The HDL Handbook: Biological Functions and Clinical Implications

The HDL Handbook Biological Functions and Clinical Implications

Tsugikazu Komoda

AMSTERDAM l BOSTON l HEIDELBERG l LONDON NEW YORK l OXFORD l PARIS l SAN DIEGO SAN FRANCISCO l SINGAPORE l SYDNEY l TOKYO Academic Press is an imprint of Elsevier

Academic Press is an imprint of Elsevier 32 Jamestown Road, London NW1 7BY, UK 30 Corporate Drive, Suite 400, Burlington, MA 01803, USA 525 B Street, Suite 1800, San Diego, CA 92101-4495, USA First edition 2010 Copyright Ó 2010 Elsevier Inc. All rights reserved No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher. Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (+ 44) (0) 1865 843830; fax (+44) (0) 1865 853333; email: [email protected]. Alternatively, visit the Science and Technology Books website at www.elsevierdirect.com/rights for further information Notice No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-12-382171-3 For information on all Academic Press publications visit our website at elsevierdirect.com Typeset by TNQ Books and Journals Pvt Ltd. www.tnq.co.in Printed and bound in China 10 11 12 13 14 15 10 9 8 7 6 5 4 3 2 1

Preface

When I started to write this book “The HDL Handbook: Biological Functions and Clinical Applications”, Professor Takashi Miida of Juntendo University in Tokyo strongly supported my plan. Of course, he is also an excellent contributor to this book. In addition, Professor David Alpers, Professor Emeritus of Washington University, School of Medicine, well revised the present HDL book. Therefore, I want to thank him for his revision of this book. Unfortunately, since planning to publish this HDL book, two and half years have passed. Some contributors immediately accepted my planning, however, half of the contributors have not been able to complete the publication of “The HDL Handbook: Biological Functions and Clinical Applications”. However, the contents of this HDL book include up-to-date progress on HDL research. The contents of this book are a crystallization of the work from all the contributors, because, despite being busy, all the contributors carefully revised their chapters under the suitable comments from eight reviewers. Therefore, if you read this book, you will be fascinated by the renewal of development of HDL researches and the present book is a very useful tool not only for basic researchers in institutes or pharmaceutical companies but also practical physicians. In addition, this book will be evaluated as an HDL Bible for medical and co-medical graduate students by their counselors. Furthermore, since this book is small, it is portable. However, the contents of this HDL book contain the latest news of HDL molecules. Finally, I believe that this book should be read to give an excellent impression of the HDL fields. Tsugikazu Komoda, MD

Contributors

Aishah Al-Jarallah, Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada G.M. Anantharamaiah, Departments of Medicine, and Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL, USA Rachelle Brunet, Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada Giovanna Catalano, INSERM UMRS939, Hoˆpital de la Pitie´, Paris, France; UPMC Universite´ Pierre et Marie Curie, Hoˆpital de la Pitie´, Paris, France Eric Chabrie`re, Institut de Recherche Biome´dicale des Arme´es-Antenne CRSSA, De´partement de Toxicologie, Groupe Bioe´purateurs Catalytiques et Re´activateurs, La Tronche, France; Architecture et Fonction des Macromole´cules Biologiques, Groupe Biocristallographie, Biotechnologie et Enzymologie Structurale, Universite´ de la Me´diterrane´e, Marseille, France Geeta Datta, Department of Medicine, and Biochemistry, University of Alabama at Birmingham, Birmingham, AL, USA Maryse Guerin, INSERM UMRS939, Hoˆpital de la Pitie´, Paris, France; UPMC Universite´ Pierre et Marie Curie, Hoˆpital de la Pitie´, Paris, France Akira Hara, Laboratory of Biochemistry, Gifu Pharmaceutical University, Japan Hiroaki Hattori, Advanced Technology and Development Division, BML Inc., 1361-1 Matoba, Kawagoe, Saitama, Japan Neil J. Hime, Centre for Vascular Research, Sydney Medical School (Pathology) and Bosch Institute, The University of Sydney, Medical Foundation Building, Camperdown, NSW, Australia Satoshi Hirayama, Department of Laboratory Medicine, Juntendo University School of Medicine, Tokyo, Japan Akihiro Inazu, Department of Laboratory Sciences, School of Health Sciences, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Ishikawa, Japan Zorana Jelic-Ivanovic, Institute of Medical Biochemistry, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia

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Contributors

Tsugikazu Komoda, Nihon Medical Science Institute, II-4 Minami-Tohrimachi, Kawagoe, Saitama, Japan Jelena Kotur-Stevuljevic, Institute of Medical Biochemistry, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia Patrick Masson, Institut de Recherche Biome´dicale des Arme´es-Antenne CRSSA, De´partement de Toxicologie, Groupe Bioe´purateurs Catalytiques et Re´activateurs, La Tronche, France Akira Matsunaga, Department of Laboratory Medicine, Fukuoka University School of Medicine, Fukuoka, Japan Toshiyuki Matsunaga, Laboratory of Biochemistry, Gifu Pharmaceutical University, Japan Takashi Miida, Department of Laboratory Medicine, Juntendo University School of Medicine, Tokyo, Japan Takanari Nakano, Department of Biochemistry, Faculty of Medicine, Saitama Medical University, Saitama, Japan; Brentwood Biomedical Research Institute, Department of Medicine, School of Medicine, University of California Los Angeles, Los Angeles, CA, USA Daniel Rochu, Institut de Recherche Biome´dicale des Arme´es-Antenne CRSSA, De´partement de Toxicologie, Groupe Bioe´purateurs Catalytiques et Re´activateurs, La Tronche, France; Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany Keijiro Saku, Department of Cardiology, Fukuoka University School of Medicine, Fukuoka, Japan Makoto Seo, Department of Biochemistry, Faculty of Medicine, Saitama Medical University, Saitama, Japan Slavica Spasic, Institute of Medical Biochemistry, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia Vesna Spasojevic-Kalimanovska, Institute of Medical Biochemistry, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia Aleksandra Stefanovic, Institute of Medical Biochemistry, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia Naoki Terasaka, Biological Research Laboratories, Daiichi Sankyo Co., Ltd, Tokyo, Japan Bernardo Trigatti, Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada Yoshinari Uehara, Department of Cardiology, Fukuoka University School of Medicine, Fukuoka, Japan Jelena Vekic, Institute of Medical Biochemistry, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia

Contributors

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C. Roger White, Department of Medicine, and Biochemistry, University of Alabama at Birmingham, Birmingham, AL, USA Aleksandra Zeljkovic, Institute of Medical Biochemistry, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia Bo Zhang, Department of Cardiology, Fukuoka University School of Medicine, Fukuoka, Japan

Chapter 1

Role of Phospholipid Transfer Protein in HDL Remodeling and Atherosclerosis Hiroaki Hattori Advanced Technology and Development Division, BML Inc., Kawagoe, Saitama, Japan

INTRODUCTION Phospholipid transfer protein (PLTP) plays an important role in the regulation of high density lipoprotein (HDL) metabolism. The regulatory role of PLTP is achieved via its two main functions, phospholipid transfer activity (Tall et al., 1983; Rao et al., 1997) and the ability to modulate HDL size and composition in a process called HDL remodeling (Rye and Barter, 1986; Tu et al., 1993; Jauhiainen et al., 1993). The regulation of HDL metabolism is achieved by the concerted action of a number of plasma and cellular factors. These include the cellular receptors, scavenger receptor class B type 1 (SR-B1) and ATP-binding cassette transporter A1 (ABC-A1), as well as plasma proteins such as cholesteryl ester transfer protein (CETP), lecithin-cholesterol acyltransferase (LCAT), and the endothelial-bound enzymes, lipoprotein lipase (LPL) and triglyceride (TG) hydrolase hepatic lipase (HL). As indicated by the inverse relationship between HDL cholesterol and incidence of coronary heart disease in many epidemiological studies (Gordon and Rifkind, 1989), the plasma HDL level has a major impact on the progression of atherosclerosis. Although the exact mechanism behind the athero-protective role of HDL is still not fully understood, the reverse cholesterol transport (RCT) hypothesis has been widely accepted (Curtiss et al., 2006). Reverse cholesterol transport is the process by which cholesterol is transported from peripheral cells to the liver for elimination (Eisenberg, 1984). Preb-HDL particles, a subpopulation of HDL, act as efficient acceptors in the efflux process of cholesterol at the plasma membrane of peripheral cells (Eisenberg, 1984). PLTP is able to generate preb-HDL particles through HDL remodeling, and has a major role also in maintaining The HDL Handbook. ISBN: 978-0-12-382171-3 Copyright Ó 2010 Elsevier Inc. All rights of reproduction in any form reserved.

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FIGURE 1.1 The physiological role of PLTP in HDL metabolism. Participation of PLTP is illustrated by bold arrows. The functions of PLTP are: (i) transfer of surface remnants (phospholipids and cholesterol) upon lipolysis of triglyceride-rich lipoproteins; (ii) generation of preb-HDL during remodeling of HDL; and (iii) transfer of a-tocopherol from HDL particles to cell membranes. Abbreviations used: VLDL, very low density lipoprotein; LDL, low density lipoprotein; IDL, intermediate density lipoprotein; HDL, high density lipoprotein; Rem, remnant; PLTP, phospholipid transfer protein; CETP, cholesteryl ester transfer protein; LCAT, lecithin-cholesterol acyltransferase; HL, hepatic lipase; LPL, lipoprotein lipase; SR-BI, scavenger receptor class B type I; ABC-A1, ATP-binding cassette transporter A1; a-T, a-tocopherol.

plasma HDL levels owing to its ability to transport surface remnants produced by lipolysis of triglyceride-rich lipoproteins (Figure 1.1). Thus, PLTP can be envisioned to play an important role in the prevention of atherosclerosis (Castro and Fielding, 1988; von Eckardstein et al., 1996; van Haperen et al., 2000; Tall and Lalanne, 2003). In spite of these effects on HDL, studies in genetically modified mouse models have suggested that systemic PLTP deficiency is athero-protective in vivo, and that PLTP overexpression is pro-atherogenic. Recent studies have focused on this apparent inconsistency, and have examined the effects of local PLTP on atherogenesis, using transplanted macrophages.

MOLECULAR BIOLOGY AND STRUCTURE OF PHOSPHOLIPID TRANSFER PROTEIN The PLTP gene is located on chromosome 20 (20q12-q13.1), and has a length of 13.3 kilobases, including 15 introns. PLTP cDNA is 1750 bp in length, and encodes a 17 amino acid hydrophobic signal peptide and a 476 amino acid mature protein (Day et al., 1994). Most tissues show expression of PLTP

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mRNA, but liver and adipose tissue are probably the major contributors to plasma PLTP (Dusserre et al., 2000). Although the predicted molecular weight mass of the mature protein is 55 kDa, plasma PLTP appears as an 80-kDa protein by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDSPAGE) under reducing and non-reducing conditions (Oka et al., 2000a). The discrepancy between the calculated molecular weight mass and the mass estimated by SDS-PAGE may be explained by the fact that PLTP has six potential N-glycosylation sites and numerous O-glycosylation sites (Day et al., 1994). As the protein additionally contains four cysteine residues, it also has the potential to form two intra-chain disulfide bonds. In contrast to apolipoproteins that are primarily hydrophilic, PLTP has a high content of hydrophobic residues scattered throughout, with over 40% of the amino acids being hydrophobic. Three other hydrophobic proteins of the lipid transfer/lipopolysaccharide binding protein family, namely lipopolysaccharide-binding protein (LBP), neutrophil bactericidal permeability-increasing protein (BPI), and CETP share structural homology with PLTP. However, these proteins also exhibit significant structural differences (Albers et al., 1996). For example, the carboxyl terminal portion of CETP is the most hydrophobic of these four proteins, and its main function is to bind and transfer neutral lipids (Tall et al., 1983; Albers et al., 1984). Although the carboxyl terminal portion of PLTP is somewhat hydrophobic, it does not have the functional capacity to transfer neutral lipids (Tollefson et al., 1988). Unlike PLTP, BPI has a very basic amino-terminal domain, which is responsible for its cytotoxic activity, whereas the hydrophobic carboxyl-terminal domain is believed to anchor the protein in the granule membrane (Gray et al., 1989). LBP and BPI share 44% amino acid sequence identity to bind lipopolysaccharide (Schumann et al., 1990). Furthermore, both proteins have similar amino-terminal amino acids (Tobias et al., 1988). Secondary structure predictions suggest that PLTP has two potential transmembrane regions spanning from residues 169 through 181, and residues 288 through 304 (Albers et al., 1996). Two potential disulfide bonds exist between cysteine residues 5 and 129, and between cysteine residues 168 and 318. The cysteine residues 146 and 185 form a disulfide bridge that is essential for the correct folding and secretion of PLTP (Huuskonen et al., 1998, 1999; Qu et al., 1999). We have shown that two forms of PLTP exist in human plasma, one with high activity (HA-PLTP) and another with low activity (LA-PLTP) (Oka et al., 2000b; Ka¨rkka¨inen et al., 2002). The LA-PLTP is associated with apoA-I, and the HA-PLTP co-purifies with apoE (Murdoch et al., 2002). LA-PLTP is located between LDL and HDL on size-exclusion chromatography, having an apparent molecular mass of 520 kDa and a Stokes diameter of 12e17 nm (Oka et al., 2000a; Murdoch et al., 2002). In contrast, HA-PLTP is associated with an average molecular mass of 160 kDa and a Stokes diameter between 7.6 and 12.0 nm. HA-PLTP but not LA-PLTP is able to remodel HDL, resulting in the formation of two types of particles, preb-HDL and large fused HDL (Vikstedt

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et al., 2007a). The mechanism by which these two HDL subpopulations are generated is not known.

PLTP-MEDIATED LIPID TRANSFER PLTP is a rather non-specific lipid transfer protein. Several studies have shown that it is able to transfer all common phospholipid types (Massey et al., 1984; Huuskonen et al., 1996; Rao et al., 1997). Diacylglyceride, a significant lipid constituent of HDL particles, is also transferred efficiently (Rao et al., 1997). The PLTP-mediated transfer of phosphatidylcholine molecules varies with their fatty acyl chain length, decreasing with increasing length, a phenomenon similar to that observed for spontaneous transfer (Huuskonen et al., 1996). PLTP functions by forming a ternary complex between donor and acceptor lipoprotein particles, and may facilitate phospholipid transfer by lowering the energy barrier for lipid monomer dissociation and/or association (Huuskonen et al., 2001). Among lipoproteins, HDL particles are the most efficient as phospholipid donors and/or acceptors in PLTP-mediated phospholipid transfer (Rao et al., 1997). Thus, PLTP appears to display specificity towards HDL in the transfer reaction. As HDLs are by far the most numerous lipoprotein particles in plasma, collisions are more likely to occur between PLTP and them than between PLTP and other lipoproteins (Huuskonen et al., 2001). Two lipidbinding pockets in PLTP protein, at the entrance to and inside the pockets, participate in the phospholipid transfer process (Huuskonen et al., 1999). It has recently been demonstrated that the amphipathic a-helix in PLTP at amino acid residues 144e163, at the tip of the N-terminal barrel, is critical for removing the lipid domain formed by ABC-A1, interacting with phospholipids, and promoting cellular cholesterol and phospholipid transfer (Oram et al., 2008). This suggests that PLTP may shuttle lipids between cells and HDL particles. PLTP also binds and transfers several other amphipathic molecules, including a-tocopherol, diacylglycerides, cerebrosides and lipopolysaccharides (Lagrost et al., 1998; Desrumaux et al., 1999).

PLTP-MEDIATED HDL REMODELING In humans, both CETP and PLTP catalyze the conversion of HDL in vitro into larger and smaller particles, including preb-HDL (Albers et al., 1995; Pulcini et al., 1995; Rye et al., 1995, 1997; Curtiss et al., 2000). The physiological significance of this process is that it increases the production of preb-HDL and thereby enhances the capacity of HDL to accept cellular cholesterol (von Eckardstein et al., 1996). HDL remodeling is also catalyzed by pig and mouse PLTP proteins (Pussinen et al., 1995; Marques-Vidal et al., 1997). PLTP has also been shown to mediate the conversion of HDL2 particles with a concomitant release of lipid-poor apoA-I (Marques-Vidal et al., 1997), and to transform reconstituted discoidal HDL particles into vesicular structures (Nishida et al.,

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1997). By using HDL particles containing phospholipids in the surface lipid layer or cholesteryl esters in the core, it has been demonstrated that PLTP rapidly transfers the surface phospholipids and reaches equilibrium prior to mixing of the cholesteryl ester core (Lusa et al., 1996). It is thought that the initial reaction in PLTP-mediated HDL remodeling is phospholipid transfer, followed by the release of lipid-poor apoA-I, and subsequent fusion of the unstable particles (Lusa et al., 1996). CETP also catalyzes HDL remodeling (Pulcini et al., 1995; Rye et al., 1997). Although particle fusion is again involved (Pulcini et al., 1995), there are several differences between the PLTP-mediated and CETP-mediated processes. No release of lipid-poor apoA-I was observed in HDL remodeling by CETP (Albers et al., 1995). Furthermore, PLTP promoted preferentially the formation of large HDL particles, whereas CETP favored the production of small HDL (Pulcini et al., 1995; Lagrost et al., 1998). The process of HDL remodeling by PLTP is controlled by at least three different factors: the HDL apolipoprotein/protein composition; the core lipid composition of HDL; and the phospholipid transfer activity of PLTP (Pussinen et al., 1997, 2001; Rye et al., 1998; Huuskonen et al., 2000a). In pig HDL, which natively contains apoA-I but not apoA-II, both the formation of large particles and the release of apoA-I were inhibited by increasing the content of apoA-II (Pussinen et al., 1997). Addition of purified serum amyloid A (SAA), an acute-phase upregulated protein, into HDL3 particles facilitated their ability to undergo HDL remodeling, despite the lower content of apoA-I in SAA-HDL (Pussinen et al., 2001). HDL particles enriched in core triglycerides exhibited enhanced PLTP-mediated HDL remodeling (Rye et al., 1998). Phospholipid transfer is a prerequisite for efficient PLTP-mediated HDL remodeling (Huuskonen et al., 2000a). PLTP can also remodel triglyceride-rich lipoproteins (TGRLPs) (Rye et al., 1998). Particularly during the postprandial phase, lipolysis of TGRLPs is intricately linked to subsequent remodeling by PLTP, which results in the integration of lipolytic surface remnants into HDL (Huuskonen et al., 2001), profoundly altering HDL speciation (Jauhiainen et al., 1993). The ensuing imbalance between lipolysis and remodeling may contribute to the toxicity of lipolyzed TGRLPs, leading to damage to vascular endothelial cells and/or macrophage apoptosis (Wehinger et al., 2007). Moreover, PLTP remodeled spherical apoE-containing HDL into large and small particles without dissociation of apoE, suggesting that apoE enhances the capacity of PLTP to remodel HDL, but reduces the ability of HDL to participate in PLTP-mediated phospholipid transfers (Settasatian et al., 2008).

GENETICALLY MODIFIED ANIMALS Plasma from mice overexpressing human PLTP was more effective in preventing acetylated LDL-induced accretion of cholesteryl ester in macrophages

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(von Eckardstein et al., 1996). Nevertheless, systemic PLTP deficiency in mice is associated with a decrease in atherosclerosis susceptibility, despite a decrease in plasma HDL levels, and overexpression of PLTP is accompanied by an increase in susceptibility to the disease (von Eckardstein et al., 1996; Jiang et al., 2001, 2002; van Haperen et al., 2002; Yang et al., 2003). Overexpression of human PLTP in transgenic mice (hPLTPtg) also increases preb-HDL generation, but decreases plasma HDL cholesterol (Jiang et al., 1996; von Eckardstein et al., 1996). Although preb-HDL is a minor HDL subpopulation, it is believed to be a very efficient acceptor of cellular cholesterol (Oram and Yokoyama, 1996). Therefore an elevated production of preb-HDL might enhance RCT (Huuskonen et al., 2001). These results suggest that PLTP may act as a pro-atherogenic, rather than an anti-atherogenic factor in vivo. Overexpression of hPLTP in LDL receptor-deficient (LDLr/) mice resulted in a dose-dependent decrease in HDL cholesterol, and a stimulation of VLDL secretion (van Haperen et al., 2002). LDLr/ and hPLTPtg/LDLr/ mice fed an atherogenic diet showed similar levels of hypercholesterolemia, but the hPLTPtg/LDLr/ mice had lower plasma HDL levels and more atherosclerosis (van Haperen et al., 2002). In LDLrþ/ mice expressing both hCETP and hPLTP, PLTP expression was accompanied by a dose-dependent decrease in HDL cholesterol and an increase in atherosclerosis, which was positively related to the level of hPLTP expression (Lie et al., 2004). These findings were confirmed and extended in apoE-deficient (apoE/) mice transfected with mouse PLTP using an adenovirus vector (Yang et al., 2003). On a chow diet, transient expression of PLTP caused a 30% reduction of HDL cholesterol and phospholipids, as well as a 20% reduction of plasma a-tocopherol content, resulting in increased susceptibility of apoB-containing lipoproteins to peroxidation in vitro (Yang et al., 2003). However, while complete deficiency of PLTP decreased both plasma HDL, apoB-secretion and atherosclerosis (Jiang et al., 2001), no such metabolic effects were produced by an approximately 50% decrease in PLTP activity in PLTPþ/ mice (Tall and Lalanne, 2003). Pronounced overexpression of PLTP increased atherosclerosis (van Haperen et al., 2002; Yang et al., 2003). However, moderate expression by two-fold in transgenics (van Haperen et al., 2002), and overexpression using low-dose adeno-associated virus, did not do so (Yang et al., 2003). Transgenic mice overexpressing a mutant PLTP, lacking phospholipid transfer activity, showed no increase in atherosclerosis lesion size (Samyn et al., 2008). Mice expressing inducible PLTP showed a strongly atherogenic lipoprotein profile, an increase of pre-existing atherosclerotic lesion size, and a decrease in lesion stability (Moreland et al., 2008). The role of PLTP deficiency in atherosclerosis has been studied in several mouse models: human apoB transgenic, apoE/ and LDLr/ mice (Jiang et al., 2001). PLTP/ mice of different genetic backgrounds showed a reduction of atherosclerosis, which was related to the reduction of apoB lipoproteins. Mechanistically, depletion of apoB lipoproteins of vitamin E by

