-II (LpA-I+A-II), typically in a molar ratio of 1–2:1 [1]. The heterogeneity in HDL size and structure is intimately related to the amphipathic highly dynamic helical structure of apoA-I. HDL particles are constantly being remodeled as they transport cholesterol between cells and other lipoproteins. HDL particles are produced as small (< 8 nm in diameter), lipid-poor (lipid content: < 30%), discoid particles made up of apolipoproteins (mainly apoA-I), which carry small amounts of lipid, mainly phospholipids, and free cholesterol (see Figure 2.1). These particles represent an excellent substrate for lecithin:cholesterol acyltransferase (LCAT), the enzyme that generates most of the cholesteryl esters present in plasma. ApoA-I mutations can cause plasma HDL deficiency, as well as LCAT deficiency, and Tangier disease is caused by mutations of the gene encoding ATP-binding cassette transporter A1 (ABCA1).

Figure 2.1 Reverse Cholesterol Transport (RCT) and the Role of High-density Lipoprotein (HDL) and ApoA-I.

RCT is initiated from arterial macrophages by the interaction of lipid-free apoA-I with cellular ABCA1 to generate nascent HDL particles. ABCG1 adds additional cholesterol to HDL, and lecithin:cholesterol acyltransferase (LCAT) esterifies HDL-cholesterol to generate mature spherical HDL particles.

2 Physiological Functions of apoA-I

As the quantitatively predominant component of HDL, apoA-I is crucial for HDL formation. Reverse cholesterol transport (RCT) is a multistep process resulting in the net movement of cholesterol from peripheral tissues back to the liver via lipoproteins. At the first rate-limiting step of RCT, apoA-I interacts with ABCA1 on the cell surface to form discoid nascent HDL (Figure 2.1). The primary substrate for ABCA1 is lipid-free monomolecular apoA-I. The C-terminal domains of apoA-I, which are characterized by the greatest affinity for lipids, seem to play a crucial role in the interaction between apoA-I and ABCA1 [2–4]. Studies on synthetic peptides that mimic apolipoproteins have indicated that the interaction between apoA-I and ABCA1 is not sequence-specific, and instead the amphipathic helices of apoA-I were identified as the key structural motifs [5,6]. ApoA-I directly interacts with residues located in two extracellular loops of ABCA1 [7]. A three-step model of the interaction between ABCA1 and apoA-I proposed by Vedhachalam and colleagues [8] explains the contribution of the interaction of apoA-I with ABCA1 and membrane lipids, as well as the heterogeneous nature of the nascent HDL particles. The binding of apoA-I by ABCA1 would induce signaling responses that would stabilize ABCA1 and enhance phospholipid translocase activity. This would lead to compression of the phospholipid molecules in the exofacial leaflet of the membrane, which would bulge in the direction of the extracellular space. Consequently, more apoA-I would bind to the membrane protrusions. Step 3 would involve membrane microsolubilization and the creation of discoid nascent HDL particles. This stage would limit the rate of the entire ABCA1/apoA-I interaction. Variations in the lipid composition of the membrane curvatures created in different membrane environments would cause heterogeneity of the nascent HDLs, particularly in terms of the free cholesterol content. The discoid nascent HDL particles are thought to comprise a cholesterol-containing phospholipid bilayer, with two copies of apoA-I wrapped around the perimeter in a largely α-helical antiparallel double-belt conformation (Figure 2.1). ApoA-I on a nascent HDL particle activates LCAT whose apolar products, cholesterol esters, move from the surface to the core of the particle, converting it to a mature core containing spherical HDL (Figure 2.1). ApoA-I is also required to activate LCAT and mediate the interactions of HDL with cell surface receptors, such as scavenger receptor B1 or plasma membrane transporters, including ABCG1 (Figure 2.1) [9,10]. In mice and rabbits, knockout of the ApoA1 gene causes HDL deficiency, and conversely, transgenic overexpression of ApoA1 increases HDL cholesterol (HDL-C) in a gene dose-dependent manner. In susceptible animals with an atherogenic lipoprotein profile, atherosclerosis is enhanced by apoA-I deficiency and decreased by transgenic overexpression of apoA-I. Human subjects with apoA-I deficiency and apoA-I-deficient mice fail to form normal HDL particles