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PLTP was proposed to account for the reduction of atherosclerosis in PLTPdeficient mice (Jiang et al., 2002). While vitamin E reduces atherosclerosis in mice, vitamin E supplementation in humans has had no demonstrable effect on atherosclerosis (Navab et al., 2004), suggesting a more prominent role of vitamin E in murine than in human atherosclerosis. In PLTP-deficient mice, decreased LDL, HDL and apoE-rich lipoproteins, as well as increased biliary cholesterol secretion and hepatic ABCG5/ABCG8 gene expression and decreased intestinal cholesterol absorption, were observed (Shelly et al., 2008). Furthermore, PLTP-deficient mice showed reduced expression of the proinflammatory genes, intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), and lower levels of interleukin-6 (IL-6), supporting the concept that PLTP may play a pro-atherogenic role. Taken together, the available data suggest that only very pronounced systemic variations of PLTP influence atherosclerosis in mice. Such variations have not been described in humans (Ja¨nis et al., 2004). Local PLTP effects have attracted attention in relation to the function of macrophages in atherosclerogenesis (Valenta et al., 2006, 2008; Liu et al., 2007; Vikstedt et al., 2007b). Bone marrow transplantation from PLTP/ mice into several background mice has been performed by three groups, yielding contradictory results (Table 1.1). In male LDLr/ mice, Valenta et al. (2006) demonstrated an increase in atherosclerosis after transplantation of PLTP/ bone marrow, suggesting an athero-protective potential of macrophage PLTP in the presence of normal apoA-I levels, but not in the presence of increased apoA-I levels. Moreover, double mutant LDLr/ PLTP/ mice transplanted with PLTP/ bone marrow showed decreased plasma total cholesterol, increased HDL cholesterol, and smaller atherosclerotic lesions (Valenta et al., 2008). In contrast, Vikstedt et al. (2007b) demonstrated a decrease in both plasma PLTP activity and atherosclerosis after transplantation of PLTP/ bone marrow in female LDLr/ mice, implying an atherogenic potential of macrophage PLTP. ApoE/ mice showed a pronounced increase in atherosclerosis after transplantation of PLTP/ bone marrow, associated with a lower secretion of apoE by macrophages, and consequently much higher plasma cholesterol levels (Liu et al., 2007). In male LDLr/ mice, macrophage-derived PLTP was athero-protective, suggesting that the influence of PLTP on atherogenesis is highly dependent upon its site of expression (Vikstedt et al., 2007b). Moreover, mechanisms affecting atherosclerosis, such as transport of vitamin E by lipoproteins, may profoundly differ between species. In C57Bl/6 mice transplanted with PLTP/ bone marrow, increased cholesterol content of the PLTP/ macrophages was observed, which was normalized by a-tocopherol supplementation, suggesting that macrophage-derived PLTP is anti-atherogenic owing to its ability to reduce cholesterol accumulation in macrophages through changes in the a-tocopherol content and antioxidative status of the cells (Valenta et al., 2008). More recently, van Haperen et al. (2008) demonstrated an atherogenic effect of PLTP

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TABLE 1.1 Differences between mouse models in the effect of macrophage PLTP on atherogenesis after bone marrow transplantation BM donors

Diet/endpoint

Effect on atherosclerosis

Reference

msapoAI/ LDLr/ huapoAItg

PLTP/ or wild type

HFD [15.8% (w/w) fat/ 1.25% (w/w) cholesterol/ no cholate] 16 weeks

Anti-atherogenic 1.3-fold decrease in PLTPwt/wt LDLr/ mice (aorta and heart valve areas); no significance in huapoAItg msapoAI LDLr/ mice

Valenta et al. (2006)

LDLr/, female

PLTP/ or wild type, male

WTD [15% fat/0.25% cholesterol] 9 weeks

Atherogenic 29% reduction in aortic root lesion in PLTP/ LDLr/ mice

Vikstedt et al. (2007b)

ApoE/

PLTP/ or wild type

WTD [0.15% cholesterol/ 20% saturated fat] 7 months

Anti-atherogenic Increased atherosclerosis Aortic arch and root (403%), and aorta (298%) in PLTP/ apoE/ mice

Liu et al. (2007)

PLTP/ LDLr/

PLTP/ or wild type

HFD [15.8% (w/w) fat/ 12.5% (w/w) cholesterol/ no cholate] 8 weeks

Anti-atherogenic 62.8% reduction in en face aortas and 48% reduction in heart sinus valve in PLTPwt/wt LDLr / mice

Valenta et al. (2008)

LDLr/

huPLTPtg/tg or huPLTPtg/wt

HFHC [0.25% cholesterol/ 15% cocoa butter/40% sucrose] 9 weeks

Atherogenic Increased atherosclerosis 2.3-fold in huPLTPtg/wt >LDLr/ 4.5-fold in huPLTPtg/tg >LDLr/

van Haparen et al. (2008)

BM, bone marrow; LDLr, LDL receptor; HFD, high fat diet; HFHC, high fat high cholesterol diet; WTD, western diet; tg, transgenic; wt, wild type

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in macrophages in LDLr/ mice transplanted with bone marrow from mice overexpressing human PLTP. An important issue is how increased PLTP expression stimulates atherosclerosis. Elevation of PLTP activity results in HDL hypercatabolism and enhanced hepatic VLDL secretion. In addition to PLTP-mediated phospholipid transfer, the effects on the transfer of cholesterol and a-tocopherol may be important in this context (Ogier et al., 2007). Moreover, O’Brien et al. (2003) suggested that PLTP may have anti-atherogenic potential by acting as a bridging protein between lipoproteins and biglycan, one of the major extracellular proteoglycans found in human atherosclerotic lesions.

PLTP AND ATHEROSCLEROSIS IN HUMANS PLTP activity is increased in subjects with several risk factors for atherosclerosis. It increases with age (Tahvanainen et al., 1999), body mass index (Dullaart et al., 1994a; Riemens et al., 1998; Huuskonen et al., 2000b; Ja¨nis et al., 2004; Kaser et al., 2004), cigarette smoking (Dullaart et al., 1994b; Cheung et al., 2006), and type 1 and type 2 diabetes mellitus (Colholm et al., 2001; Jonkers et al., 2003; Attia et al., 2007). Studying coronary artery disease (CAD) patients with stable angina, unstable angina and acute myocardial infarction in a crosssectional study, Schlitt et al. (2003) found that increased plasma PLTP activity is a risk factor for CAD. In a prospective study of CAD patients, however, PLTP activity was not predictive of future events (Rao et al., 1997). In a prospective Japanese study, plasma PLTP concentration was negatively associated with CAD (Yatsuya et al., 2004). Finally, a small cross-sectional study, comparing normolipidemic CAD patients with controls matched for plasma lipoproteins, found a non-significant trend for increased PLTP activity in the cases (Ruhling et al., 199). Schgoer et al. (2008) demonstrated that low PLTP activity was significantly decreased in peripheral artery disease (PAD) patients with no history of smoking or diabetes mellitus, and that the magnitude of the decrease increased with increasing PAD stage. PLTP activity was also a determinant of carotid intima media thickness in type 2 diabetes independent of plasma lipids, insulin resistance and C-reactive protein (de Vries et al., 2006). In human atherosclerotic lesions, PLTP was immunohistochemically colocalized in plaques with macrophages and smooth muscle foam cells (Desrumaux et al., 2003; Laffitte et al., 2003; O’Brien et al., 2003). In the atherosclerotic segments, PLTP had accumulated in the extracellular matrix, co-localizing with apoA-I, apoE and biglycan, suggesting that PLTP might promote HDL retention on extracellular matrix proteoglycans and reverse cholesterol transport (O’Brien et al., 2003). It has recently been demonstrated that PLTP-mediated remodeling of apoE-containing HDL generates large as well as small particles, which are potential acceptors of cellular phospholipids and cholesterol (Settasatian et al., 2008). This remodeling may be important for enhancing plasma cholesterol transport, especially under circumstances in

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which the activity of CETP is inhibited and the levels of apoE-containing HDL in plasma are increased. The PLTP-mediated remodeling of apoE-containing HDL may offset the deleterious effects of increased PLTP activity in type 2 diabetes (Dallinga-Thie et al., 2006; Tan et al., 2006). The relationship of elevated PLTP activity to human atherosclerosis needs to be clarified.

CONCLUSION Numerous studies in humans, animals and in vitro have addressed the importance of RCT and cholesterol efflux in atherogenesis. It is possible that augmentation of RCT and cholesterol efflux could be therapeutically useful. Potential major strategies include accelerating RCT and cholesterol efflux, which may be achieved by increasing HDL and apoA-I levels, or by stimulating PLTP or CETP. However, uncertainties remain about the impact of PLTP on RCT, cholesterol efflux and cardiovascular disease. Elevated activities in subjects with diabetes mellitus and CAD may result from partly elevated rates of VLDL turnover, inhibition of VLDL synthesis and increase of HDL catabolism. The regulation of PLTP and lipolytic enzyme levels, and their impacts on atherosclerosis, are not clear. Since the function of PLTP may influence both pro-atherogenic and anti-atherogenic factors, the net effect of these on plasma lipoproteins and atherosclerosis is not predictable in the current state of knowledge, and further investigations are warranted.

REFERENCES Albers, J. J., Tollefson, J. H., Chen, C. H., & Steinmetz, A. (1984). Isolation and characterization of human plasma lipid transfer proteins. Atherosclerosis, 4, 49e58. Albers, J. J., Tu, A. Y., Wolfbauer, G., Cheung, M. C., & Marcovina, S. M. (1996). Molecular biology of phospholipid transfer protein. Curr Opin Lipidol, 7, 88e93. Albers, J. J., Wolfbauer, G., Cheung, M. C., Day, J. R., Ching, A. F., & Tu, A. Y. (1995). Functional expression of human and mouse phospholipid transfer protein: effect of recombinant and plasma PLTP on HDL subspecies. Biochim Biophys Acta, 1258, 27e34. Attia, N., Nakbi, A., Smaoui, M., et al. (2007). Increased phospholipid transfer protein activity associated with the impaired cellular cholesterol efflux in type 2 diabetic subjects with coronary artery disease. Tohoku J Exp Med, 213, 129e137. Castro, G. R., & Fielding, C. J. (1988). Early incorporation of cell-derived cholesterol into prebeta-migrating high-density lipoprotein. Biochemistry, 27, 25e29. Cheung, M. C., Brown, B. G., Marino Larsen, E. K., Frutkin, A. D., O’Brien, K. D., & Albers, J. J. (2006). Phospholipid transfer protein activity is associated with inflammatory markers in patients with cardiovascular disease. Biochim Biophys Acta, 1762, 131e137. Colholm, H. M., Scheek, L. M., Rubens, M. B., et al. (2001). Lipid transfer protein activities in type I diabetic patients without renal failure and nondiabetic control subjects and their association with coronary artery calcification. Diabetes, 50, 652e659. Curtiss, L. K., Bonnet, D. J., & Rye, K. A. (2000). The conformation of apolipoprotein A-I in highdensity lipoproteins is influenced by core lipid composition and particle size: a surface plasmon resonance study. Biochemistry, 39, 5712e5721.

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Curtiss, L. K., Velenta, D. T., Hime, N. J., & Rye, K. A. (2006). What is so special about apolipoprotein AI in reverse cholesterol transport? Arteroscler Thromb Vasc Biol, 26, 12e19. Dallinga-Thie, G. M., van Tol, A., Hattori, H., Rensen, P. C., & Sijbrands, E. J. (2006). Plasma phospholipid transfer protein activity is decreased in type 2 diabetes during treatment with atrovastatin: a role for apolipoprotein E? Diabetes, 55, 1491e1496. Day, J. R., Albers, J. J., Lofton-Day, C. E., et al. (1994). Complete cDNA encoding human phospholipid transfer protein from human endotherial cells. J Biol Chem, 269, 9388e9391. de Vries, R., Dallinga-Thie, G. M., Smit, A. J., Wolffenbuttel, B. H., van Tol, A., & Dullaart, R. P. (2006). Elevated plasma phospholipid transfer protein activity is a determinant of carotid intima-media thickness in type 2 diabetes mellitus. Diabetologia, 49, 398e404. Desrumaux, C., Deckert, V., Athias, A., et al. (1999). Plasma phospholipid transfer protein prevents vascular endothelium dysfunction by delivering alpha-tocopherol to endothelial cells. FASEB J, 13, 883e892. Desrumaux, C. M., Mak, P. A., Boisvert, W. A., et al. (2003). Phospholipid transfer protein is present in human atherosclerotic lesions and is expressed by macrophages and foam cells. J Lipid Res, 44, 1453e1461. Dullaart, R. P., Hoogenberg, K., Dikkeschei, B. D., & van Tol, A. (1994b). Higher plasma lipid transfer protein activities and unfavorable lipoprotein changes in cigarette-smoking men. Arterioscler Thromb, 14, 1581e1585. Dullaart, R. P., Sluiter, W. J., Dikkeschei, L. D., Hoogenberg, K., & van Tol, A. (1994a). Effect of adiposity on plasma lipid transfer protein activities: a possible link between insulin resistance and high density lipoprotein metabolism. Eur J Clin Invest, 24, 188e194. Dusserre, E., Moulin, P., & Vidal, H. (2000). Differences in mRNA expression of the proteins secreted by adipocytes in human subcutaneous and visceral adipose tissues. Biochim Biophys Acta, 1500, 88e96. Eisenberg, S. (1984). High density lipoprotein metabolism. J Lipid Res, 25, 1017e1058. Gordon, D. J., & Rifkind, B. M. (1989). High-density lipoprotein e the clinical implications of recent studies. N Engl J Med, 321, 1311e1316. Gray, P. W., Flaggs, G., Leong, S. R., et al. (1989). Cloning of the cDNA of a human neutrophil bacterial protein. Structure and functional correlations. J Biol Chem, 264, 9505e9509. Huuskonen, J., Ekstrom, M., Tahvanainen, E., et al. (2000b). Quantification of human plasma phospholipid transfer protein (PLTP): relationship between PLTP mass and phospholipid transfer activity. Atherosclerosis, 151, 451e461. Huuskonen, J., Jauhiainen, M., Ehnholm, C., & Olkkonen, V. M. (1998). Biosynthesis and secretion of human plasma phospholipid transfer protein. J Lipid Res, 39, 2021e2030. Huuskonen, J., Olkkonen, V. M., Ehnholm, C., Metso, J., Julkunen, I., & Jauhiainen, M. (2000a). Phospholipid transfer is a prerequisite for PLTP-mediated HDL conversion. Biochemistry, 39, 16092e16098. Huuskonen, J., Olkkonen, V. M., Jauhiainen, M., & Ehnholm, C. (2001). The impact of phospholipid transfer protein (PLTP) on HDL metabolism. Atherosclerosis, 155, 269e281. Huuskonen, J., Olkkonen, V. M., Jauhiainen, M., Metso, J., Somerharju, P., & Ehnholm, C. (1996). Acyl chain and headgroup specificity of human plasma phospholipid transfer protein. Biochim Biophys Acta, 1303, 207e214. Huuskonen, J., Wohlfahrt, G., Jauhiainen, M., Ehnholm, C., Teleman, O., & Olkkonen, V. M. (1999). Structure and phospholipid transfer activity of human PLTP: analysis by molecular modeling and site-directed mutagenesis. J Lipid Res, 40, 1123e1130.

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Ja¨nis, M. T., Siggins, S., Tahvanainen, E., et al. (2004). Active and low-active forms of serum phospholipid transfer protein in a normal Finnish population sample. J Lipid Res, 45, 2303e2309. Jauhiainen, M., Metso, J., Pahlman, R., Blomqvist, S., van Tol, A., & Ehnholm, C. (1993). Human plasma phospholipid transfer protein causes high density lipoprotein conversion. J Biol Chem, 268, 4032e4036. Jiang, X. C., Francone, O. L., Bruce, R., et al. (1996). Increased prebeta-high density lipoprotein, apolipoprotein AI, and phospholipid in mice expressing the human phospholipid transfer protein and human apolipoprotein AI transgenes. J Clin Invest, 98, 2373e2380. Jiang, X. C., Qin, S., Qiao, C., et al. (2001). Apolipoprotein B secretion and atherosclerosis are decreased in mice with phospholipid-transfer protein deficiency. Nat Med, 7, 847e852. Jiang, X. C., Tall, A. R., Qin, S., et al. (2002). Phospholipid transfer protein deficiency protects circulating lipoproteins from oxidation due to the enhanced accumulation of vitamin E. J Biol Chem, 277, 31850e31856. Jonkers, I. J., Smelt, A. H., Hattori, H., et al. (2003). Decreased PLTP mass but elevated PLTP activity linked to insulin resistance in HTG: effects of bezafibrate therapy. J Lipid Res, 44, 1462e1469. Ka¨rkka¨inen, M., Oka, T., Olkkonen, V. M., et al. (2002). Isolation and partial characterization of the inactive and active forms of human plasma phospholipid transfer protein (PLTP). J Biol Chem, 277, 15413e15418. Kaser, S., Laimer, M., Sandhofer, A., Salzmann, K., Ebenbichler, C. F., & Patsch, J. R. (2004). Effects weight loss on PLTP activity and HDL particle size. Int J Obes Relat Metab Disord, 28, 1280e1282. Laffitte, B. A., Joseph, S. B., Chen, M., et al. (2003). The phospholipid transfer protein gene is a liver X receptor target expressed by macrophages in atherosclerotic lesions. Mol Cell Biol, 23, 2182e2191. Lagrost, L., Desrumaux, C., Masson, D., Deckert, V., & Gambert, P. (1998). Structure and function of the plasma phospholipid transfer protein. Curr Opin Lipidol, 9, 203e209. Lie, J., de Crom, R., van Gent, T., et al. (2004). Elevation of plasma phospholipid transfer protein increases the risk of atherosclerosis despite lower apolipoprotein B containing lipoproteins. J Lipid Res, 45, 805e811. Liu, R., Hojjati, M. R., Devlin, C. M., Hansen, I. H., & Jiang, X. C. (2007). Macrophage phospholipid transfer protein deficiency and ApoE secretion: impact on mouse plasma cholesterol levels and atherosclerosis. Arterioscler Thromb Vasc Biol, 27, 190e196. Lusa, S., Jauhiainen, M., Metso, J., Somerharju, P., & Ehnholm, C. (1996). The mechanism of human plasma phospholipid transfer protein-induced enlargement of high-density lipoprotein particles: evidence for particle fusion. Biochem J, 313, 275e282. Marques-Vidal, P., Jauhiainen, M., Metso, J., & Ehnholm, C. (1997). Transformation of high density lipoprotein 2 particles by hepatic lipase and phospholipid transfer protein. Atherosclerosis, 133, 87e95. Massey, J. B., Hickson, D., She, H. S., et al. (1984). Measurement and prediction of the rates of spontaneous transfer of phospholipids between plasma lipoproteins. Biochim Biophys Acta, 794, 274e280. Moreland, M., Samyn, H., van Gent, T., et al. (2008). Acute elevation of plasma PLTP activity strongly increases pre-existing atherosclerosis. Arterioscler Thromb Vasc Biol, 28, 1277e1282. Murdoch, S. J., Kahn, S. E., Albers, J. J., Brunzell, J. D., & Purnell, J. Q. (2003). PLTP activity decreases with weight loss: changes in PLTP are associated with changes in subcutaneous fat and FFA but not IAF or insulin sensitivity. J Lipid Res, 44, 1705e1712.

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Murdoch, S. J., Wolfbauer, G., Kennedy, H., Marcovina, S. M., Carr, M. C., & Albers, J. J. (2002). Differences in reactivity of antibodies to active versus inactive PLTP significantly impacts PLTP measurement. J Lipid Res, 43, 281e289. Navab, M., Ananthramaiah, G. M., Reddy, S. T., et al. (2004). The oxidation hypothesis of atherogenesis: the role of oxidized phospholipids and HDL. J Lipid Res, 45, 993e1007. Nishida, H. I., Klock, D. G., Guo, Z., Jakstys, B. P., & Nishida, T. (1997). Phospholipid transfer protein can transform reconstituted discoidal HDL into vesicular structures. Biochim Biophys Acta, 1349, 222e232. O’Brien, K. D., Vuletic, S., McDonald, T. O., et al. (2003). Cell-associated and extracellular phospholipid transfer protein in human coronary atherosclerosis. Circulation, 108, 270e274. Ogier, N., Klein, A., Deckert, V., et al. (2007). Cholesterol accumulation is increased in macrophages of phospholipid transfer protein-deficient mice: normalization by dietary alphatocopherol supplementation. Arterioscler Thromb Vasc Biol, 27, 2407e2412. Oka, T., Kujiraoka, T., Ito, M., et al. (2000a). Measurement of human plasma phospholipid transfer protein by sandwich ELISA. Clin Chem, 46, 1357e1364. Oka, T., Kujiraoka, T., Ito, M., et al. (2000b). Distribution of phospholipid transfer protein in human plasma: presence of two forms of phospholipid transfer protein, one catalytically active and the other inactive. J Lipid Res, 41, 1651e1657. Oram, J. F., Wolfbauer, G., Tang, C., Davidson, W. S., & Albers, J. J. (2008). An amphipathic helical region of the N-terminal barrel of phospholipid transfer protein is critical for ABCA1dependent cholesterol efflux. J Biol Chem, 283, 11541e11549. Oram, J. F., & Yokoyama, S. (1996). Apolipoprotein-mediated removal of cellular cholestrol and phospholipids. J Lipid Res, 37, 2473e2491. Pulcini, T., Terru, P., Sparrow, J. T., Pownall, H. J., & Ponsin, G. (1995). Plasma factors affecting the in vivo conversion of high-density lipoproteins labeled with a non-transferable marker. Biochim Biophys Acta, 1254, 13e21. Pussinen, P., Jauhiainen, M., & Ehnholm, C. (1997). ApoA-II/apoA-I molar ratio in the HDL particle influences phospholipid transfer protein-mediated HDL interconversion. J Lipid Res, 38, 12e21. Pussinen, P., Jauhiainen, M., Metso, J., Tyynela, J., & Ehnholm, C. (1995). Pig plasma phospholipids transfer protein facilitates HDL interconversion. J Lipid Res, 36, 975e985. Pussinen, P., Malle, E., Metso, J., Sattler, W., Raynes, J. G., & Jauhiainen, M. (2001). Acute-phase HDL in phospholipid transfer protein (PLTP)-mediated HDL conversion. Atherosclerosis, 155, 297e305. Qu, S. J., Fan, H. Z., Kilinc, C., & Pownall, H. J. (1999). Role of cysteine residues in human plasma phospholipid transfer protein. J Protein Chem, 18, 193e198. Rao, R., Albers, J. J., Wolfbauer, G., & Pownall, H. J. (1997). Molecular and macromolecular specificity of human plasma phospholipid transfer protein. Biochemistry, 36, 3645e3653. Riemens, S. C., van Tol, A., Sluiter, W. J., & Dullaart, R. P. (1998). Plasma phospholipid transfer protein activity is related to insulin resistance: impaired acute lowering by insulin in obese Type II diabetic patients. Diabetologia, 41, 929e934. Ruhling, K., Lang, A., Richard, F., et al. (1999). Net mass transfer of plasma cholesteryl esters and lipid transfer proteins in normolipidemic patients with peripheral vascular disease. Metabolism, 48, 1361e1366. Rye, K. A., & Barter, P. J. (1986). Changes in the size and density of human high-density lipoproteins promoted by a plasma-conversion factor. Biochim Biophys Acta, 875, 429e438. Rye, K. A., Hime, N. J., & Barter, P. J. (1995). The influence of cholesteryl ester transfer protein on the composition, size, and structure of spherical, reconstituted high density lipoproteins. J Biol Chem, 270, 189e196.

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Rye, K. A., Hime, N. J., & Barter, P. J. (1997). Evidence that cholesteryl ester transfer proteinmediated reductions in reconstituted high density lipoprotein size involve particle fusion. J Biol Chem, 272, 3953e3960. Rye, K. A., Jauhiainen, M., Barter, P. J., & Ehnholm, C. (1998). Triglyceride-enrichment of high density lipoproteins enhances their remodeling by phospholipid transfer protein. J Lipid Res, 39, 613e622. Samyn, H., Moreland, M., van Gent, T., et al. (2008). Plasma phospholipid transfer activity is essential for increased atherogenesis in PLTP transgenic mice: a mutation-inactivation study. J Lipid Res, 49, 2504e2512. Schgoer, W., Mueller, T., Jauhiainen, M., et al. (2008). Low phospholipids transfer protein (PLTP) is a risk factor for peripheral atherosclerosis. Atherosclerosis, 196, 219e226. Schlitt, A., Bickel, C., Thumma, P., et al. (2003). High plasma phospholipid transfer protein levels as a risk factor for coronary artery disease. Arterioscler Thromb Vasc Biol, 23, 1857e1862. Schumann, R. R., Leong, S. R., Flaggs, G. W., et al. (1990). Structure and function of lipopolysaccharide binding protein. Science, 249, 1429e1431. Settasatian, N., Barter, P. J., & Rye, K. A. (2008). Remodeling of apolipoprotein E-containing spherical reconstituted high density lipoproteins by phospholipid transfer protein. J Lipid Res, 49, 115e126. Shelly, L., Royer, L., Sand, T., Jensen, H., & Luo, Y. (2008). Phospholipid transfer protein deficiency ameliorates diet-induced hypercholesterolemia and inflammation in mice. J Lipid Res, 49, 773e781. Tahvanainen, E., Jauhiainen, M., Funke, H., Vartiainen, E., Sundvall, J., & Ehnholm, C. (1999). Serum phospholipid transfer protein activity and genetic variation of the PLTP gene. Atherosclerosis, 146, 107e115. Tall, A. R., Abreu, E., & Shuman, J. (1983). Separation of plasma phospholipid transfer protein from cholesterol ester/phospholipid exchange protein. J Biol Chem, 258, 2174e2180. Tall, A. R., & Lalanne, F. (2003). Phospholipid transfer protein and atherosclerosis. Arteroscler Thromb Vasc Biol, 23, 1484e1485. Tan, K. C., Shiu, S. W., Wong, Y., Wong, W. K., & Tam, S. (2006). Plasma apolipoprotein E concentration is an important determinant of phospholipid transfer protein activity in type 2 diabetes mellitus. Diabetes Metab Res Rev, 22, 307e312. Tobias, P. S., Mathison, J. C., & Ulevitch, R. J. (1988). A family of lipopolysaccharide binding proteins involved in responses to gram-negative sepsis. J Biol Chem, 263, 13479e13481. Tollefson, J. H., Ravnik, S., & Albers, J. J. (1988). Isolation and characterization of a phospholipid transfer protein (LTP-II) from human plasma. J Lipid Res, 29, 1593e1602. Tu, A. Y., Nishida, H. I., & Nishida, T. (1993). High density lipoprotein conversion mediated by human plasma phospholipid transfer protein. J Biol Chem, 268, 23098e23105. Valenta, D. T., Bulgrien, J. J., Bonnet, D. J., & Curtiss, L. K. (2008). Macrophage PLTP is atheroprotective in LDLr-deficient mice with systemic PLTP deficiency. J Lipid Res, 49, 24e32. Valenta, D. T., Ogier, N., Bradshaw, G., et al. (2006). Atheroprotective potential of macrophagederived phospholipid transfer protein in low-density lipoprotein receptor-deficient mice is overcome by apolipoprotein AI overexpression. Arterioscler Thromb Vasc Biol, 26, 1572e1578. van Haperen, R., Samyn, H., Moerland, M. et al. (2008). Elevated expression of phospholipid transfer protein in bone marrow derived cells causes Atherosclerosis. PLoS ONE, 3:e2255. van Haperen, R., van Tol, A., van Gent, T., et al. (2002). Increased risk of atherosclerosis by elevated plasma levels of phospholipid transfer protein. J Biol Chem, 277, 48938e48943.

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van Haperen, R., van Tol, A., Vermeulen, P., et al. (2000). Human plasma phospholipid transfer protein increases the anti-atherogenic potential of high density lipoproteins in transgenic mice. Arterioscler Thromb Vasc Biol, 20, 1082e1088. Vikstedt, R., Metso, J., Hakala, J., Olkkonen, V. M., Ehnholm, C., & Jauhiainen, M. (2007a). Cholesterol efflux from macrophage foam cells is enhanced phospholipid transfer protein through generation of two types of acceptor particles. Biochemistry, 46, 11979e11986. Vikstedt, R., Ye, D., Metso, J., et al. (2007b). Macrophage phospholipid transfer protein contributes significantly to total plasma phospholipid transfer activity and its deficiency leads to diminished atherosclerotic lesion development. Arterioscler Thromb Vasc Biol, 27, 578e586. von Eckardstein, A., Jauhiainen, M., Huang, Y., et al. (1996). Phospholipid transfer protein mediated conversion of high density lipoproteins generates pre beta 1-HDL. Biochim Biophys Acta, 1301, 255e262. Wehinger, A., Tancevski, I., Schgoer, W., et al. (2007). Phospholipid transfer protein augments apoptosis in THP-1-derived macrophages induced by lipolyzed hypertriglyceridemic plasma. Arterioscler Thromb Vasc Biol, 27, 908e915. Yang, X. P., Yan, D., Qiao, C., et al. (2003). Increased atherosclerotic lesions in apo E mice with plasma phospholipid transfer protein overexpression. Arterioscler Thromb Vasc Biol, 23, 1601e1607. Yatsuya, H., Tamakoshi, K., Hattori, H., et al. (2004). Serum phospholipid transfer protein mass as a possible protective factor for coronary heart diseases. Circ J, 68, 11e16.

Chapter 2

The Role of Cholesteryl Ester Transfer Protein (CETP) in HDL Metabolism Neil J. Hime Centre for Vascular Research, Sydney Medical School (Pathology) and Bosch Institute, The University of Sydney, Medical Foundation Building, Camperdown, NSW, Australia

INTRODUCTION Cholesteryl ester transfer protein (CETP) exchanges neutral lipids (triglycerides and cholesteryl esters) and phospholipids between lipoproteins. Through the in vitro observance of the capacity of lipoprotein-deficient plasma from rabbits (Zilversmit et al., 1975) and humans (Barter and Lally, 1978; Pattnaik et al., 1978) to transfer cholesteryl esters between lipoproteins, the existence of CETP was known long before crystallization enabled a detailed description of the protein (Qiu et al., 2007). In between these discoveries, many studies have used isolated preparations of CETP to shed light on the role of CETP in high density lipoprotein (HDL) metabolism. These studies will be addressed in this chapter. CETP-mediated exchange of neutral lipids results in the net mass transfer of triglycerides from triglyceride-rich very low density lipoproteins (VLDL) to HDL and the net transfer of cholesteryl esters from HDL to VLDL1. This was first observed with lipoprotein-deficient plasma in vitro (Hopkins and Barter, 1980) and subsequently shown to be facilitated by the one protein (Morton and Zilversmit, 1982). It is now known that these CETP-mediated net mass transfers occur in the lipoprotein milieu in blood. The CETP-mediated exchange of neutral lipids changes the molecular structure of lipoproteins. Because HDL is smallest in size of the various lipoprotein classes, and therefore has fewer neutral lipids per particle, it is not surprising that the movement of neutral lipids in and 1 Low density lipoprotein (LDL) can substitute for HDL as the cholesteryl ester donor and triglyceride recipient in net transfers (Albers et al., 1984).

The HDL Handbook. ISBN: 978-0-12-382171-3 Copyright Ó 2010 Elsevier Inc. All rights of reproduction in any form reserved.

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out of these particles has particularly profound effects. These effects change HDL structure and consequently function and metabolism. Not all animals that transport lipids in the aqueous environment of plasma via lipoproteins have similar cholesteryl ester-triglyceride exchange activity. Those animals that are deficient in CETP activity include: rat, mouse, pig, cow, sheep, guinea pig and dog, while those that have high CETP activity include: rabbit, monkey and human (Barter and Lally, 1978; Barter et al., 1979; Rajaram et al., 1980; Ha and Barter, 1982; Tall, 1986). Of the latter three, rabbit lipoprotein-deficient plasma has the highest cholesteryl ester transfer activity when added to radiolabeled lipoprotein substrates. The comparative lipoprotein profiles and susceptibility to atherosclerosis of various species offer important clues as to the role of CETP in HDL metabolism and cardiovascular disease. These comparisons are discussed in this chapter. Much use has been made of genetic manipulation of CETP expression in animal models to elucidate the role of CETP in HDL metabolism. Also, the very few individuals that are known to be CETP-deficient have been intensively studied. These analyses are addressed in the following pages. If the effect of the action of CETP on HDL had to be described in as few words as possible, it is that it reduces the concentration of HDL cholesterol in the plasma. Given that a low concentration of HDL cholesterol is clearly recognized as an independent risk factor for cardiovascular disease, CETP inhibition has been targeted as a strategy to prevent/reduce disease. The results of attempts to inhibit CETP in humans and the current status of this novel therapy are discussed at the end of this chapter.

MOLECULAR BIOLOGY CETP is a 476 residue hydrophobic glycoprotein that has sequence similarity and identical exoneintron boundaries to other lipid-binding proteins: phospholipid transfer protein, lipopolysaccharide-binding protein and bactericidal/permeability-increasing protein (Bruce et al., 1998). This indicates a common ancestral protein and, although these proteins have different physiological functions, they can each bind lipopolysaccharides and phospholipids, and facilitate lipid transport in the bloodstream (Beamer et al., 1997; Yu et al., 1997). CETP can facilitate the bi-directional exchange of the same neutral lipid or phospholipid between lipoproteins, so called homoexchange, without any change in net lipid transfer. The physiological significance of such an exchange is unclear. What is clearly of physiological relevance is the heteroexchange, net mass transfer of cholesteryl ester and triglyceride between lipoproteins. The exchange of lipids appears to be a passive process with the rate of transfer influenced by the concentrations of respective lipids in lipoproteins (Morton and Steinbrunner, 1990). Therefore the net result of heteroexchange on triglyceride-rich lipoproteins (i.e., VLDL) is a reduction in triglycerides and an increase in cholesteryl esters. Conversely, the effect on cholesteryl ester-rich lipoproteins (i.e., HDL) is

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a reduction in cholesteryl esters and an increase in triglycerides. As will be discussed in the following pages, this has considerable effect on the structure of HDL particles and on the ability of HDL to protect from atherosclerosis. Binding of CETP to VLDL, LDL and HDL is reversible and saturable, and is necessary for lipid transfer (Morton, 1985). Binding and cholesteryl ester transfer rate is influenced by modifications to lipoproteins that change surface charge (Nishida et al., 1993). The mechanism by which CETP facilitates lipid transfer has been postulated and various models proposed. One model proposes that CETP shuttles lipids between lipoproteins by binding to a lipoprotein particle, “picking up” lipid(s), dissociating from the lipoprotein particle and then binding to another lipoprotein particle to off-load the lipid. This model fits well with experimental kinetic data (Barter and Jones, 1980; Epps et al., 1995). The “shuttle” model assumes that, in plasma, CETP exists in two forms: (1) freely circulating, and (2) bound to a single lipoprotein particle. This model also only fits well with the kinetic data if esterified cholesterol in HDL is more “available” for transfer than esterified cholesterol in LDL. This may well be the case since CETP associates more avidly with HDL than LDL (Pattnaik and Zilversmit, 1979). A second model of CETP-mediated lipid transfer involves the formation of a ternary complex consisting of CETP and two lipoprotein particles. This ternary complex is the transition state for lipid exchange. This model also fits with experimental kinetic data (Ihm et al., 1982). Experiments examining CETP-mediated structural changes in HDL also support the concept of the generation of a ternary complex (Rye et al., 1997). ˚ resolution The recent determination of the structure of CETP at 2.2-A has provided more definitive evidence of how CETP mediates lipid exchange (Qiu et al., 2007). The crystal structure of human CETP was solved in complex with four bound lipid molecules. The overall shape of CETP is akin to that of a boomerang with a concave and a convex side. Traversing the length of the protein is a tunnel with two distinct openings that are large enough to permit lipid access. In the crystallized protein, this tunnel was occupied by two cholesteryl esters and two phospholipid molecules. The ability of different combinations of lipids to occupy this tunnel may account for conflicting models of lipid exchange based on kinetic data. Synthesis of proteins with mutated tunnels suggests that lipids are required to traverse the tunnel in order to be transferred between lipoproteins (Qiu et al., 2007). The openings of the tunnel are on the concave side of the protein making this surface the most likely to bind lipoproteins. The concave surface of the crystallized protein complements a 10 nm diameter sphere. This fits well with biochemical studies showing CETP to have high binding affinity for 10 nm diameter discoidal HDL (Bruce et al., 1995). CETP structure does allow for the possibility of conformational change to accommodate larger VLDL (Qiu et al., 2007). The structure of the concave surface indicates that CETP can only bind one lipoprotein particle at a time. This only supports the “shuttling” mechanism of lipid transfer. It is suggested that each time CETP picks up neutral lipid from lipoproteins in the tunnel, the

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tunnel openings are filled with phospholipid molecules to permit the protein to return to the polar environment of the plasma (Qiu et al., 2007).

EFFECT OF CETP ACTION ON HDL STRUCTURE In human plasma, most CETP is found associated with HDL (Pattnaik and Zilversmit, 1979). From analysis of binding capacities of various lipoproteins for CETP and respective plasma concentrations of lipoproteins, it has been estimated that 74% of plasma CETP is bound to HDL (Nishida et al., 1993). This increased association between CETP and HDL compared to other lipoprotein classes may reflect complementing structures as discussed above and/or higher electrostatic attraction. In the simplistic environment of a test tube incubation (up to 24 hours), CETP-mediated transfer of cholesteryl esters from HDL to VLDL and LDL results in a reduction in HDL particle size and a dissociation of apolipoprotein A-I in a lipid-poor form (Liang et al., 1994). With the addition of other plasma factors, this CETP-generated lipid-poor apolipoprotein A-I results in the formation of new HDL particles (Clay et al., 1992). Interestingly, in an 8-hour incubation of HDL with CETP and VLDL, hepatic lipase is required to achieve a reduction in HDL particle size (Clay et al., 1992). This may relate to HDL enriched in triglyceride being the preferred substrate for hepatic lipase. In hypertriglyceridemic mice, the presence of a CETP transgene also decreases HDL particle size (Hayek et al., 1993). Plasma HDL are a heterogeneous group of particles that vary in chemical and physical modalities. In order to get a more detailed description of the changes in HDL structure mediated by CETP, use has been made of homogeneous, reconstituted HDL (rHDL) assembled in the laboratory. Although rHDL are not the same as any given plasma HDL particle, there is sufficient experimental data to suggest that the interactions of rHDL with plasma proteins is equivalent to that of plasma HDL. When rHDL are incubated with CETP and Intralipid (a source of triglycerides), the loss of cholesteryl esters from rHDL is greater than the gain of triglycerides (Rye et al., 1995). This results in a loss of core lipids from the rHDL, a decrease in particle size, and a redistribution of apolipoprotein A-I molecules from three per particle to two. As there is no loss in rHDL constituents in this process, it results in a 50% increase in the number of rHDL particles (Rye et al., 1997).

EFFECT OF CETP ACTIVITY ON PLASMA HDL LEVELS AND ATHEROSCLEROTIC DISEASE CETP activity reduces the plasma concentration of HDL cholesterol. In mice, an animal normally deficient in CETP and with a high concentration of plasma HDL relative to humans, the expression of a human CETP transgene results in a reduction in HDL cholesterol (Agellon et al., 1991). In humans, genetic

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deficiency in CETP results in marked elevation in HDL cholesterol among homozygotes and moderate elevation in heterozygotes (Inazu et al., 1990, 1994; Cefalu` et al., 2009). Mutations in the CETP gene leading to CETP deficiency are particularly prevalent in Japanese people and CETP deficiency is the most common cause of hyperalphalipoproteinemia (elevated HDL) in the Japanese (Nagano et al., 2004). The strong inverse association between HDL cholesterol concentration and atherosclerosis risk (Gordon et al., 1989) has fuelled the study of CETP as a modulator of cardiovascular disease. The expression of CETP in wild type, LDL receptor knockout and apolipoprotein E knockout, atherosclerosis prone, mice decreases plasma HDL cholesterol and results in an increase in atherosclerosis (Marotti et al., 1993; Plump et al., 1999). However, expression of CETP in mice deficient in the scavenger receptor BI (SR-BI) and mice expressing human lecithin:cholesterol acyltransferase protects against atherosclerosis (Fo¨ger et al., 1999; Harder et al., 2007). Polymorphisms in the CETP gene that result in reduced CETP expression are associated with higher plasma HDL cholesterol concentrations, a lower prevalence of cardiovascular disease and increased longevity in humans (Ordovas et al., 2000; Barzilai et al., 2003; Koropatnick et al., 2008). A prospective case-control study showed that elevated CETP levels occur with an increased risk of future coronary artery disease but only in people with high plasma triglyceride levels (Boekholdt et al., 2004). In another case-control study there was actually a trend towards high CETP levels reducing cardiovascular disease risk when triglyceride levels were low (Borggreve et al., 2007). The effect of triglyceride concentration on the association between CETP and atherosclerosis risk is important for triglyceride levels will influence the level of CETP activity for a given plasma CETP concentration. This highlights the need to make distinctions between CETP activity and CETP mass when assessing trial data. Intima-media thickness, a marker for subclinical atherosclerosis, is positively associated with plasma cholesteryl ester transfer rates in diabetic and non-diabetic subjects, however, no association is observed for CETP mass (de Vries et al., 2005). A comparison of species that have or lack CETP activity offers some clues as to the relationship between CETP, HDL and atherosclerosis. Relative to humans, monkeys, and rabbits, mice (strain C57BL6) have only a trace of cholesteryl ester transfer activity (Ha and Barter, 1982; Marotti et al., 1993). Mice have approximately two- and ten-fold greater concentration of plasma HDL cholesterol than humans and rabbits, respectively. Mice are highly resistant to diet-induced atherosclerosis compared to humans and rabbits. Thus, at least with these species, there is a consistent negative association between CETP activity and HDL cholesterol concentration, and a positive correlation between CETP activity and atherosclerosis susceptibility. So is it as simple as CETP activity lowers HDL which increases atherosclerosis risk? Unfortunately, this is too simplistic and there are examples that destroy this nice hypothesis. For example, CETP activity is low in rat, sheep and pig but HDL

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The HDL Handbook

cholesterol is not particularly high in these species (Ha and Barter, 1982). However, as cautioned above, it needs to be considered that the plasma CETP activity in these species was measured ex vivo. Furthermore, hyperalphalipoproteinemia associated with CETP deficiency is, in certain circumstances, associated with atherosclerosis (Hirano et al., 1995). What is clear is that CETP activity can, although not always, lower plasma HDL concentrations. Also, CETP activity can change HDL in ways that affect the functionality of these particles. This effect of CETP is poorly understood but is likely to affect the anti-atherogenic properties of HDL.

EFFECT OF CETP ACTIVITY ON HDL FUNCTION AND CONSEQUENCES FOR ATHEROSCLEROTIC DISEASE The primary mechanisms by which HDL are understood to protect against atherosclerotic disease are defined as anti-inflammatory (Barter et al., 2004) and reverse cholesterol transport (Glomset, 1968; Rader et al., 2009). The antiinflammatory properties of HDL include the ability of HDL to inhibit endothelial cell adhesion molecules and chemokines. Also, HDL-associated proteins, such as paraoxonase-1, can act as antioxidants. These properties undoubtedly are influenced by the form in which HDL reside, but equally important may be the residence time of individual HDL particles in the plasma. Put simply, the higher the concentration of HDL the greater the anti-inflammatory potential. The anti-inflammatory properties of HDL are not well understood and, at present, it is difficult to speculate how CETP may affect these properties. Although CETP activity decreases HDL cholesterol, it also has the potential to increases the number of HDL particles (Clay et al., 1992; Rye et al., 1997). In increasing the number of HDL particles, rather than increasing the concentration of any one HDL constituent, this may increase the sum anti-inflammatory potential. Reverse cholesterol transport, on the other hand, may depend less on the absolute concentration of HDL at any given time and more on the forms of HDL present. Reverse cholesterol transport refers to the ability of HDL to transport excess cholesterol from peripheral tissues to the liver for excretion. It has been demonstrated that the rate of reverse cholesterol transport from cholesterol-loaded macrophages predicts atherosclerosis severity better than the concentration of HDL cholesterol (Rader et al., 2009). The rate-limiting factor in cholesterol efflux from cholesterol-laden cells is the availability of extracellular acceptors of cholesterol. The major acceptor of cellular cholesterol is lipid-poor apolipoprotein A-I (the major apolipoprotein of HDL). As mentioned above, the action of CETP on HDL can result in the dissociation of apolipoprotein A-I. It has been hypothesized that this dissociated apolipoprotein A-I may then be available for acceptance of cellular cholesterol (Curtiss et al., 2006). As an anti-atherogenic mechanism of CETP, this would be particularly relevant when the CETP is expressed by macrophages in

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FIGURE 2.1 Hypothesised mechanism by which the actions of CETP (and other proteins) on HDL in the interstitium promote removal of cholesterol from atherosclerotic lesions. (from Curtiss, L.K. et al Arterioscler Thromb Vasc Biol. 2006;26:12-19.) This illustration depicts cholesterol (yellow)-loaded macrophages (green) in an atherosclerotic lesion in the arterial intima. The circular inset shows the schema whereby the proteins: CETP, phospholipid transfer protein (PLTP) and hepatic lipase (HL) expressed by macrophages, act upon HDL to generate lipid-poor apolipoprotein A-I (preb-HDL). This lipid-poor apolipoprotein A-I accepts cholesterol from macrophages resulting in the generation of new HDL particles that move from the interstitium to the plasma. The net effect is a reduction in cholesterol in atherosclerotic lesions. From Curtiss, L.K., Valenta, D.T., Hime, N.J. and Rye, K-A. What is so special about apolipoprotein AI in reverse cholesterol transport? Arterioscler Thromb Vasc Biol 2006;26:12-19. Reprinted with permission. The original figure was drawn by D.T. Valenta.

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The HDL Handbook

atherosclerotic lesions. However, transfer of bone marrow from CETP transgenic mice into atherosclerosis-prone LDL receptor-deficient mice actually increases atherosclerotic lesion size (Van Eck et al., 2007). This is despite bone marrow-derived CETP increasing lipid-poor HDL three-fold (as measured by immunoelectrophoresis). Bone marrow-derived CETP induces a pro-atherogenic lipoprotein profile with an increase in VLDL/LDL cholesterol and a decrease in HDL cholesterol. This change in lipoprotein profile may well offset any advantage to be gained from the generation of lipid-poor HDL. Cellular cholesterol efflux to either HDL or apolipoprotein A-I does not differ between CETP expressing and non-CETP expressing macrophages (Van Eck et al., 2007). However, these studies were conducted ex vivo using isolated human HDL and apolipoprotein A-I. It is feasible that, in certain environments, CETP and other plasma factors could generate sufficient acceptors of cellular cholesterol to decrease atherosclerosis. CETP-mediated neutral lipid transfer between VLDL and HDL generates triglyceride-rich HDL, the preferred substrate of hepatic lipase. Hydrolysis of triglyceride-enriched HDL lipids by hepatic lipase results in the dissociation of apolipoprotein A-I from HDL (Clay et al., 1991). The net result is that the combined actions of CETP and hepatic lipase on HDL promote the shedding of apolipoprotein A-I that becomes available for the formation of new HDL particles by associating with lipid (Clay et al., 1992). This lipid may come from cholesterol-loaded macrophages in the arterial intima and be transported to apolipoprotein A-I via the ATP-binding cassette transporter A1 (ABC-A1) e the first step in the reverse cholesterol transport pathway. All the necessary components are available in the intima for this to happen (Figure 2.1). HDL are able to enter the intimal space. CETP is present in atherosclerotic lesions (Zhang et al., 2001) and hepatic lipase is expressed by macrophages (Gonza´lezNavarro et al., 2002). Whether these processes decrease atherosclerosis is not known, however, it is interesting that bone marrow-derived hepatic lipase reduces atherosclerosis but only when CETP is present (Hime et al., 2008). HDL from CETP-deficient subjects have been shown to enhance cholesterol efflux from macrophages compared to control HDL (Matsuura et al., 2006). This cholesterol efflux is dependent on ABC-G1, a cell membrane transporter that transports cellular lipid to mature HDL. Thus, while CETP may be a promoter of cellular cholesterol efflux to lipid-poor apolipoprotein A-I, it may lessen the amount of cellular cholesterol efflux to mature HDL. If this were the case, the effect on reverse cholesterol transport could depend on the relative contributions of these two processes to the overall transport of cholesterol from peripheral tissues to the liver. Cholesterol movement from macrophages to HDL may not be the only stage of the reverse cholesterol transport under the influence of CETP. CETP could have a role in the transfer of cholesterol from HDL to the liver for excretion. Adenovirus expression of CETP in mouse hepatocytes enhances uptake of cholesteryl esters from HDL independent of lipoprotein receptors

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(Gauthier et al., 2005). The integral membrane protein SR-BI facilitates the uptake of cholesteryl esters from HDL in the liver. Cholesteryl esters from HDL isolated from mice expressing CETP are taken up more readily via SR-BI than cholesteryl esters from HDL from control mice (Collet et al., 1999). The remodeling of HDL involving CETP-mediated neutral lipid exchange and hepatic lipase lipid hydrolysis also enhances uptake of HDL cholesteryl esters by SR-BI (Collet et al., 1999). The overall effect of CETP on the rate of reverse cholesterol transport is complex. While CETP would appear to enhance some reverse cholesterol transport processes, the reduction in HDL cholesterol through CETP-mediated neutral lipid exchange with other lipoproteins may be detrimental to the efficient movement of cholesterol from the vasculature to the liver. Beyond the scope of this chapter but important nonetheless, CETP activity influences the clearance rate of cholesterol via apolipoprotein B-containing lipoproteins and the LDL receptor. The effect of CETP on models of impaired and intact apolipoprotein B lipoprotein clearance is well summarized in a review by Rader et al. (2009). As has been discussed, animal models have provided valuable information regarding the association between CETP, HDL and atherosclerosis. Human CETP expression in atherosclerosis prone apolipoprotein E and LDL receptor deficient mice increases atherosclerosis (Plump et al., 1999). These two mouse models of atherosclerosis have high concentrations of atherogenic LDL/VLDL particles and low levels of HDL. The presence of CETP exacerbates this atherogenic lipid profile, and any change in the functionality of HDL is likely to be of little influence with regard to atherosclerosis in a background of such an atherogenic lipid profile. The C57BL6 strain of mice fed an atherogenic diet also have a lipid profile that is more atherogenic relative to chow fed mice. CETP expression in atherogenic diet fed C57BL6 mice also increases atherosclerosis (Marotti et al., 1993). In hypertriglyceridemic human apolipoprotein CIII transgenic mice, expression of CETP reduces diet-induced atherosclerosis (Hayek et al., 1995). It is possible that, in situations where CETP activity promotes generation of lipid-poor apolipoprotein A-I, atherosclerosis is reduced, whereas when this is not the case and CETP activity reduces HDL levels, then atherosclerosis is increased (Table 2.1). As discussed above, the combined activity of CETP and hepatic lipase on HDL can generate lipid-poor apolipoprotein A-I. In a case-controlled study there is evidence that a reduction in CETP and hepatic lipase activity is associated with atherosclerosis, even when accompanied by high levels of HDL (Hirano et al., 1995). Pharmacological CETP-inhibition in atherogenic diet-fed rabbits significantly reduces atherosclerosis (Morehouse et al., 2007). This is a model in which CETP inhibition is particularly effective at raising HDL. Furthermore, since rabbits do not have hepatic lipase activity, CETP-mediated triglyceride enrichment of HDL might not promote generation of lipid-poor apolipoprotein A-I. Thus, this is a scenario where CETP inhibition may be particularly beneficial.

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The HDL Handbook

TABLE 2.1 Effects of CETP on HDL metabolism and the potential for these effects to influence atherosclerosis risk

Effect of CETP on HDL metabolism

Potential change in atherosclerosis risk due to CETP-mediated effect*

Decrease in HDL cholesteryl esters (Agellon et al., 1991; Inazu et al., 1990, 1994; Ordovas et al., 2000; Barzilai et al., 2003; Nagano et al., 2004; Koropatnick et al., 2008; Cefalu` et al., 2009

Increase

Reduction in HDL particle size (Clay et al., 1992; Hayek et al., 1993; Liang et al., 1994; Rye et al., 1995)

Increase or decrease

Dissociation of apolipoprotein A-I (Clay et al., 1992; Liang et al., 1994)

Decrease

Formation of new HDL particles (Clay et al., 1992; Rye et al., 1997)

Decrease

* The specified change in atherosclerosis risk is that speculated by the author and does not represent the opinion of the authors of the references.

POLYMORPHISMS OF THE CETP GENE AND INTERACTIONS BETWEEN ENVIRONMENTAL INFLUENCES AND HDL A variety of polymorphisms in the human CETP gene have been identified that result in a decrease in CETP activity and an increase in HDL cholesterol (Thompson et al., 2008). Associations between these polymorphisms and a reduction in cardiovascular disease risk are generally weak. This may be because the increases in HDL cholesterol are modest (3e5%) or it may be that not all effects of CETP gene polymorphisms are beneficial with regards to cardiovascular disease. Another confounding factor is that most studies examining CETP gene polymorphisms have not involved the thousands of participants necessary to fully appreciate relatively modest changes. A polymorphism in intron 1 of the CETP gene (Drayna and Lawn, 1987), denoted TaqIB because of the name of the restriction enzyme used to detect the polymorphism, has been most intensively studied. This polymorphism was one of the first genetic variations associated with HDL levels (Kondo et al., 1989). One reason why this particular polymorphism has been studied more than others is that the less common variant of the polymorphism (B2), that results in lower CETP levels, occurs at a high frequency of approximately 40% in populations so far studied. A meta-analysis has clearly demonstrated that the B2 variant is associated with a small but highly statistically significant increase

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27

in HDL cholesterol (Boekholdt et al., 2005). After adjustment for study, sex, age, smoking, body mass index, diabetes, systolic blood pressure, LDL cholesterol and use of alcohol, the B2 variant was significantly associated with a reduction in coronary artery disease. The effect on disease may be predominantly associated with HDL levels as statistical significance is lost when adjustment is made for HDL cholesterol. This meta-analysis was confined to white subjects only. A variety of environmental factors, including alcohol consumption, dietary fat intake, smoking and exercise, are known to influence HDL levels. Several studies have examined the interaction between the CETP gene TaqIB polymorphism, environmental factors and HDL. The association between the TaqIB polymorphism and HDL (lower HDL with the B1 genotype and higher HDL with the B2 genotype) has been shown to be more evident in diabetic patients with a high intake of fat in their diet than those with a low fat intake (Li et al., 2007). Alcohol consumption is associated with an increase in HDL cholesterol and this association is stronger for individuals with the B2B2 genotype (Tsujita et al., 2007; Jensen et al., 2008). Smoking is associated with decreased HDL levels and increased CETP activity and multiple regression analysis suggests that the decreased HDL with smoking is explained by the increase in CETP activity (Dullaart et al., 1994). Despite evidence that the effect of the TaqIB polymorphism on HDL levels is limited to non-smokers and ex-smokers (Kondo et al., 1989; Freeman et al., 1994), this polymorphism does influence the increased risk of myocardial infarction associated with smoking (Goldenberg et al., 2007). Smoking is associated with a reduction in the age of onset of first myocardial infarction in B1B1 and B1B2 carriers but not in B2B2 carriers. Associations between CETP gene polymorphisms, HDL levels and exercise are not clear. Polymorphisms aside, long-term exercise training is known to increase HDL and one study showed that endurance exercise training decreased plasma CETP mass (Seip et al., 1993).

INHIBITORS OF CETP ACTIVITY AS THERAPY There is not a single clinically used drug that effectively raises HDL cholesterol. It has been thought that such a drug could be used either where cholesterol lowering drugs are ineffective or used in combination with LDL cholesterol lowering drugs to improve efficacy (Hausenloy and Yellon, 2008). There has been great hope that inhibitors of CETP could fill this void. While CETP inhibition (torcetrapib) in combination with statin therapy (atorvastatin) effectively raises HDL cholesterol, it does not efficaciously reduce atherosclerosis in either hypercholesterolemic patients or patients with established coronary disease (Kastelein et al., 2007; Nissen et al., 2007). A post hoc analysis of the latter of these two studies found that there was a relationship between torcetrapib-mediated change in HDL cholesterol and atherosclerosis. Patients with regression of atheroma volume had greater increases in HDL

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The HDL Handbook

cholesterol (Nicholls et al., 2008). This supports HDL raising as a therapeutic target, but leaves undecided the effectiveness of CETP inhibitors. The phase III clinical trial ILLUMINATE (Investigation of Lipid Level Management to Understand its Impact on Atherosclerotic Events) was prematurely terminated because of an increase in mortality in the torceptrapib/atorvastatin treatment arm compared with atorvastatin treatment alone (Barter et al., 2007). The increased mortality may relate to associated increases in blood pressure with torceptrapib treatment. It has recently been shown that torceptrapib induces aldosterone and cortisol production independent of CETP inhibition (Hu et al., 2009). Another CETP inhibitor (anacetrapib) raises HDL cholesterol and shows no signs of blood pressure raising (Bloomfield et al., 2009). Thus, there is justification for further investigations of CETP inhibitors as therapy. It is, however, unclear what effect CETP inhibitors have on the many functions of HDL.

CONCLUSION A combination of data obtained from transgenic research animals, different animal species, CETP-deficient humans, epidemiology, inhibitors of CETP and in vitro studies provides ample evidence that CETP activity has fundamental effects on HDL metabolism. The most consistent effect is that CETP activity reduces plasma HDL cholesteryl ester concentration via neutral lipid exchange with other lipoproteins. This is primarily viewed as an atherosclerosispromoting effect due to the independent inverse association between HDL cholesterol and atherosclerosis risk. However, the link between CETP, HDL metabolism and atherosclerosis is not independent of other modulators of lipid metabolism and atherogenesis. It remains to be seen whether CETP inhibitors are effective anti-atherosclerotic agents. Regardless of the outcome of CETP inhibitors as therapy, these drugs will provide valuable information on the effect of CETP on individual HDL functions; this is an area that is poorly understood.

REFERENCES Agellon, L. B., Walsh, A., Hayek, T., et al. (1991). Reduced high density lipoprotein cholesterol in human cholesteryl ester transfer protein transgenic mice. J Biol Chem, 266, 10796e10801. Albers, J. J., Tollefson, J. H., Chen, C. H., & Steinmetz, A. (1984). Isolation and characterization of human lipid transfer proteins. Arteriosclerosis, 4(1), 49e58. Barter, P. J., Caufield, M., Eriksson, M., et al. for the ILLUSTRATE Investigators. (2007). Effects of torcetrapib in patients at high risk for coronary events. N Engl J Med, 357, 2109e2122. Barter, P. J., Gooden, J. M., & Rajaram, O. V. (1979). Species differences in the activity of a serum triglyceride transferring factor. Atherosclerosis, 33, 165e169.

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Barter, P. J., & Jones, M. E. (1980). Kinetic studies of the transfer of esterified cholesterol between human plasma low and high density lipoproteins. J Lipid Res, 21, 238e249. Barter, P. J., & Lally, J. I. (1978). The activity of an esterified cholesterol transferring factor in human and rat serum. Biochim Biophys Acta, 531, 233e236. Barter, P. J., Nicholls, S., Rye, K.-A., Anantharamaiah, G. M., Navab, M., & Fogelman, A. M. (2004). Antiinflammatory properties of HDL. Circ Res, 95, 764e772. Barzilai, N., Atzmon, G., Schechter, C., et al. (2003). Unique lipoprotein phenotype and genotype associated with exceptional longevity. J Am Med Assoc, 290, 2030e2040. Beamer, L. J., Carroll, S. F., & Eisenberg, D. (1997). Crystal structure of human BPI and two bound phospholipids at 2.4 angstrom resolution. Science, 276, 1861e1864. Bloomfield, D., Carlson, G. L., Sapre, A., et al. (2009). Efficacy and safety of the cholesteryl ester transfer protein inhibitor anacetrapib as monotherapy and coadministered with atorvastatin in dyslipidemic patients. Am Heart J, 157, 352e360. Boekholdt, S. M., Kuivenhoven, J.-A., Wareham, N. J., et al. (2004). Plasma levels of cholesteryl ester transfer protein and the risk of future coronary artery disease in apparently healthy men and women: the prospective EPIC (European Prospective Investigation into Cancer and Nutrition) e Norfolk population study. Circulation, 110, 1418e1423. Boekholdt, S. M., Sacks, F. M., Jukema, J. W., et al. (2005). Cholesteryl ester transfer protein TaqIB variant, high-density lipoprotein cholesterol levels, cardiovascular risk, and efficacy of pravastatin treatment: individual patient meta-analysis of 13 677 subjects. Circulation, 111, 278e287. Borggreve, S. E., Hillege, H. L., Dallinga-Thie, G. M., et al. on behalf of the PREVEND study group. (2007). High plasma cholesteryl ester transfer protein levels may favour reduced incidence of cardiovascular events in men with low triglycerides. Eur Heart J, 28, 1012e1018. Bruce, C., Beamer, L. J., & Tall, A. R. (1998). The implications of the structure of the bactericidal/ permeability-increasing protein on the lipid-transfer function of the cholesteryl ester transfer protein. Curr Opin Struct Biol, 8, 426e434. Bruce, C., Davidson, W. S., Kussie, P., et al. (1995). Molecular determinants of plasma cholesteryl ester transfer protein binding to high density lipoproteins. J Biol Chem, 270, 11532e11542. Cefalu`, A. B., Noto, D., Magnolo, L., et al. (2009). Novel mutations of CETP gene in Italian subjects with hyperalphalipoproteinemia. Atherosclerosis, 204, 202e207. Clay, M. A., Newnham, H. H., & Barter, P. J. (1991). Hepatic lipase promotes a loss of apolipoprotein A-I from triglyceride-enriched human high density lipoproteins during incubation in vitro. Arterioscler Thromb Vasc Biol, 11, 415e422. Clay, M. A., Newnham, H. H., Forte, T. M., & Barter, P. J. (1992). Cholesteryl ester transfer protein and hepatic lipase activity promote shedding of apo A-I from HDL and subsequent formation of discoidal HDL. Biochim Biophys Acta, 1124, 52e58. Collet, X., Tall, A. R., Serajuddin, H., et al. (1999). Remodeling of HDL by CETP in vivo and by CETP and hepatic lipase in vitro results in enhanced uptake of HDL CE by cells expressing scavenger receptor B-I. J Lipid Res, 40, 1185e1193. Curtiss, L. K., Valenta, D. T., Hime, N. J., & Rye, K.-A. (2006). What is so special about apolipoprotein AI in reverse cholesterol transport? Arterioscler Thromb Vasc Biol, 26, 12e19. de Vries, R., Perton, F. G., Dallinga-Thie, G. M., et al. (2005). Plasma cholesteryl ester transfer is a determinant of intima-media thickness in type 2 diabetic and nondiabetic subjects: role of CETP and triglycerides. Diabetes, 54, 3554e3559. Drayna, D., & Lawn, R. (1987). Mutiple FRLPs at the human cholesteryl ester transfer protein (CETP) locus. Nucleic Acids Res, 15, 4698.

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Dullaart, R. P., Hoogenberg, K., Dikkeschei, B. D., & van Tol, A. (1994). Higher plasma lipid transfer protein activities and unfavorable lipoprotein changes in cigarette-smoking men. Arterioscler Thromb Vasc Biol, 14, 1581e1585. Epps, D. E., Greenlee, K. A., Harris, J. S., et al. (1995). Kinetics and inhibition of lipid exchange catalyzed by plasma cholesteryl ester transfer protein (lipid transfer protein). Biochemistry, 34, 12560e12569. Fo¨ger, B., Chase, M., Amar, M. J., et al. (1999). Cholesteryl ester transfer protein corrects dysfunctional high density lipoproteins and reduces aortic atherosclerosis in lecithin cholesterol acyltransferase transgenic mice. J Biol Chem, 274, 36912e36920. Freeman, D. J., Griffin, B. A., Holmes, A. P., et al. (1994). Regulation of plasma HDL cholesterol and subfraction distribution by genetic and environmental factors. Associations between the TaqI B RFLP in the CETP gene and smoking and obesity. Arterioscler Thromb Vasc Biol, 14, 336e344. Gauthier, A., Lau, P., Zha, X., Milne, R., & McPherson, R. (2005). Cholesteryl ester transfer protein directly mediates selective uptake of high density lipoprotein cholesteryl esters by the liver. Arterioscler Thromb Vasc Biol, 25, 2177e2184. Glomset, J. A. (1968). The plasma lecithin:cholesterol acyltransferase reaction. J Lipid Res, 9, 155e167. Goldenberg, I., Moss, A. J., Block, R., et al. (2007). Polymorphism in the cholesteryl ester transfer protein gene and the risk of early onset myocardial infarction among cigarette smokers. Ann Noninvasive Electrocardiol, 12, 364e374. Gonza´lez-Navarro, H., Nong, Z., Freeman, L., Bensadoun, A., Peterson, K., & SantamarinaFojo, S. (2002). Identification of mouse and human macrophages as a site of synthesis of hepatic lipase. J Lipid Res, 43, 671e675. Gordon, D. J., Probstfield, J. L., Garrison, R. J., et al. (1989). High-density lipoprotein cholesterol and cardiovascular disease: four prospective American studies. Circulation, 79, 8e15. Ha, Y. C., & Barter, P. J. (1982). Differences in plasma cholesteryl ester transfer activity in sixteen vertebrate species. Comp Biochem Physiol, 71B, 265e269. Harder, C., Lau, P., Meng, A., Whitman, S. C., & McPherson, R. (2007). Cholesteryl ester transfer protein (CETP) expression protects against diet induced atherosclerosis in SR-BI deficient mice. Arterioscler Thromb Vasc Biol, 27, 858e864. Hausenloy, D. J., & Yellon, D. M. (2008). Targeting residual cardiovascular risk: raising highdensity lipoprotein cholesterol levels. Heart, 94, 706e714. Hayek, T., Azrolan, N., Verdery, R. B., et al. (1993). Hypertriglyceridemia and cholesteryl ester transfer protein interact to dramatically alter high density lipoprotein levels, particle sizes, and metabolism: studies in transgenic mice. J Clin Invest, 92, 1143e1152. Hayek, T., Masucci-Magoulas, L., Jiang, X., et al. (1995). Decreased early atherosclerotic lesions in hypertriglyceridemic mice expressing cholesteryl ester transfer protein transgene. J Clin Invest, 96, 2071e2074. Hime, N. J., Black, A. S., Bulgrien, J. J., & Curtiss, L. K. (2008). Leukocyte-derived hepatic lipase increases HDL and decreases en face aortic atherosclerosis in LDLr/ mice expressing CETP. J Lipid Res, 49, 2113e2123. Hirano, K.-i., Yamashita, S., Kuga, Y., et al. (1995). Atherosclerotic disease in marked hyperalphalipoproteinemia: combined reduction of cholesteryl ester transfer protein and hepatic triglyceride lipase. Arterioscler Thromb Vasc Biol, 15, 1849e1856. Hopkins, G. J., & Barter, P. J. (1980). Transfers of esterified cholesterol and triglyceride between high density and very low density lipoproteins: in vitro studies of rabbits and humans. Metabolism, 29, 546e550.

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Hu, X., Dietz, J. D., Xia, C., et al. (2009). Torcetrapib induces aldosterone and cortisol production by an intracellular calcium-mediated mechanism independently of cholesteryl ester transfer protein (CETP) inhibition. Endocrinology, 150, 2211e2219. Ihm, J., Quinn, D. M., Busch, S. J., Chataing, B., & Harmony, J. A. K. (1982). Kinetics of plasma protein-catalyzed exchange of phosphatidylcholine and cholesteryl ester between plasma lipoproteins. J Lipid Res, 23, 1328e1341. Inazu, A., Brown, M. L., Hesler, C. B., et al. (1990). Increased high-density lipoprotein levels caused by a common cholesteryl-ester transfer protein gene mutation. N Engl J Med, 323, 1234e1238. Inazu, A., Jiang, X.-C., Haraki, T., et al. (1994). Genetic cholesteryl ester transfer protein deficiency caused by two prevalent mutations as a major determinant of increased levels of high density lipoprotein cholesterol. J Clin Invest, 94, 1872e1882. Jensen, M. K., Mukamal, K. J., Overvad, K., & Rimm, E. B. (2008). Alcohol consumption, TaqIB polymorphism of cholesteryl ester transfer protein, high-density lipoprotein cholesterol, and risk of coronary heart disease in men and women. Eur Heart J, 29, 104e112. Kastelein, J. J. P., van Leuven, S. I., Burgess, L., et al. for the RADIANCE 1 Investigators. (2007). Effect of torcetrapib on carotid atherosclerosis in familial hypercholesterolemia. N Engl J Med, 356, 1e11. Kondo, I., Berg, K., Drayna, D., & Lawn, R. (1989). DNA polymorphism at the locus for human cholesteryl ester transfer protein (CETP) is associated with high density lipoprotein cholesterol and apolipoprotein levels. Clin Genet, 35, 49e56. Koropatnick, T. A., Kimbell, J., Chen, R., et al. (2008). A prospective study of high-density lipoprotein cholesterol, cholesteryl ester transfer protein gene variants, and healthy aging in very old Japanese-American men. J Gerontol, 63A, 1235e1240. Li, T. Y., Zhang, C., Asselbergs, F. W., et al. (2007). Interaction between dietary fat intake and the cholesterol ester transfer protein TaqIB polymorphism in relation to HDL-cholesterol concentrations among US diabetic men. Am J Clin Nutr, 86, 1524e1529. Liang, H.-Q., Rye, K.-A., & Barter, P. J. (1994). Dissociation of lipid-free apolipoprotein A-I from high density lipoproteins. J Lipid Res, 35, 1187e1199. Marotti, K. R., Castle, C. K., Boyle, T. P., Lin, A. H., Murray, R. W., & Melchlor, G. W. (1993). Severe atherosclerosis in transgenic mice expressing simian cholesteryl ester transfer protein. Nature, 364, 73e75. Matsuura, F., Wang, N., Chen, W., Jiang, X.-C., & Tall, A. R. (2006). HDL from CETP-deficient subjects shows enhanced ability to promote cholesterol efflux from macrophages in an apoEand ABCG1-dependent pathway. J Clin Invest, 116, 1435e1442. Morehouse, L. A., Sugarman, E. D., Bourassa, P.-A., et al. (2007). Inhibition of CETP activity by torcetrapib reduces susceptibility to diet-induced atherosclerosis in New Zealand white rabbits. J Lipid Res, 48, 1263e1272. Morton, R. E. (1985). Binding of plasma-derived lipid transfer protein to lipoprotein substrates: the role of binding in the lipid transfer process. J Biol Chem, 260, 12593e12599. Morton, R. E., & Steinbrunner, J. V. (1990). Concentration of neutral lipids in the phospholipid surface of substrate particles determines lipid transfer protein activity. J Lipid Res, 31, 1559e1567. Morton, R. E., & Zilversmit, D. B. (1982). Purification and characterization of lipid transfer protein(s) from human lipoprotein-deficient plasma. J Lipid Res, 23, 1058e1067. Nagano, M., Yamashita, S., Hirano, K.-i., et al. (2004). Molecular mechanisms of cholesteryl ester transfer protein deficiency in Japanese. J Athero Thromb, 11, 110e121. Nicholls, S. J., Tuzcu, E. M., Brennan, D. M., Tardif, J.-C., & Nissen, S. E. (2008). Cholesteryl ester transfer protein inhibition, high-density lipoprotein raising, and progression of coronary

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atherosclerosis: insights from ILLUSTRATE (Investigation of Lipid Level Management Using Coronary Ultrasound to Assess Reduction of Atherosclerosis by CETP Inhibition and HDL Elevation). Circulation, 118, 2506e2514. Nishida, H. I., Arai, H., & Nishida, T. (1993). Cholesterol ester transfer mediated by lipid transfer protein as influenced by changes in the charge characteristics of plasma lipoproteins. J Biol Chem, 268, 16352e16360. Nissen, S. E., Tardif, J.-C., Nicholls, S. J., et al. for the ILLUSTRATE Investigators. (2007). Effect of torcetrapib on the progression of coronary atherosclerosis. N Engl J Med, 356, 1304e1316. Ordovas, J. M., Cupples, L. A., Corella, D., et al. (2000). Association of cholesteryl ester transfer protein-TaqIB polymorphism with variations in lipoprotein subclasses and coronary heart disease risk: the Framingham study. Artieroscler Thromb Vasc Biol, 20, 1323e1329. Pattnaik, N. M., Montes, A., Hughes, L. B., & Zilversmit, D. B. (1978). Cholesteryl ester exchange protein in human plasma isolation and characterization. Biochim Biophys Acta, 530, 428e438. Pattnaik, N. M., & Zilversmit, D. B. (1979). Interaction of cholesteryl ester exchange protein with human plasma lipoproteins and phospholipid vesicles. J Biol Chem, 254, 2782e2786. Plump, A. S., Masucci-Magoulas, L., Bruce, C., Bisgaier, C. L., Breslow, J. L., & Tall, A. R. (1999). Increased atherosclerosis in apoE and LDL receptor gene knock-out mice as a result of human cholesteryl ester transfer protein transgene expression. Arterioscler Thromb Vasc Biol, 19, 1105e1110. Qiu, X., Mistry, A., Ammirati, M. J., et al. (2007). Crystal structure of cholesteryl ester transfer protein reveals a long tunnel and four bound lipid molecules. Nature Struct Mol Biol, 14, 106e113. Rader, D. J., Alexander, E. T., Weibel, G. L., Billheimer, J., & Rothblat, G. H. (2009). The role of reverse cholesterol transport in animals and humans and relationship to atherosclerosis. J Lipid Res, 50, S189eS194. Rajaram, O. V., White, G. H., & Barter, P. J. (1980). Partial purification and characterization of a triacylglycerol-transfer protein from rabbit serum. Biochim Biophys Acta, 617, 383e392. Rye, K.-A., Hime, N. J., & Barter, P. J. (1995). The influence of cholesteryl ester transfer protein on the composition, size, and structure of spherical, reconstituted high density lipoproteins. J Biol Chem, 270, 189e196. Rye, K.-A., Hime, N. J., & Barter, P. J. (1997). Evidence that cholesteryl ester transfer proteinmediated reductions in reconstituted high density lipoprotein size involve particle fusion. J Biol Chem, 272, 3953e3960. Seip, R. L., Moulin, P., Cocke, T., et al. (1993). Exercise training decreases plasma cholesteryl ester transfer protein. Arterioscler Thromb Vasc Biol, 13, 1359e1367. Tall, A. R. (1986). Plasma lipid transfer proteins. J Lipid Res, 27, 361e367. Thompson, A., Di Angelantonio, E., Sarwar, N., et al. (2008). Association of cholesteryl ester transfer protein genotypes with CETP mass and activity, lipid levels, and coronary risk. J Am Med Assoc, 299, 2777e2788. Tsujita, Y., Nakamura, Y., Zhang, Q., et al. (2007). The association between high-density lipoprotein cholesterol level and cholesteryl ester transfer protein TaqIB gene polyporphism is influenced by alcohol drinking in a population-based sample. Atherosclerosis, 191, 199e205. Van Eck, M., Ye, D., Hildebrand, R. B., et al. (2007). Important role for bone marrow-derived cholesteryl ester transfer protein in lipoprotein cholesterol redistribution and atherosclerotic lesion development in LDL receptor knockout mice. Circ Res, 100, 678e685. Yu, B., Hailman, E., & Wright, S. D. (1997). Lipopolysaccharide binding protein and soluble CD14 catalyze exchange of phospholipids. J Clin Invest, 99, 315e324.

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Zhang, Z., Yamashita, S., Hirano, K.-i., et al. (2001). Expression of cholesteryl ester transfer protein in human atherosclerotic lesions and its implication in reverse cholesterol transport. Atherosclerosis, 159, 67e75. Zilversmit, D. B., Hughes, L. B., & Balmer, J. (1975). Stimulation of cholesterol ester exchange by lipoprotein-free rabbit plasma. Biochim Biophys Acta, 409, 393e398.

Chapter 3

Plasma Cholesteryl Ester Transfer Protein (CETP) in Relation to Human Pathophysiology Akihiro Inazu Department of Laboratory Sciences, School of Health Sciences, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Ishikawa, Japan

INTRODUCTION Plasma low density lipoprotein (LDL) transports cholesterol from liver to peripheral tissues including the adrenal glands and gonads. On the other hand, high density lipoprotein (HDL) transports cholesterol from peripheral tissues including atheroma to liver, subsequently to bile and feces via the so-called reverse cholesterol transport (RCT) pathway (Figure 3.1). The structure of cholesterol is resistant to enzymatic degradation in the human body, hydroxylation is the only pathway to modify cholesterol for excretion from the body. In humans, HDL consists of particles heterogeneous in size, density and apolipoprotein composition. HDL is a vehicle for cholesterol, triglyceride, and phospholipids. Also, HDL has several apolipoproteins and enzymes on its surface that either promote or inhibit triglyceride or phospholipid lipolysis, inhibit hydroperoxidation of lipids, and promote lipid transfer among lipoproteins. In addition, HDL may be a platform for complement regulation, coagulation and inflammation (Scanu and Edelstein, 2008). Plasma HDL content levels are usually measured as cholesterol level concentration, but its particle numbers are better assessed by apolipoprotein A-I levels. This distinction may be due in part to the fact that as interindividual differences of plasma HDL-cholesterol, HDL2-cholesterol levels appeared to be highly variable, but HDL3 remains constant. Smoking and male sex decrease HDL2 levels, but alcohol intake and exercise increase them. HDL2 levels were determined from the catabolic rate of apolipoprotein The HDL Handbook. ISBN: 978-0-12-382171-3 Copyright Ó 2010 Elsevier Inc. All rights of reproduction in any form reserved.

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CM VLDL LPL ApoA-I

CE

CETP

TG

PLTP

HL HDL1

HDL2 ApoE

Cells ABCA1

HDL3

FC, PL

LCAT ApoA-II α 1 HDL α 2-3 HDL Preβ1-HDL

FIGURE 3.1 Schema for HDL metabolism. Plasma cholesteryl ester transfer protein (CETP) facilitates exchange of neutral lipids of CE and TG between chylomicron (CM)/VLDL and HDL2. HDL-TG is provided by CETP, and it is subsequently hydrolyzed by hepatic lipase (HL). The synthetic rate of preb1-HDL is positively correlated with lipoprotein lipase (LPL)-mediated lipolysis or PLTP-mediated PL/FC transfer and increased cholesterol efflux by the ABCA1 transporter. On the other hand, the catabolic rate of preb1-HDL is correlated with cholesterol esterification rate by lecithin:cholesterol acyltransferase (LCAT). Thus, preb1-HDL levels are determined by activities of LPL, PLTP, ABCA1, and LCAT.

A-I and A-II rather than altered synthesis rate. The catabolic rate of HDL apolipoproteins is determined by HDL particle size. The smaller HDL tends to be catabolized faster in the kidney or other tissues. One of the determinants of HDL neutral lipid composition is plasma cholesteryl ester transfer protein (CETP). In incubated human plasma, transfer and equilibration of lecithin:cholesterol acyltransferase (LCAT)-generated CE is found, but the transfer of CE among lipoproteins was not found in rat (Barter and Lally, 1978). Similarly, mice, dogs, and pigs are members of a group of low plasma CETP activity, but rabbits and monkeys belong to a group of high CETP activity. Humans, hamsters, guinea pigs and chickens belong to a group with intermediate CETP activity. Interestingly, more phospholipid transfer protein activity is found in plasmas of low CETP activity animals (Ha and Barter, 1982; Cheung et al., 1996). Plasma CETP binds neutral lipids (cholesteryl ester (CE) or triglyceride (TG)) and phospholipid (PL) on HDL3, but CETP selectively promotes an exchange of CE and TG among lipoproteins. On the one hand, HDL-TG can be hydrolyzed by hepatic lipase and, on the other hand, plasma CETP decreases HDL particle size via CE/TG exchange between chylomicron/VLDL and HDL. Thus, CETP thereby accelerates the catabolic rate of HDL apolipoproteins (Lamarche et al., 1999).

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STRUCTURE OF CETP Plasma CETP was initially isolated as a highly purified 74 kD protein (Pattnaik et al., 1978). The human CETP gene is located at chromosome 16q13, near the locus of the LCAT gene. The CETP gene consists of 16 exons, spanning 25 kb (Agellon et al., 1990). The CETP mRNA encodes 476 amino acids (Drayna et al., 1987). The mature CETP contains four N-linked sugars (88, 240, 341, and 396) with a variable glycosylation site of 341Asn (Stevenson et al., 1993). CETP mRNA is expressed in various tissues, but liver cells, adipocytes and macrophages are abundant sources. Exon 9 works a cassette exon to generate short mRNA missing the sequences in frame in addition to full-length mRNA, but the splice-out variant is not efficiently secreted (Inazu et al., 1992). The C-terminal 26 amino acids of CETP form an amphipathic helix. Hydrophic residues bind to surface lipoproteins, and hydrophobic residues such as Leu, Phe are essential for binding neutral lipids such as CE and TG (Wang et al., 1993). The crystal structure of CETP shows that CETP forms a long tunnel occupied by four lipid molecules, two of CE or TG located inside the tunnel and two of PL plugging both sides of the tunnel openings. CETP is one of the lipopolysaccharide binding protein (LBP) family members. CETP exhibits an elongated boomerang shape located on the lipoprotein surface. Based on molecular size, CETP might prefer CE transfer rather than TG because of steric hindrance to TGs at the tunnel neck around residues 433, 443, 457, and 459 (Qiu et al., 2007).

REGULATION OF CETP EXPRESSION Cholesterol-rich and saturated fat-rich diets increased CETP expression via a liver X receptor (LXR) element in the promoter, a direct repeat of a nuclear receptor binding sequence separated by four nucleotides (DR4) (Luo and Tall, 2000). Among drugs that lower lipid levels, probucol increased plasma CETP activity (þ20%), but pravastatin decreased it (20%) (Inazu et al., 1999). Statins decrease both cholesterol and oxysterol, the latter being a ligand for LXRa activity (Masson et al., 2004). Thus, statins could decrease CETP mRNA levels through diminished LXR activity. However, the molecular effect of probucol on CETP expression is unknown, but it may be associated with increased cholesterol content in the liver by the remnant pathway. Unlike bezafibrate, fenofibrate decreased plasma CETP activity (20e30%) (Guerin et al., 1996; Watts et al., 2006). Since a putative peroxisome proliferator response element (PPRE) is located just upstream of the LXRasite, peroxisome proliferator-activated receptor a (PPARa) could suppress CETP promoter activity by antagonizing LXR activity (Cheema et al., 2005).

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Nicotinic acid is a well-established lipid-lowering agent. Side effects such as flushing may restrict drug usage, however, recent identification of a G protein-coupled receptor GPR109A and of a prostaglandin 2 (PGD2) receptor antagonist (laropiprant) may provide strategies to control side effects. Nicotinic acid is a powerful inhibitor of fat-mobilizing lipolysis via hormone-sensitive lipase in adipose tissue, and therefore limits free fatty acid (FFA) flux into the liver. Nicotinic acid lowers TG as well as Lp(a) levels, and increases HDL-C levels by z20e40%. Nicotinic acid may induce PPARg expression but it is also reported to lower CETP activity. Nicotinic acid increased HDL cholesterol levels by reducing hepatic CETP mRNA only in mice expressing the human CETP transgene (Hernandez et al., 2007; Van der Hoorn et al., 2008).

FUNCTION OF CETP LCAT promotes free cholesterol (FC) esterification in HDL3 and CETP transfers newly-esterified CE from HDL3 to VLDL or chylomicrons. Thus, these tandem reactions appear to be physiological. However, it is unclear whether or not CETP is pro-atherogenic, but it is likely that its atherogenicity is dependent on the metabolic context of lipoprotein receptors expressed in the liver, which are major determinants of RCT pathways to the liver.

LCAT, cholesterol esterification, and CE transfer rate Table 3.1 shows simultaneously determined plasma exogenous LCAT activity and cholesterol esterification rate (CER) and exogenous CETP activity in

TABLE 3.1 Plasma endogenous and exogenous LCAT activity and exogenous CETP activity in young women (n ¼ 38) HDL enzyme/protein

Method

Mean (SD)

Range

LCAT, endogenous

Self substrate method (Nagasaki and Akanuma, 1977)

110 (20)

80e180

LCAT, exogenous

Common substrate method (Manabe et al., 1987)

590 (110)

330e900

CETP

NBD-cholesteryl ester transfer activity between proteoliposome to VLDL

210 (30)

150e290

All units are nmol/ml/h. Endogenous LCAT activity of cholesterol esterification rate is only z20% of exogenous LCAT activity, the latter is correlated with plasma LCAT mass. Endogenous LCAT activity is only z50% of plasma CETP activity, therefore HDL-FC/CE ratio could be altered in heterozygous CETP deficiency.

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young women. Plasma CER is an endogenous LCAT reaction, which shows only z20% of maximal enzymatic activity of LCAT. Net CE transfer rate from HDL to VLDL is only 20e50 nmol/ml/h despite exogenous CETP activity of z200 nmol/ml/h (Pruneta et al., 1999). Since the net CE transfer rate is smaller than that of CER, CE in HDL needs to be directly catabolized in the liver. Therefore, plasma VLDL levels appear to be a rate-limiting step of net CE transfer rate in the fasting state. However, in the post-prandial state, net CE transfer is accelerated because the increased VLDL/chylomicrons provide increased CE acceptor capacity, and the clearance of LDL-CE or remnant-CE is dependent on LDL-receptor activity or remnant receptor (LRP) in the liver. Since HDL-FC is more rapidly catabolized in the liver than HDL-CE in a monkey study, selective uptake of FC without endocytosis of HDL apolipoproteins appears to be a predominant pathway of HDL-C catabolism in the liver (Scobey et al., 1989). Thus, HDL-CE pathways play minor roles in human HDL-cholesterol catabolism (Schwartz et al., 2004). Also, FC from HDL is efficiently secreted in bile, but not from other lipoproteins (Robins and Fasulo, 1997). Thus, cholesterol esterification is not necessarily required for the selective uptake of HDL-cholesterol in the liver via hepatic lipase and scavenger receptor class BI (SR-BI) mediated RCT pathways. The lipoprotein phenotype of high HDL and the low CER appear to be anti-atherogenic as long as efficient RCT is maintained in the liver.

Modulators of lipid transfer CETP-mediated lipid transfer is not preferably directed toward a specific lipoprotein in a reconstituted system. Because CE is generated in HDL via the LCAT reaction, higher CE concentrations are found in HDL. Therefore, net CE transfer operates from HDL to other lipoproteins in vivo. Similarly, because chylomicrons and VLDL are rich in TG, net TG transfer is found from chylomicron/VLDL to other lipoproteins via heteroexchange of CE and TG. In addition, some specific apolipoproteins and TG lipolysis occurring during the post-prandial state would modify the direction of lipid transfer among lipoproteins. As a modulator of CE transfer, apoF was identified as lipid transfer inhibitor protein (LTIP). LTIP inhibits CE transfer between VLDL and LDL, whereas it increases CE transfer from HDL to VLDL (Wang et al., 1999). Phospholipid transfer protein (PLTP) promotes PL transfer from VLDL to HDL, in addition to the PL transfer activity of CETP. Also, PLTP possesses free cholesterol and vitamin E transfer activity. As CE acceptors of the CE transfer reaction, VLDL and chylomicrons are active when lipolysis has occurred. VLDL-bound LPL and FFA levels may have a positive effect on the binding between CETP and lipoproteins, thereby accelerating CE mass transfer.

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Effects of CETP on LDL subclass remodeling An association of large LDL and low CETP activity with TaqIB polymorphism was found in men, but not in women in the genetic epidemiological survey of the Framingham Study (Ordovas et al., 2000). In remodeling of apoBcontaining lipoproteins, addition of CE increases lipoprotein size and deletion of PL and FC decrease its size, resulting in two homogeneous LDL subclasses (Musliner et al., 1991). Complete CETP deficiency produced unique characteristics of broad LDL band with (at least five) distinct IDL-LDL subclasses on a native polyacrylamide gel (Sakai et al., 1991), but partial CETP deficiency increased LDL size.

Effects of CETP on macrophage-specific RCT in mice and hamsters In radioactive cholesterol-labeled macrophage methodology, Rader et al. (Tanigawa et al., 2007) have shown direct RCT from peripheral macrophages to liver, bile and feces. In LDLR-KO mice, CETP cDNA adeno-associated virus mediated transfection promotes cholesterol transport to the liver, but not to bile and feces. In contrast, in SRBI-KO mice, CETP cDNA transfection increased cholesterol loss in the feces, indicating induction of overall RCT via active LDL-R activity despite diminished selective uptake of HDL-CE (or FC) in the liver (Tanigawa et al., 2007). The former model is similar to the setting of familial hypercholesterolemia (FH) or downregulated LDL receptor activity by a saturated-fat diet, while the latter model of decreased SR-BI activity reflects conditions found in hormone replacement therapy (Zhang et al., 2007). Thus, macrophage-specific RCT is dependent on CETP activity and active LDL receptors in the liver, and the efficacy of fecal sterol excretion is compatible with effects of anti-oxidative agents such as vitamin E and probucol, which would increase plasma CETP activity, in cholesterol-fed rabbits (Jeon et al., 2005). In hamsters, torcetrapib, a CETP inhibitor, elevated both HDL-C levels and the amounts of cholesterol and bile acids secreted in feces, indicating an overall increase in RCT (Tchoua et al., 2008). Such a difference may be explained (at least in part) by the presence of natural CETP activity and the inducible CYP7A gene found in hamsters (Zhang et al., 2004).

Effects on cholesterol efflux and preb-HDL formation Subjects with complete CETP deficiency have more preb-HDL despite less remodeling from large HDL to small subclasses via CETP (Asztalos et al., 2004). Thus, increased preb-HDL levels are caused by impaired maturation to large HDL due to decreased endogenous LCAT activity (Oliveira et al., 1997) or increased lipolysis of TG-rich lipoproteins (Miyazaki et al., 2009). Since LCAT mass and exogenous LCAT activity remain at normal levels, impaired LCAT activity is

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explained either by: (1) end-product inhibition, namely excess CE in large HDL or (2) altered phospholipid composition, such as sphingomyelin (SM) levels, in HDL. Plasma cholesterol esterification rate was decreased in CETP deficiency, which is compatible with altered lipid composition found in HDL fractions of homozygous CETP deficiency; i.e., high CE/TG ratio and low PL/FC ratio (Koizumi et al., 1991). Since CER is inversely associated with SM/PC ratio in HDL, SM itself may be an unsuspected link between low cholesterol esterification rate and low CETP activity (Noguchi and Inazu, unpublished data). However, SM-rich lipoproteins are not always pro-atherogenic because SM avidly binds cholesterol, and HDL with increased SM levels may be good acceptors for cholesterol efflux from atherosclerotic plaques (Fournier et al., 1997). Increased HDL levels found in CETP deficiency had no beneficial effect on the ABCA1-mediated cholesterol efflux but did enhance SR-BI-mediated efflux (Miwa et al., 2009).

Lipoprotein metabolism in CETP deficiency from a kinetic study Initially, Ikewaki et al. reported delayed catabolism of apoA-I and apoA-II in human subjects with CETP deficiency (Ikewaki et al., 1993). Also, they reported an increased catabolic rate of LDL-apoB in addition to a decreased production rate of VLDL-apoB (Ikewaki et al., 1995). In a CETP-deficient dog, Ouguerram et al. reported that VLDL and LDL CE metabolism was coupled to apoB catabolism without enrichment of CE during VLDL-LDL conversion and that 60% of HDL CE turnover was mediated by a selective uptake pathway (Bailhache et al., 2004; Ouguerram et al., 2004). As compared to other CETP-deficient animals, dogs have higher selective uptake of HDLCE (60% vs. 25e30% in rats and mice). The cholesterol esterification rate of dog plasma is 160 nmol/ml/h, which is between the rates in humans (30e80 nmol/ml/h) and in rats (300 nmol/ml/h). Thus, dogs may have an efficient RCT due to high activities of SR-BI and LCAT in addition to CETP deficiency.

ROLE OF CETP IN APOE-RICH HDL FORMATION Structure of apoE-rich HDL Plasma HDL is classified as HDL1 (density 1.08e1.09 g/ml), HDL2 (1.09e1.15), and HDL3 (1.15e1.18). HDL1 is apoE-rich with a diameter of 13e19 nm and increased LCAT activity compared with HDL2 and HDL3 (Schmitz and Assmann, 1982). HDL1 is also identified in cholesterol-fed CETP-deficient animals such as dogs and pigs.

Function of apoE-rich HDL In cultured smooth muscle cells, cholesterol from HDLc, lipoproteins with apoE only (density 1.006e1.02), present in cholesterol-fed canine plasma,

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The HDL Handbook

was efficiently delivered to the cells as well as LDL (Mahley et al., 1977). ApoE-rich HDL appears in various situations such as genetic dyslipidemia, but its characteristics may not be uniform. In cholesterol-fed canines, plasma cholesterol increases, HDL loses apoA-I but it gains apoE. HDLc appears (as well as occurrence of LDL and b-migrating VLDL) when cholesterol exceeds 700 mg/dl. Thus, HDL1 and HDLc appeared to suppress apoB-containing lipoprotein formation in the liver, such as LDL and b-VLDL. Thus, one would predict that these lipoproteins would inhibit atherogenesis in canine models. ApoE-rich HDL have dual roles in atherogenicity. ApoE can serve as an LRP ligand, and therefore canine HDLc inhibits clearance of chylomicrons (Hussain et al., 1995). However, post-prandial lipemia is diminished in homoand heterozygous CETP deficiency (Inazu et al., 2008). VLDL lipolysis and hepatic uptake of CM/VLDL remnant appear to be increased probably due to apoE transfer from HDL to CM/VLDL during post-prandial periods (Krimbou et al., 2003). None the less, apoE-rich HDL reduces LPL-mediated retention of LDL by subendothelial matrix, and therefore could play an anti-atherogenic role in artery walls. Also, apoE-rich lipoproteins protect cells from apoptosis via the LRP signaling pathway (Hayashi et al., 2007). In SB-BI knockout mice, LCAT activity was impaired and oxidative stress was increased in large HDL (Lee et al., 2007; Van Eck et al., 2007). Remnant-like particle cholesterol (RLP-C) levels reflect cholesterol levels (10e15 mg/dl) of large apoE-rich HDL (probably apoE only particles) in homozygous CETP deficiency (Inazu et al., 2008). The apoE-rich HDL contains apoA-IV as well as apoA-I (Bisgaier et al., 1991), but the RLP fraction of homozygous CETP deficiency had large amounts of apoE with a trace of apoA-I and apoA-IV.

MOLECULAR GENETICS AND THE ETHNIC DIFFERENCE IN THE FREQUENCY OF HUMAN CETP DEFICIENCY Plasma CETP deficiency was originally reported in Japanese siblings with hyperalphalipoproteinemia (HALP) (Koizumi et al., 1985). The first mutation was found in a splice donor site mutation in intron 14 (intron 14 G(þ1)-to-A), resulting in non-translation of exon 14 and production of a stop codon in the fourth codon encoded by exon 15. These changes resulted in decreased mRNA levels to one-third of controls and a truncated protein that appeared to be rapidly degraded (Brown et al., 1989; Gotoda et al., 1997). So far, 20 different mutations have been found both in Asian and Caucasian populations, but predominantly in Asians (Nagano et al., 2004; Thompson et al., 2009). Two mutations were found in both ethnic groups (R268X and intron 14 G(þ1)-to-A), suggesting multiple origins of these mutations (de novo mutations) (Ai et al., 2009). Both mutations indeed have CpG sequences as mutational hot spots for deamination of the cytosine. Although many mutations

Chapter j 3

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43

are nonsense or splicing mutations, four missense mutations are reported to be associated with decreased CETP activity (L151P, L261R, R282C, and D442G). Only one promoter mutation was reported at e69G>A. Large differences in the frequency of CETP deficiency in various populations appear to be related to the frequency of two variants. The intron 14 G (þ1)-to-A mutation is the Japanese-type mutation with the higher gene frequency (0.8% in the general population of Japan). Homozygotes of this mutation were reported in >50 cases reflecting relatively higher frequency of consanguinity in Japan in past generations.

CLINICAL CHEMISTRY OF LDL-CHOLESTEROL AND HDL-CHOLESTEROL IN CETP DEFICIENCY Homozygotic mutations result in complete CETP deficiency with a phenotype of very high HDL-C levels and relatively low LDL-C levels (mean levels of 164 mg/dL and 77 mg/dL, respectively) (Inazu et al., 1990). Heterozygotes have a moderate increase in HDL-C (mean 66 mg/dL) and a decrease in plasma CETP levels (mean 1.4 mg/L) as compared to unaffected controls (53 mg/dL and 2.3 mg/L). Asp 442 Gly (D442G) is another highly prevalent mutation in Japan (3.4% in the general population of Japan) as well as in other Asian populations (1.7e5.9%), although it is only partially defective in CETP activity (Inazu et al., 1994). The compound heterozygotes of intron 14 G(þ1)to-A and D442G produce a less severe phenotype of CETP deficiency (n ¼ 9, CETP 0.9  0.3 (SD) mg/L, HDL-C 130  24 mg/dL) as compared to mean levels of plasma CETP 1.8  0.6 mg/L (SD) in Japanese men and 2.0  0.5 in women (Kiyohara et al., 1998).

LDL-C measurement The Friedewald formula, LDL-C ¼ TC  HDL-C  (TG/5), is used for estimation of LDL-C, but accurate measurement for HDL-C is required. For the precipitation method for HDL-C, the Cholesterol Reference Method Laboratory Network (CRMLN) using a heparin, Mn2þ supernatant cholesterol of plasma d > 1.006 (Centers for Disease Control and Prevention (CDC)) is better than the Designed Comparison Method (DCM) using dextran-sulfate, Mg2þ supernatant cholesterol levels, since the latter precipitates apoE-rich HDL in addition to apoB-containing lipoproteins, but the former does not. Even if accurate measurement of HDL-C is accomplished, cholesterol levels in VLDL are relatively decreased in CETP deficiency (Koizumi et al., 1991). Therefore, the Friedewald formula would underestimate LDL-C. However, since the density between 1.019 and 1.063 includes apoE-rich large HDL such as HDL1, the LDL-C separated by ultracentrifugation would overestimate LDL-C. Suitability for homogeneous LDL-C assays has not been reported in homozygous CETP deficiency.

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ApoE-rich HDL-C determination As a more suitable precipitation method for HDL-C in CETP deficiency, Chiba et al. reported that 13% polyethylene glycol (PEG) allows recovery of total HDL in the supernatant (Chiba et al., 1997). In that study, patients with complete CETP deficiency had a mean HDL-C level of 121 mg/dL detected by a commercial polyanionic reagent (dextran sulfate, sodium phosphotungstate, Mg2þ), but 176 mg/dL of total HDL-C using supernatants produced by the PEG method. The difference (z55 mg/dL) may indicate cholesterol levels in apoErich HDL.

EPIDEMIOLOGY OF INCREASED HDL CHOLESTEROL LEVELS AND CETP DEFICIENCY HDL-cholesterol could be excreted from bile as consequence of reverse cholesterol transport (RCT) involving HDL maturation from preb-HDL to apoE-rich HDL. However, CETP would bypass the cholesterol flow from HDL to VLDL-LDL without involving the liver. Thus, CETP-mediated CE transfer would increase indirect cholesterol transport to the liver via VLDL-IDL-LDL through LDL receptor or remnant receptor pathways. In addition, HDLcholesterol is directly transported to the liver by selective uptake of HDL-CE or FC via hepatic lipase and/or SR-BI pathway. Thus, the role of the CETP pathway appears to be anti-atherogenic when LDL levels are low and TRL clearance is rapid on a low-fat diet. However, the Western-type saturated-fat diet would suppress LDL receptor expression, and the flow of HDL-derived cholesterol back to the liver would be diminished via the LDL pathway. Indeed, subjects with high CETP activity may manifest lower coronary risk in the presence of low plasma TG levels (Borggreve et al., 2007). The role of CETP in LDL-receptor deficiency (familial hypercholesterolemia) is controversial, since double heterozygotes with FH and CETP deficiency are not protected from CHD (Haraki et al., 1997). It remains to be discussed whether slightly increased HDL-C (60 mg/ dL vs. 46 mg/dL) is not sufficient to prevent CHD or whether lower CETP is disadvantageous in FH.

Epidemiology of HDL cholesterol In earlier studies by Gofman et al. (Gofman et al., 1966), levels of HDL2 and HDL3 were significantly decreased in patients with CHD, but HDL1 levels were not changed. In heparin-sepharose chromatography, HDL-apoE levels were significantly decreased in survivors with myocardial infarction (Wilson et al., 1993). In a proteomic study of HDL proteins, apoE levels in the HDL3 fraction were increased in patients with CHD (Vaiser et al., 2007), but unfortunately no data were available on HDL2 or VLDL.

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Inconsistency of anti-atherogenicity of HDL might be explained by how much large HDL or apoE-rich HDL are increased, as these particles are believed to have less anti-atherogenic effects compared to small HDL. When apoA-I and apoB are kept constant, HDL-C and HDL particle size may confer risk at very high values (van der Steeg et al., 2008). On the contrary, apoA-I is a negative risk factor even when corrected for HDL-C and apoB, suggesting that HDL number assessed by apoA-I concentration is statistically more important than HDL size for anti-atherogenicity effects. The debate over whether HDL size or its components are more important for atherogenesis should be answered by measuring specific HDL-related lipid component levels such as sphingomyelin, sphingosine-1-phosphate, and dolichol in HDL of various dyslipidemias (Kontush et al., 2007).

The role of confounding factors in increased HDL state In many reports, a high HDL-C state is a negative risk factor for CHD and stroke (Kurth et al., 2007). Since low HDL-C is inversely associated with increased TG levels, low HDL appeared to be a marker for disturbed TG metabolism (Schaefer et al., 1994). However, some reports suggest a U-shape relationship with HDL-C and vascular events (Chien et al., 2002). Such a relationship may be associated with some confounding factors associated with increased HDL-C levels: alcohol, estrogen and exercise (Williams, 1996). There are reports of an adverse interaction between alcohol and hypertension on stroke (Leppala et al., 1999). Others have suggested an interaction between increased levels of TG and HDL-C on CHD (Jeppesen et al., 1998). A large genetic epidemiological survey is warranted to find associations between CETP or hepatic lipase polymorphisms and CHD events, especially by interacting with environmental factors such as alcohol consumption and hormone replacement therapy. Since lower activities of hepatic lipase and SR-BI and higher CETP are characteristics of premenopausal women, consideration of gender difference is necessary in unraveling the interactions between HDL and CHD (Jansen et al., 2002) (Table 3.2).

TABLE 3.2

Gender difference in HDL-associated biological activities Transfer proteins, enzyme, receptor

Lipoprotein phenotype

CETP

PLTP

Hepatic lipase

SR-BI

HDL2b

preb-HDL

Men

Low

High

High

High

Low

High

Women*

High

Low

Low

Low

High

Low

LPL activity is not different between men and women. * Premenopausal state

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The HDL Handbook

The role of CETP mutations and polymorphisms on CHD risk A meta-analysis of studies including CETP gene single nucleotide polymorphisms (SNPs) of TaqIB2, -629C>A and Ile 405 Val (I405V) showed that the genotypes with low CETP may have anti-atherogenic effects (Thompson et al., 2008). Our data suggested that e1337C>T is responsible for the antiatherogenicity of the well-investigated TaqIB2 allele in the Japanese population (Lu et al., 2003; Takata et al., 2006). Similarly, anti-atherogenicity of lower CETP levels was also suggested in heterozygous CETP deficiency (Curb et al., 2004). CHD prevalence appears to be low in homozygous CETP deficiency, which is compatible with findings of the Kochi Study cross-sectional survey of disease prevalence stratified by increased HDL-cholesterol levels >80 mg/dL and >100 mg/dL (Moriyama et al., 1998). Three hundred subjects with HDL-C > 100 mg/dL were found in that paper, but no case with CHD was found. Indeed, high HDL-cholesterol and intron 14 G(þ1)>A variant may increase the odds for healthy aging in the Honolulu Heart Program Study (Koropatnick et al., 2008). Consistently, recent case reports of Caucasian CETP deficiency have shown the rarity of atherosclerotic disease even though Western diets were consumed (Teh et al., 1998; Rhyne et al., 2006). However, some investigators believe pro-atherogenicity in some cases with homozygous CETP deficiency (Nagano et al., 2005). In contrast, there is no defined CHD, but two cases with cerebrovascular disease were found in our cohort of homozygous CETP deficiency (n ¼ 53).

Malignancy association In earlier studies, Keys suggested a possible association between increased HDL-C levels and malignancy incidence (Keys, 1983). The failure of torcetrapib is a reminder of such a possible association. This issue should be assessed in studies using other CETP inhibitors.

DEVELOPMENT OF A CETP INHIBITOR Three compounds are currently in clinical trials, torcetrapib (CP-529414), anacetrapib (MK-859) and dalcetrapib (JTT-705/ Roche R1658). Phase III of torcetrapib was terminated in December, 2006 due to an unexpected excess of mortality in the torcetrapib arm. The early termination was partially explained by hypertension due to aldosterone excess. However, the role of CETP inhibition on the increased mortality was not clearly shown, but it may be rather associated with infection or malignancy than CHD (Barter et al., 2007). The vascular endpoints of carotid atherosclerosis and coronary atheroma volume assessed by intravascular ultrasound showed no benefit from torcetrapib over a background of atorvastatin treatment, despite increased levels

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47

of HDL and further decreased levels of LDL and TG (Kastelein et al., 2007; Nissen et al., 2007). HDL-cholesterol might be excreted from bile as consequence of reverse cholesterol transport (RCT) involving HDL maturation from preb-HDL to apoE-rich HDL. Using a CETP inhibitor, CE uptake in liver was not decreased in rabbits, but fecal sterol excretion was not increased in patients taking torcetrapib, indicating that overall RCT was not significantly induced (Brousseau et al., 2005; Kee et al., 2006; Catalano et al., 2009). However, torcetrapib did increase overall RCT assessed by cholesterol and bile acids in feces of hamsters (Tchoua et al., 2008). Such a difference in the response to CETP inhibitors definitely needs to be clarified.

Effects on small HDL subclasses Hyperalphalipoproteinemia (HALP) caused by prednisone plus cyclosporine was ineffective in producing HDL acceptors for cholesterol efflux. The ABCA1-dependent efflux was maintained, but the non-ABCA1-dependent route appeared to be impaired (Sviridov et al., 2006). CETP inhibition may disturb apoA-I liberation from HDL in atherosclerotic lesions. Therefore, ABCA1-mediated cholesterol efflux activity to small HDL or liberated apoA-I could be compromised. However, recent studies suggest that the ABCG1 transporter may favorably induce cholesterol efflux from cells to large HDL (Yvan-Charvet et al., 2007). Torcetrapib would increase this large HDL level, which is an active cholesterol acceptor for ABCG1 or SR-BImediated efflux, although the role of SR-BI-mediated cholesterol efflux remains controversial (Yvan-Charvet et al., 2008). Although small HDL, such as HDL3 subclass, is known to protect LDL from oxidation (Davidson et al., 2009), HDL3 levels were not increased in genetic CETP deficiency, but they were moderately increased in patients with CETP inhibitors.

Effects on apoB-containing lipoproteins By inhibiting neutral lipid transfer among lipoproteins, CE transfer from HDL to VLDL in exchange with TG was diminished. Therefore, relatively CE-poor, TG-rich VLDLs were lipolyzed to LDL and VLDL-IDL-LDL were rapidly removed from the circulation probably due to LDL-receptor upregulation (Millar et al., 2006). In LDL subclasses, small-and-dense LDL levels were decreased but large LDL levels were increased in patients with torcetrapib (Brousseau et al., 2004), which is compatible with a phenotype in low CETP subjects with a TaqIB2 polymorphism (Ordovas et al., 2000). Plasma Lp(a) levels were decreased in CETP deficiency, and z50% reduction of plasma Lp(a) levels was achieved by anacetrapib (Bloomfield et al., 2009). Large HDL contains multiple apoE molecules, but such lipoproteins could be efficiently removed from the circulation via increased LDL receptor

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expression. Thus, increased levels of apoE-rich HDL produced by a CETP inhibitor could be offset by combination therapy with a statin, which induces LDL receptor expression and increases hepatic uptake of apoE-rich HDL. Increased RCT was found when both CETP and LDL receptor are upregulated in the liver. Thus, combination therapy with a CETP inhibitor and a statin would result in opposite responses in the RCT pathway, a finding that would be compatible with a proposed adverse pharmacogenetic interaction between a statin and a CETP inhibitor (Regieli et al., 2008).

ROLE OF CETP IN AGING AND LONGEVITY CETP enhances HDL remodeling from large HDL to small subclasses including pre-HDL. However, CETP deficiency would decrease cholesterol esterification rate, thereby inhibiting maturation of preb-HDL to a-migrating spherical HDL. Therefore, in CETP deficiency, large-to-small HDL remodeling is decreased and preb-HDL catabolism is also decreased. The levels of prebHDL were increased in homozygous CETP deficiency, but were decreased in the heterozygotes (Asztalos et al., 2004), indicating that maturation of the small HDL subclass is preserved in heterozygotes, but not in homozygotes. The difference is dependent on the magnitude of low CER and low ABCA1mediated efflux activity (Figure 3.2). Recent studies suggested PAF-AH (lipoprotein-associated phospholipase A2) inhibitors could inhibit sdLDL formation, thereby preventing atherosclerosis in animal models and humans. Plasma paraoxonase activity was decreased in HALP with hepatic lipase deficiency (Kontush et al., 2004). Thus, the antioxidant activity of HDL needs to be evaluated in patients treated with CETP inhibitors.

Effects on aging and Alzheimer’s disease A promising effect on longevity has been reported in Ashkenazi Jews, as increased homozygosity of I405V was found in offspring of individuals with exceptional longevity (mean age 98 years). These subjects had high HDL, low LDL and large LDL size, and low prevalence of hypertension and metabolic syndrome (Barzilai et al., 2003). A different CETP polymorphism (D442G) may have a protective effect against the development of Alzheimer’s disease (AD), especially in apoE4 carriers in the Chinese population (Chen et al., 2008). But, the opposite relationship between another CETP polymorphism (I405V) and AD was found in the Dutch population (Arias-Vasquez et al., 2007). Indeed, the CETP gene haplotype was associated with both markers of cholesterol synthesis and degradation in the cerebrospinal fluid and CETP may have neuronal repair effects through PL transfer activity. Data regarding possible AD associations between genes for CETP and apoE are conflicting, and need to be resolved.

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CETP and Atherosclerosis

A

CM VLDL FC, PL

TG

CE

Cells

ApoA-I

HDL2

HDL2

HDL3 ApoA-II

α 1 HDL

α 2-3 HDL

Preβ1-HDL

B CM VLDL FC, PL ApoA-I

CE

HDL1

HDL2

ApoE

Cells

TG

HDL3 ApoA-II

α 1 HDL

α 2-3 HDL

Preβ1-HDL

FIGURE 3.2 Differential metabolic fate of HDL in heterozygous and homozygous CETP deficiency. (A) In heterozygotes, both CM/VLDL lipolysis and cellular ABCA1-mediated FC/PL efflux are maintained. Also, LCAT reaction is relatively preserved. Thus, decrease in preb1-HDL indicates that HDL maturation is not disturbed. (B) In homozygotes, CM/VLDL lipolysis is enhanced and the cellular efflux is diminished. Also, LCAT reaction is severely suppressed. Thus, preb1-HDL is accumulated in plasma.

Susceptibility to infectious disease As CETP belongs to the lipopolysaccharide binding protein (LBP) gene family, a role for CETP has been suggested in infection. Since LBP and lipoprotein may be associated with the detoxification of endotoxin, apoB-containing lipoprotein levels may reflect the efficacy of LBP function (Vreugdenhil et al., 2001). Along with an increase of LBP, plasma cholesterol, PL, LDL-C, HDL-C

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The HDL Handbook

decrease, whereas plasma TG, VLDL and apoE-rich HDL tend to be increased after intravenous endotoxin (Hudgins et al., 2003; Li et al., 2008). In experimental endotoxemia, increased CRP levels are found with reciprocal decreases of LCAT and CETP activities (Levels et al., 2007). CETP expression was suppressed by cytokines of TNFa and IL-1. PLTP deficiency led to a significant increase in LPS-induced mortality in mice (Gautier et al., 2008). Thus, we need to consider possible disadvantages of CETP deficiency in terms of endotoxemia because CETP would enhance the LPS binding to HDL/ LDL. The liver uptake of LPS was greater in CETP-transgenic mice than controls, suggesting accelerated clearance of LPS from the circulation (Cazita et al., 2008). On the other hand, large HDL found in CETP deficiency might be protective against Schistosoma japonicum (Okamura-Noji et al., 2001).

ROLE OF CETP IN DYSLIPIDEMIA ASSOCIATED WITH DIABETES AND METABOLIC SYNDROME In hyperlipidemic patients, increased production of VLDL and/or decreased catabolism of LDL are major risk factors in addition to low HDL-C. Decreased production rate of VLDL appears to be associated with decreased CETP activity in patients with metabolic syndrome treated by fenofibrate (Watts et al., 2006). A CETP inhibitor may be especially useful for combined hyperlipidemia of high VLDL and low HDL levels. LDL catabolic rate is increased with CETP inhibitors, but effects on VLDL production rate have been less established. Plasma CETP levels are increased in metabolic syndrome (Sandhofer et al., 2006). Plasma PLTP levels are increased, but the increase in CETP is somewhat controversial in diabetes (Dallinga-Thie et al., 2007). Plasma CETP activity was positively related with CETP mass, and negatively to HbA1c (Dullaart et al., 2004). In diabetes, decreased sterol regulatory element binding protein (SREBP) expression may lead to lower CETP expression in the liver (MacLean et al., 2005). Phosphoinositide 3-kinase activity is decreased in diabetes, and liver SRBI expression is decreased (Shetty et al., 2006). Since increased CETP activity may be beneficial in diabetes as shown in a db/db mouse study (MacLean et al., 2003), such a complex relationship should be examined in human diabetes. In the Copenhagen City Heart Study, elevated HDL-C caused by an I405V polymorphism is a risk for CHD in women without hormone replacement therapy but not in men (Agerholm-Larsen et al., 2000). Atherogenicity of CETP may be related to SR-BI expression in terms of RCT to bile cholesterol excretion. Hepatic SR-BI is induced by a diet rich in polyunsaturated fat, but suppressed by cholesterol, vitamin E and estradiol. As shown in knock-out mice, SR-BI deficiency is associated with increased HDL levels but it is proatherogenic (Trigatti et al., 1999). Thus, specific attention may be required for diabetic and female patients when a CETP inhibitor is considered, because reduced SR-BI expression is

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51

assumed in those conditions. Also, usefulness of combination therapy with a statin (HMG-CoA reductase inhibitor) and a CETP inhibitor needs to be validated experimentally.

CLINICAL TRIALS OF CETP INHIBITORS More studies are needed for development of HDL intervention through inhibiting plasma CETP activity. Especially, it is important to assess how to suppress plasma CETP activity. Antisense CETP therapy is of greater interest than chemical compounds because the antisense therapy would decrease plasma CETP mass. In protection of neuronal disease, more study of cerebrospinal fluid lipoprotein is needed in terms of compositions of apoE and lipid transfer proteins and pharmaceutical changes in those lipoproteins. In addition to CE/TG transfer, CETP may transfer estrogen-ester and retinyl-ester but not vitamin E. Antioxidative local effects of CETP may be more important under oxidative stress or combined metabolic conditions of PTLP deficiency, which is defective in vitamin E transport. Thus, the role of CETP in terms of lipoprotein oxidation needs to be clarified in various settings of concurrent hyperlipoproteinemia or hormonal exposure such as estrogen. LPS is associated not only with endotoxemia, but also with vascular oxidative stress and inflammation. Therefore, the role of CETP in LPS metabolism needs to be clarified in CETP deficiency and patients treated with CETP inhibitors.

LOW CETP STATUS, GENETIC OR ENVIRONMENTAL? Recent publications suggested that low CETP mass is associated with lipidlowering drugs, history of myocardial infarction, diabetes, smoking, and inflammation with elevations of CRP and IL-6 (Ritsh et al., 2010). Indeed, CETP expression was decreased in leukocytes and macrophages in acute coronary syndrome (Ye et al., 2008), suggesting downregulation of CETP expression during acute inflammation. In addition, Vasan et al. have shown that low CETP activity was associated with greater cardiovascular risk in a prospective study of the Framingham Heart Study (Vasan et al., 2009). Thus, the cause of low CETP activity needs to be clarified to insight conflicting data between CETP activity and cardiovascular risk. Thus, both genetic and environmental factors need to be assessed in a cardiovascular health study including plasma CETP mass or activity.

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Agerholm-Larsen, B., Nordestgaard, B. G., Steffensen, R., Jensen, G., & Tybjaerg-Hansen, A. (2000). Elevated HDL cholesterol is a risk fator for ischemic heart disease in white women when caused by a common mutation in the cholesteryl ester transfer protein gene. Circulation, 101, 1907e1912. Ai, M., Tanaka, A., Shimokado, K., et al. (2009). A deficiency of cholesteryl ester transfer protein whose serum remnant-like particle (RLP)-triglyceride significantly increased, but RLPcholesterol did not after an oral fat load. Ann Clin Biochem, 46, 457e463. Arias-Vasquez, A., Isaacs, A., Aulchenko, Y. S., et al. (2007). The cholesteryl ester transfer protein (CETP) gene and the risk of Alzheimer’s disease. Neurogenetics, 8, 189e193. Asztalos, B., Horvath, K. V., Kajinami, K., et al. (2004). Apolipoprotein composition of HDL in cholesteryl ester transfer protein deficiency. J Lipid Res, 45, 448e455. Bailhache, E., Briand, F., Nguyen, P., Krempf, M., Magot, T., & Ouguerram, K. (2004). Metabolism of cholesterol ester of apolipoprotein B100-containing lipoproteins in dogs: evidence for disregarding cholesterol ester transfer. Eur J Clin Invest, 34, 527e534. Barter, P. J., Caulfield, M., Eriksson, M., et al., for the ILLUMINATE Investigators. (2007). Effects of torcetrapib in patients at high risk for coronary events. N Engl J Med, 357, 2109e2122. Barter, P. J., & Lally, J. I. (1978). The activity of an esterified cholesterol transferring factor in human and rat serum. Biochim Biophys Acta, 531, 233e236. Barzilai, N., Atzmon, G., Schechter, C., et al. (2003). Unique lipoprotein phenotype and genotype associated with exceptional longevity. J Am Med Assoc, 290, 2030e2040. Bisgaier, C. L., Siebenkas, M. V., Brown, M. L., et al. (1991). Familial cholesteryl ester transfer protein deficiency is associated with triglyceride-rich low density lipoproteins containing cholesteryl esters of probable intracellular origin. J Lipid Res, 32, 21e33. Bloomfield, D., Carlson, G. L., Sapre, A., et al. (2009). Efficacy and safety of the cholesteryl ester transfer protein inhibitor anacetrapib as monotherapy and coadministered with atorvastatin in dyslipidemic patients. Am Heart J, 157, 352e360. Borggreve, S. E., Hillege, H. L., Dallinga-Thie, G. M., et al., on behalf of the PREVEND Study Group. (2007). High plasma cholesteryl ester transfer protein levels may favour reduced incidence of cardiovascular events in men with low triglycerides. Eur Heart J, 28(8), 1012e1018. Brousseau, M. E., Diffenderfer, M. R., Millar, J. S., et al. (2005). Effects of cholesteryl ester transfer protein inhibition on high-density lipoprotein subspecies, apolipoprotein A-I metabolism, and fecal sterol excretion. Arterioscler Thromb Vasc Biol, 25, 1e8. Brousseau, M. E., Schaefer, E. J., Wolfe, M. L., et al. (2004). Effects of an inhibitor of cholesteryl ester transfer protein on HDL cholesterol. N Engl J Med, 350, 1505e1515. Brown, M. L., Inazu, A., Hesler, C. B., et al. (1989). Molecular basis of lipid transfer protein deficiency in a family with increased high-density lipoproteins. Nature, 342(6248), 448e451. Catalano, G., Julia, Z., Frisdal, E., et al. (2009). Torcetrapib differentially modulates the biological activities of HDL2 and HDL3 particles in the reverse cholesterol transport pathway. Arterioscler Thromb Vasc Biol, 29, 268e275. Cazita, P. M., Barbeiro, D. F., Moretti, A. I. S., Quitao, E. C. R., & Soriano, F. G. (2008). Human cholesteryl ester transfer protein expression enhances the mouse survival rate in an experimental systemic inflammation model: a novel role for CETP. Shock, 30, 590e595. Cheema, S. K., Agarwal-Mawal, A., Murray, C. M., & Tucher, S. (2005). Lack of stimulation of cholesteryl ester transfer protein by cholesterol in the presence of a high-fat diet. J Lipid Res, 46, 2356e2366.

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Chen, D. W., Yang, J. F., Tang, Z., et al. (2008). Cholesteryl ester transfer protein polymorphism D442G associated with a potential decreased risk for Alzheimer’s disease as a modifier for APOE 34 in Chinese. Brain Res, 1187, 52e57. Cheung, M. C., Wolfbauer, G., & Albers, J. J. (1996). Plasma phospholipids mass transfer rate: relationship to plasma phospholipid and cholesteryl ester transfer activities and lipid parameters. Biochim Biophys Acta, 1303, 103e110. Chiba, H., Akita, H., Tsuchihashi, K., et al. (1997). Quantitative and compositional changes in high density lipoprotein subclasses in patients with various genotypes of cholesteryl ester transfer protein deficiency. J Lipid Res, 38, 1204e1216. Chien, K. L., Sung, F. C., Hsu, H. C., Su, T. C., Lin, R. S., & Lee, Y. T. (2002). Apolipoprotein A-I and B and stroke events in a community-based cohort in Taiwan. Report of the Chin-Shan Community Cardiovascular Study. Stroke, 33, 39e44. Curb, J. D., Abbott, R. D., Rodriguez, B. L., et al. (2004). A prospective study of HDL-C and cholesteryl ester transfer protein gene mutations and the risk of coronary heart disease in the elderly. J Lipid Res, 45, 948e953. Dallinga-Thie, G. M., Dullaart, R. P. F., & van Tol, A. (2007). Concerted actions of cholesteryl ester transfer protein and phospholipid transfer protein in type 2 diabetes: effects of apolipoproteins. Curr Opin Lipidol, 18, 251e257. Davidson, W. S., Silva, R. A. G. D., Chantepie, S., Lago, W. R., Chapman, M. J., & Kontush, A. (2009). Proteomic analysis of defined HDL subpopulations reveals particle-specific protein clusters. Arterioscler Thromb Vasc Biol, 29, 870e876. Drayna, D., Jarnagin, A. S., McLean, J., et al. (1987). Cloning and sequencing of human cholesteryl ester transfer protein cDNA. Nature, 327, 632e634. Dullaart, R. P. F., de Vries, R., Scheek, L., et al. (2004). Type 2 diabetes mellitus is associated with differential effects on plasma cholesteryl ester transfer protein and phospholipid transfer protein activities and concentrations. Scand J Clin Lab Invest, 64, 205e216. Fournier, N., Paul, J. L., Atger, V., et al. (1997). HDL phospholipid content and composition as a major factor determining cholesterol efflux capacity from Fu5AH cells to human serum. Arterioscler Thromb Vasc Biol, 17, 2685e2691. Gautier, T., Klein, A., Deckert, V., et al. (2008). Effect of plasma phospholipid transfer protein deficiency on lethal endotoxemia in mice. J Biol Chem, 283, 18702e18710. Gofman, J. W., Young, W., & Tandy, R. (1966). Ischemic heart disease, atherosclerosis, and longevity. Circulation, 34, 679e697. Gotoda, T., Kinoshita, M., Ishibashi, S., et al. (1997). Skipping of exon 14 and possible instability of both the mRNA and the resultant truncated protein underlie a common cholesteryl ester transfer protein deficicency in Japan. Arterioscler Thromb Vasc Biol., 17, 1376e1381. Guerin, M., Bruckert, E., Dolphin, P. J., Turpin, G., & Chapman, M. J. (1996). Fenofibrate reduces plasma cholesteryl ester transfer from HDL to VLDL and normalizes the atherogenic, dense LDL profile in combined hyperlipidemia. Arterioscler Thromb Vasc Biol., 16, 763e772. Ha, Y. C., & Barter, P. J. (1982). Differences in plasma cholesteryl ester transfer activity in sixteen vertebrate species. Comp Biochem Physiol, 71B, 265e269. Haraki, T., Inazu, A., Yagi, K., Kajinami, K., Koizumi, J., & Mabuchi, H. (1997). Clinical characteristics of double heterozygotes with familial hypercholesterolemia and cholesteryl ester transfer protein deficiency. Atherosclerosis, 132, 229e236. Hayashi, H., Campenot, R. B., Vance, D. E., & Vance, J. E. (2007). Apolipoprotein E-containing lipoproteins protect neurons from apoptosis via a signaling pathway involving low-density lipoprotein receptor-related protein-1. J Neurosci, 27, 1933e1941.

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Hernandez, M., Wright, S. D., & Cai, T. Q. (2007). Critical role of cholesterol ester transfer protein in nicotinic acid-mediated HDL elevation in mice. Biochem Biophys Res Commun, 355, 1075e1080. Hudgins, L. C., Parker, T. S., Levine, D. M., et al. (2003). A single intravenous dose of endotoxin rapidly alters serum lipoproteins and lipid transfer proteins in normal volunteers. J Lipid Res, 44, 1489e1498. Hussain, M. M., Innerarity, T. L., Brecht, W. J., & Mahley, R. W. (1995). Chylomicron metabolism in normal, cholesterol-fed, and Watanabe heritable hyperlipidemic rabbits. J Biol Chem, 270, 8578e8587. Ikewaki, K., Nishiwaki, M., Sakamoto, T., et al. (1995). Increased catabolic rate of low density lipoproteins in humans with cholesteryl ester transfer protein deficiency. J Clin Invest, 96, 1573e1581. Ikewaki, K., Rader, D. J., Sakamoto, T., et al. (1993). Delayed catabolism of high density lipoprotein apolipoprotein A-I and A-II in human cholesteryl ester transfer protein deficiency. J Clin Invest, 92, 1650e1658. Inazu, A., Brown, M. L., Hesler, C. B., et al. (1990). Increased high-density lipoprotein levels caused by a common cholesteryl-ester transfer protein gene mutation. N Engl J Med, 323(18), 1234e1238. Inazu, A., Jiang, X. C., Haraki, T., et al. (1994). Genetic cholesteryl ester transfer protein deficiency caused by two prevalent mutations as a major determinant of increased levels of high density lipoprotein cholesterol. J Clin Invest, 94(5), 1872e1882. Inazu, A., Koizumi, J., Kajinami, K., Kiyohara, T., Chichibu, K., & Mabuchi, H. (1999). Opposite effects on serum cholesteryl ester transfer protein levels between long-term treatments with pravastatin and probucol in patients with primary hypercholesterolemia and xanthoma. Atherosclerosis, 145(2), 405e413. Inazu, A., Nakajima, K., Nakano, T., et al. (2008). Decreased post-prandial triglyceride response and diminished remnant lipoprotein formation in cholesteryl ester transfer protein (CETP) deficiency. Atherosclerosis, 196, 953e957. Inazu, A., Quinet, E. M., Wang, S., et al. (1992). Alternative splicing of the mRNA encoding the human cholesteryl ester transfer protein. Biochemistry, 31(8), 2352e2358. Jansen, H., Verhoeven, A. J. M., & Sijbrands, E. J. G. (2002). Hepatic lipase: a pro- or antiatherogenic protein? J Lipid Res, 43, 1352e1362. Jeon, S.-M., Park, Y. B., Kwon, O. S., et al. (2005). Vitamin E supplementation alters HDLcholesterol and paranoxase activity in rabbits fed high cholesterol diet: comparison with probucol. J. Biochem Molecular Taxicology, 19, 336e346. Jeppesen, J., Hein, H. O., Suadicani, P., & Gyntelberg, F. (1998). Triglyceride concentration and ischemic heart disease. An eight-year follow-up in the Copenhagen Male Study. Circulation, 97, 1029e1036. Kastelein, J. J. P., van Leuven, S. I., Burgess, L., et al., for the RADIANCE 1 Investigators. (2007). Effects of torcetrapib on carotid atherosclerosis in familial hypercholesterolemia. N Engl J Med, 356, 1620e1630. Kee, P., Caiazza, D., Rye, K. A., Barrett, P. H. R., Morehouse, L. A., & Barter, P. J. (2006). Effects of inhibiting cholesteryl ester transfer protein on the kinetics of high-density lipoprotein cholesteryl ester transport in plasma. In vivo studies in rabbits. Arterioscler Thromb Vasc Biol, 26, 884e890. Keys, A. (1983). Lipoprotein profile e its value in prediction. Prev Med, 12, 25e31. Kiyohara, T., Kiriyama, R., Zamma, S., Inazu, A., Koizumi, J., Mabuchi, H., & Chichibu, K. (1998). Enzyme immunoassay for cholesteryl ester transfer protein in human serum. Clin Chim Acta, 271(2), 109e118.

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Koizumi, J., Inazu, A., Yagi, K., et al. (1991). Serum lipoprotein lipid concentration and composition in homozygous and heterozygous patients with cholesteryl ester transfer protein deficiency. Atherosclerosis, 90(2-3), 189e196. Koizumi, J., Mabuchi, H., Yoshimura, A., et al. (1985). Deficiency of serum cholesteryl-ester transfer activity in patients with familial hyperalphalipoproteinaemia. Atherosclerosis, 58, 175e186. Kontush, A., de Faria, E. C., Chantepie, S., & Chapman, M. J. (2004). Antioxidative activity of HDL particle subspecies is impaired in hyperalphalipoproeinemia: Relevance of enzymatic and physicochemical properties. Arterioscler Thromb Vasc Biol, 24, 526e533. Kontush, A., Therond, P., Zerrad, A., et al. (2007). Preferential sphingosine-1-phosphate enrichment and sphingomyelin depletion are key features of small dense HDL3 particles. Arterioscler Thromb Vasc Biol, 27, 1843e1849. Koropatnick, T. A., Kimbell, J., Chen, R., et al. (2008). A prospective study of high-density lipoprotein cholesterol, cholesteryl ester transfer protein gene variants, and healthy aging in very old Japanese-American men. J Gerontol, 63A, 1235e1240. Krimbou, L., Marcil, M., Chiba, H., & Genest, J., Jr. (2003). Structural and functional properties of human plasma high density-sized lipoprotein containing only apoE particles. J Lipid Res, 44, 884e892. Kurth, T., Everett, B. M., Buring, J. E., Kase, C. S., Ridker, P. M., & Gaziano, J. M. (2007). Lipid levels and the risk of ischemic stroke in women. Neurology, 68, 556e562. Lamarche, B., Uffelman, K. D., Carpentier, A., et al. (1999). Triglyceride enrichment of HDL enhances in vivo metabolic clearance of HDL apoA-I in healthy men. J Clin Invest, 103, 1191e1199. Lee, J. Y., Badeau, R. M., Mulya, A., et al. (2007). Functional LCAT deficiency in human apolipoprotein A-I transgenic, SR-BI knockout mice. J Lipid Res, 48, 1052e1061. Leppala, J. M., Paunio, M., Virtamo, J., et al. (1999). Alcohol consumption and stroke incidence in male smokers. Circulation, 100, 1209e1214. Levels, J. H. M., Pajkrt, D., Schultz, M., et al. (2007). Alterations in lipoprotein homeostasis during human experimental endotoxemia and clinical sepsis. Biochim Biophys Acta, 1771, 1429e1438. Li, L., Thompson, P. A., & Kitchens, R. L. (2008). Infection induces a positive acute phase apolipoprotein E response from a negative acute phase gene: role of hepatic LDL receptors. J Lipid Res, 49, 1782e1793. Lu, H., Inazu, A., Moriyama, Y., et al. (2003). Haplotype analyses of cholesteryl ester transfer protein gene promoter: a clue to an unsolved mystery of TaqIB polymorphism. J Mol Med, 81 (4), 246e255. Luo, Y., & Tall, A. R. (2000). Sterol upregulation of human CETP expression in vitro and in transgenic mice by an LXR element. J Clin Invest, 105, 513e520. MacLean, P. S., Bower, J. F., Vadlamudi, S., et al. (2003). Cholesteryl ester transfer protein expression prevents diet-induced atherosclerosis lesions in male db/db mice. Arterioscler Thromb Vasc Biol, 23, 1412e1415. MacLean, P. S., Vadlamudi, S., MacDonald, K. G., Pories, W. J., & Barakat, H. A. (2005). Suppression of hepatic cholesteryl ester transfer protein expression in obese humans with the development of type 2 diabetes mellitus. J Clin Endocrinol Metab, 90, 2250e2258. Mahley, R. W., Innerarity, T. L., Weisgraber, K. H., & Fry, D. L. (1977). Canine hyperlipoproteinemia and atherosclerosis. Am J Pathol, 87, 205e226. Manabe, M., Abe, T., Nozawa, M., Maki, A., Hirata, M., & Itakura, H. (1987). New substrate for determination of serum lecithin:cholesterol acyltransferase. J Lipid Res, 28, 1206e1215. Masson, D., Staels, B., Gautier, T., et al. (2004). Cholesteryl ester transfer protein modulates the effect of liver X receptor agonists on cholesterol transport and excretion in the mouse. J Lipid Res, 45, 543e550.

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Millar, J. S., Brousseau, M. E., Diffenderfer, M. R., et al. (2006). Effects of cholesteryl ester transfer protein inhibitor torcetrapib on apolipoprotein B100 metabolism in humans. Arterioscler Thromb Vasc Biol, 26, 1350e1356. Miwa, K., Inazu, A., Kawashiri, M., et al. (2009). Cholesterol efflux from J774 macrophages and Fu5AH hepatoma cells to serum is preserved in CETP-deficient patients. Clin Chim Acta, 402, 19e24. Miyazaki, O., Fukamachi, I., Mori, A., et al. (2009). Formation of prebeta1-HDL during lipolysis of triglyceride-rich lipoprotein. Biochem Biophys Res Commun, 379, 55e59. Moriyama, Y., Okamura, T., Inazu, A., et al. (1998). A low prevalence of coronary heart disease in subjects with increased high-density lipoprotein cholesterol levels including those with plasma cholesteryl ester transfer protein deficiency. Prev Med, 27(5Pt1), 659e667. Musliner, T. A., Long, M. D., Forte, T. M., & Krauss, R. M. (1991). Size transformations of intermediate and low density lipoproteins induced by unesterified fatty acids. J Lipid Res, 32, 903e915. Nagano, M., Nakamura, M., Kobayashi, N., Kamata, J., & Hiramori, K. (2005). Effort angina in a middle-aged woman with abnormally high levels of serum high-density lipoprotein cholesterol. Circ J, 69, 609e612. Nagano, M., Yamashita, S., Hirano, K. I., et al. (2004). Molecular mechanisms of cholesteryl ester transfer protein deficiency in Japanese. J Atheroscler Thromb, 11, 110e121. Nagasaki, T., & Akanuma, Y. (1977). A new colorimetric method for the determination of plasma lecithin-cholesterol acyltransferase activity. Clin Chim Acta, 75, 371e375. Nissen, S. E., Tardif, J. C., Nicholls, S. J., et al., for the ILLUSTRATE Investigators. (2007). Effects of torcetrapib on the progression of coronary atherosclerosis. N Engl J Med, 356, 1304e1316. Okamura-Noji, K., Sasai, K., Zhan, R., et al. (2001). Cholesteryl ester transfer protein deficiency causes slow egg embryonation of Schistosoma japonicum. Biochim Biophys Res Commun, 286, 305e310. Oliveira, H. C. F., Ma, L., Milne, R., et al. (1997). Cholesteryl ester transfer protein (CETP) activity enhances plasma cholesteryl ester formation: studies in CETP transgenic mice and human genetic CETP deficiency. Arterioscler Thromb Vasc Biol, 17(6), 1045e1052. Ordovas, J. M., Cupples, L. A., Corella, D., et al. (2000). Association of cholesteryl ester transfer protein-TaqIB polymorphism with variations in lipoprotein subclasses and coronary heart disease risk: the Framingham study. Arterioscler Thromb Vasc Biol., 20(5), 1323e1329. Ouguerram, K., Nguyen, P., Krempf, M., et al. (2004). Selective uptake of high density lipoproteins cholesteryl ester in the dog, a species lacking in cholesteryl ester transfer protein activity. An in vivo approach using stable isotopes. Comparative Biochem Physiol, Part B, 138, 339e345. Pattnaik, N. M., Montes, A., Hughes, L. B., & Zilversmit, D. B. (1978). Cholesteryl ester exchange protein in human plasma isolation and characterization. Biochim Biophys Acta, 530, 428e438. Pruneta, V., Pulcini, T., Lalanne, F., et al. (1999). VLDL-bound lipoprotein lipase facilitates the cholesteryl ester transfer protein-mediated transfer of cholesteryl esters from HDL to VLDL. J Lipid Res, 40, 2333e2339. Qiu, X., Mistry, A., Ammirati, M. J., et al. (2007). Crystal structure of cholesteryl ester transfer protein reveals a long tunnel and four bound lipid molecules. Nature Struct Molec Biol, 14(2), 106e113. Regieli, J. J., Jukema, J. W., Grobbee, D. E., et al. (2008). CETP genotype predicts increased mortality in statin-treated men with proven cardiovascular disease: an adverse pharmacogenetic interaction. Eur Heart J, 29, 2792e2799.

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Rhyne, J., Ryan, M. J., White, C., Chimonas, T., & Miller, M. (2006). The two novel CETP mutations Gln87X and Gln165X in a compound heterozygous state are associated with marked hyperalphalipoproteinemia and absence of significant coronary artery disease. J Mol Med, 84(8), 647e650. Ritsch, A., Scharnagl, H., Eller, P., et al. (2010). Cholesteryl ester transfer protein and mortality in patients undergoing coronary angiography. The Ludwigshafen Risk and Cardiovascular Health Study. Circulation, 121, 366e374. Robins, S. J., & Fasulo, J. M. (1997). High density lipoproteins, but not other lipoproteins, provide a vehicle for sterol transport to bile. J Clin Invest, 99, 380e384. Sakai, N., Matsuzawa, Y., Hirano, K., et al. (1991). Detection of two species of low density lipoprotein particles in cholesteryl ester transfer protein deficiency. Arterioscler Thromb, 11, 71e79. Sandhofer, A., Kaser, S., Ritsch, A., et al. (2006). Cholesteryl ester transfer protein in metabolic syndrome. Obesity, 14, 812e818. Scanu, A. M., & Edelstein, C. (2008). HDL: bridging past and present with a look at the future. FASEB J, 22, 4044e4054. Schaefer, E. J., Lamon-Fava, S., Ordovas, J. M., et al. (1994). Factors associated with low and elevated plasma high density lipoprotein cholesterol and apolipoprotein A-I levels in the Framingham Offspring Study. J Lipid Res, 35, 871e882. Schmitz, G., & Assmann, G. (1982). Isolation of human serum HDL1 by zonal ultracentrifugation. J Lipid Res, 23, 903e910. Schwartz, C. C., VandenBroek, J. M., & Cooper, P. S. (2004). Lipoprotein cholesteryl ester production, transfer, and output in vivo in humans. J Lipid Res, 45, 1594e1607. Scobey, M. W., Johnson, F. L., & Rudel, L. L. (1989). Delivery of high-density lipoprotein free and esterified cholesterol to bile by the perfused monkey liver. Am J Physiol, 257(4 Pt 1), G644e52. Shetty, S., Eckhardt, E. R. M., Post, S. R., & van der Westhuyzen, D. R. (2006). Phosphatidylinositol-3-kinase regulates scavenger receptor class B type I subcellular localization and selective lipid uptake in hepatocytes. Arterioscler Thromb Vasc Biol, 26, 2125e2131. Stevenson, S., Wang, S., Deng, L., & Tall, A. R. (1993). Human plasma cholesteryl ester transfer protein consists of a mixture of two forms reflecting variable glycosylation at Asparagine341. Biochemistry, 32, 5121e5126. Sviridov, D., Chin-Dusting, J., Nestel, P., et al. (2006). Elevated HDL cholesterol is functionally ineffective in cardiac transplant recipients: Evidence for impaired reverse cholesterol transport. Transplantation, 81, 361e366. Takata, M., Inazu, A., Katsuda, S., et al. (2006). CETP (cholesteryl ester transfer protein) promoter -1337 C>T polymorphism protects against coronary atherosclerosis in Japanese patients with heterozygous familial hypercholesterolaemia. Clin Sci (Lond), 111(5), 325e331. Tanigawa, H., Billheimer, J. T., Tohyama, J., Zhang, Y., Rothblat, G., & Rader, D. J. (2007). Expression of cholesteryl ester transfer protein in mice promotes macrophage reverse cholesterol transport. Circulation, 116(11), 1267e1273. Tchoua, U., D’Souza, W., Mukhamedova, N., et al. (2008). The effect of cholesteryl ester transfer protein overexpression and inhibition on reverse cholesterol transport. Cardiovasc Res, 77, 732e739. Teh, E. M., Dolphin, P. J., Breckenridge, W. C., & Tan, M. H. (1998). Human plasma CETP deficiency: identification of a novel mutation in exon 9 of the CETP gene in a Caucasian subject from North America. J Lipid Res, 39, 442e456.

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Thompson, A., Angelantonio, E. D., Sarwar, N., et al. (2008). Association of cholesteryl ester transfer protein genotypes with CETP mass and activity, lipid levels, and coronary risk. J Am Med Assoc, 299, 2777e2788. Thompson, J. F., Reynolds, J. M., Williams, S. P., Wood, L. S., Paciga, S. A., & Lloyd, D. B. (2009). Frequency and function of CETP variants among individuals of Asian ancestry. Atherosclerosis, 202, 241e247. Trigatti, B., Rayburn, H., Vinals, M., et al. (1999). Influence of the high density lipoprotein receptor SR-BI on reproductive and cardiovascular pathophysiology. Proc Natl Acad Sci USA, 96, 9322e9327. Vaisar, T., Pennathur, S., Green, P. S., et al. (2007). Shotgun proteomics implicates protease inhibition and complement activation in the anti-inflammatory properties of HDL. J Clin Invest, 117, 746e756. Van der Hoorn, J. W. A., de Haan, W., Berbee, J. F. P., et al. (2008). Niacin increases HDL by reducing hepatic expression and plasma levels of cholesteryl ester transfer protein in APOE*3Leiden.CETP mice. Arterioscler Thromb Vasc Biol, 28, 2016e2022. van der Steeg, W. A., Holme, I., Boekholdt, S. M., et al. (2008). High-density lipoprotein cholesterol, high-density lipoprotein particle size, and apolipoprotein A-I: Significance for cardiovascular risk. J Am Med Assoc, 51, 634e642. Van Eck, M., Hoekstra, M., Hildebrand, R. B., et al. (2007). Increased oxidative stress in scavenger receptor BI knockout mice with dysfunctional HDL. Arterioscler Thromb Vasc Biol, 27, 2413e2419. Vasan, R. S., Pencina., M. J., Robins, S. J., et al. (2009). Association of circulating cholesteryl ester transfer protein activity with incidence of cardiovascular disease in the community. Circulation, 120, 2414e2420. Vreugdenhil, A. C. E., Snoek, A. M. P., van’t Veer, C., Greve, J. W. M., & Buurman, W. A. (2001). LPS-binding protein circulates in association with apoB-containing lipoproteins and enhances endotoxin-LDL/VLDL interaction. J Clin Invest, 107, 225e234. Wang, S., Wang, X., Deng, L., Rassart, E., Milne, R. W., & Tall, A. R. (1993). Point mutagenesis of carboxyl-terminal amino acids of cholesteryl ester transfer protein. J Biol Chem, 268, 1955e1959. Wang, X., Driscoll, D. M., & Morton, R. E. (1999). Molecular cloning and expression of lipid transfer inhibitor protein reveals its identity with apolipoprotein F. J Biol Chem, 274, 1814e1820. Watts, G. F., Ji, J., Chan, D. C., et al. (2006). Relationships between changes in plasma lipid transfer proteins and apolipoprotein B-100 kinetics during fenofibrate treatment in the metabolic syndrome. Clin Sci, 111, 193e199. Williams, P. T. (1996). High-density lipoprotein cholesterol and other risk factors for coronary heart disease in female runners. N Engl J Med, 334, 1298e1303. Wilson, H. M., Patel, J. C., Russell, D., & Skinner, E. R. (1993). Alterations in the concentration of an apolipoprotein E-containing subfraction of plasma high density lipoprotein in coronary heart disease. Clin Chim Acta, 220, 175e187. Ye, D., Kraaijeveld, A. O., Grauss, R. W., et al. (2008). Reduced leucocyte cholesteryl ester transfer protein expression in acute coronary syndromes. J Intern Med, 246, 571e585. Yvan-Charvet, L., Matsuura, F., Wang, N., et al. (2007). Inhibition of cholesteryl ester transfer protein by torcetrapib modestly increases macrophage cholesterol efflux to HDL. Arterioscler Thromb Vasc Biol, 27, 1132e1138. Yvan-Charvet, L., Pagler, T. A., Wang, N., et al. (2008). SR-BI inhibits ABCG1-stimulated net cholesterol efflux from cells to plasma HDL. J Lipid Res, 49, 107e114.

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Zhang, A., Gao, S., Fan, J., Huang, W., Zhao, T., & Liu, G. (2004). Increased plasma HDL cholesterol levels and biliary cholesterol excretion in hamsters by LCAT overexpression. FEBS Letters, 570, 25e29. Zhang, X., Moor, A., Merkler, K., Liu, Q., & McLean, M. (2007). Regulation of alternative splicing of liver scavenger receptor class B gene by estrogen and the involved regulatory splicing factors. Endcrinology, 148, 5295e5304.

Chapter 4

HDL and Reverse Cholesterol Transport: Physiological Modulation Giovanna Catalano and Maryse Guerin INSERM UMRS939, Hoˆpital de la Pitie´, Paris, France; UPMC Universite´ Pierre et Marie Curie, Hoˆpital de la Pitie´, Paris, France

INFLUENCE OF AGING ON THE REVERSE CHOLESTEROL TRANSPORT PATHWAY The incidence of cardiovascular disease (CVD) due to atherosclerosis increases with aging (Grundy, 1995). Aging is characterized by the occurrence of several physical and biochemical modifications that may affect high density lipoprotein (HDL) structure and functions (Walter, 2009). Analysis of the effects of aging on the reverse cholesterol transport (RCT) process revealed that HDL3 from elderly subjects displayed a reduced capacity to mediate free cholesterol efflux via the ATP-binding cassette A1 (ABCA-1) pathway from THP-1 cells as compared to HDL3 from younger subjects, as a result of decreased HDL bilayer fluidity, increased apolipoprotein A (apoA-I) oxidation, decreased phosphatidylcholine to sphingomyeline ratio (Berrougui, 2007). In elderly subjects, endogenous vitamin E levels in native HDL particles were reduced as compared with those in HDL isolated from young and middle aged subjects (Khalil, 1998). This reduced vitamin E content in HDL could be responsible for an increased susceptibility of these particles to oxidation. HDL antioxidant activity decreases with aging as a consequence of decreased paraoxonase 1 protection effect towards low density lipopreotein (LDL) peroxidation (Seres et al., 2004; Jaouad et al., 2006).

INFLUENCE OF HORMONES ON THE REVERSE CHOLESTEROL TRANSPORT PATHWAY That atherosclerosis development and reverse cholesterol transport (RCT) are related to gender is evident by the distinct incidence of coronary death between men and women. The epidemiology of coronary heart disease demonstrates The HDL Handbook. ISBN: 978-0-12-382171-3 Copyright Ó 2010 Elsevier Inc. All rights of reproduction in any form reserved.

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a striking sex difference in its age-specific incidence and severity even independently of environmental risk factors. If atherogenesis is one of the consequences of altered RCT, then sex difference in RCT could explain the different predisposition of men and women to develop cardiovascular disease. The net effect of hormones on protection from atherosclerosis is difficult to assess since these effects depend on whether associations with endogenous or exogenous hormones have been investigated. Much of the evidence for sex hormone effects on lipid metabolism comes from studies of changes associated with administration of synthetic gonadal steroids or with changes in gonadal function. Studies on differences in lipoprotein metabolism in normal men and women are extremely limited.

Estrogens-mediated effect Numerous epidemiological studies suggest that estrogens protect women against cardiovascular disease before the age of menopause. After menopause, CVD risk for women becomes progressively closer to that of men, suggesting an atheroprotective role for estrogens (Arnal et al., 2004). There are three main naturally occurring estrogens: 17b estradiol (E2), estrone and estriol. E2 is mainly produced by the ovary whereas estrone and estriol are produced in the liver from estradiol (Gruber et al., 2002). Estriol is also produced by the placenta during pregnancy. Women usually display higher HDL cholesterol (HDL-C) levels than men (Schaefer et al., 1982). Prepubertal boys and girls do not differ significantly in their serum lipid levels and lipoprotein profile. In contrast with girls, in whom HDL-C levels do not change with puberty despite the rise in estrogen concentrations, sexually maturing boys display a decrease in HDL-C and an increase in both LDL-C and triglyceride (TG) levels (Godsland et al., 1987) which occur concomitantly with an elevation in testosterone levels. Evidence of how sex hormones influence lipid metabolism is given by studies in pregnant women throughout the gestational period (Sattar et al., 1997). Estradiol elevation during pregnancy favors an increase in VLDL particles, especially the VLDL2 subpopulation, a decreased hepatic lipase (HL) activity and variations in plasma cholesteryl ester transfer protein (CETP) activity (Iglesias et al., 1994). During pregnancy LDL-C, HDL-C and TG rise progressively. LDL-C remains elevated until after delivery, but TGs fall to baseline at this time (LaRosa, 1992). CETP activity has been shown to increase significantly during the second trimester of pregnancy before declining just before delivery (Silliman et al., 1993). In vivo studies conducted in intact female rats demonstrated that physiological doses of E2 increased apoA-I and HDL-C levels (Parini et al., 2000). An increase in plasma HDL-C and apoA-I levels in women as compared to men has been also described (Patsch et al., 1980). Equally, in vitro studies demonstrated that secretion of apoA-I-containing particles from HepG2 cells was stimulated by estradiol as a result of increased transcription rate of apoA-I mRNA

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(Jin et al., 1998). These observations are consistent with in vivo studies demonstrating that increased HDL levels in postmenopausal women under estrogen therapy result primarily from an increase of HDL production rate rather than from a decrease of HDL catabolism (Schaefer et al., 1982; Kauma et al., 1996). Concerning CETP activity, a clear sex difference was observed in the genotype effect on HDL-C level. HDL-C levels were strongly associated with TaqIB polymorphism (Kauma et al., 1996) in women and weakly in postmenopausal women without hormone replacement therapy. Attenuation of the gene dosage effect after menopause suggests that the gender difference in HDL-C may be due in part to sex hormones. Furthermore, a study conducted in hCETP transgenic rats suggests that sex steroids potentiate the regulation of CETP expression even at low plasma levels of estrogen (Vadlamudi et al., 1998). Estrogen therapy in atherosclerotic mice has consistently demonstrated a dose dependent inhibitory effect of E2 on lesion initiation and progression in ovariectomized females. When these mice were treated with E2 in a physiological range, atheroma plaques rarely progressed beyond uncomplicated fatty streaks. In the apoE
/
mouse, castration is followed by an increase of lesion area and E2 administration prevents fatty streak deposition but only at concentrations similar to those in the gestational period. However, owing to high doses of estrogens used in animal studies, it is unclear whether estrogen effects are physiologically relevant. The different predisposition between men and premenopausal women in developing atherosclerosis, suggests hormonal replacement therapy as a tool for prevention in postmenopausal women. Treatment of rats with pharmacological doses of E2 increased hepatic LDL-R protein and mRNA (Parini et al., 1997). In fasting women, LDL-R expression was positively correlated with plasma estrone concentration (Nanjee et al., 1990). In rat liver, SR-BI expression is suppressed by estrogen administration as well as in cultured human hepatic cells (Landschulz et al., 1996; Fluiter et al., 1998; Graf et al., 2001). Recently, it has been demonstrated that estrogen regulation of SR-BI expression is due to differential modulation of SR-BI RNA splicing and glycosylation, a process that is important in folding and export of SR-BI to the membrane (Zhang et al., 2007). By contrast with results obtained in hepatic cells, estrogen treatment increases SR-BI expression and selective lipid uptake in adrenal and ovarian luteal cells as well as in macrophages (Fluiter et al., 1998). SR-BII is an alternatively spliced product of the SR-BI gene that differs only in the C-terminal cytoplasmic domain. Concomitant with SR-BI downregulation, estrogen induces an increase in SR-BII expression. Both non-ovariectomized female rats and ovariectomized estrogen-treated female rats present an increased expression of SR-BII with a nearly irrelevant expression of SR-BI in the liver, while ovariectomized female and male rats showed an opposite profile of expression (Graf et al., 2001). Hepatic lipase (HL) is also subject to hormonal regulation. Its activity is decreased after the peak of estrogen in the reproductive cycle (Tikkanen et al., 1986) and women have lower HL than men (Bersot et al., 1999)

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which may account for the higher HDL-C levels in women. Estrogen-mediated inhibition of HL has been recently demonstrated to occur via repression of the HL gene promoter (Jones et al., 2002).

Androgens-mediated effect Testosteron (T) can exert its metabolic effects either directly or through its metabolites E2 and dihydro-testosterone. The effect of T and E2, in particular, can be either additive or counter regulatory. T is clearly associated with a lowering effect on HDL-C. Zitzmann et al. (2001) found significant correlation between HDL-C levels and the number of CAG repeat polymorphisms in the androgen receptor gene, which determines the sensitivity of target cells towards testosterone. T upregulates SR-BI and HL expression in macrophages, even if upregulation of HL in HepG2 cells was observed only with supraphysiological concentration of T. Macrophages with increased testosterone-induced SR-BI expression, showed enhanced cholesterol efflux capacity that could reflect facilitated RCT in vivo (Langer et al., 2002). Castration or T suppression in men with prostate cancer was found to increase HDL-C by about 20%. The effect of T on HDL metabolism is marked on the large HDL subclass but the mechanism by which T regulates HDL metabolism is not well understood.

Thyroid-mediated effect Thyroid hormones affect serum cholesterol by altering lipid metabolism. Hypothyroidism frequently causes hyperlipidemia with increased TC, TG, LDL-C and apoB levels. On the contrary, hyperthyroidism is responsible for low levels of TC and LDL-C and increased HDL-C levels. Changes in LDL-C are mainly due to altered clearance of LDL as a result of the presence of a thyroid hormone responsive element in the LDL-r gene promoter. The effects on HDL-C level are mainly due to modification of CETP and HL activities, that decrease with hypothyroidism and increase with hyperthyroidism (Kuusi et al., 1988). In hyperthyroid states, HDL decrease is mainly observed in the HDL2b subfraction (Muls et al., 1982). Changes in apoA-I in hyper- and hypothyroidism are mainly caused by changes in the concentration of LpAI rather than LpAI:AII particles since apoA-II levels remain unaltered. HL appears to be the main factor influencing HDL concentrations in these patients but it is not clear if modulation of HDL levels results from variations in HDL synthesis or catabolism. ApoA-I mRNA synthesis is reduced in hypothyroidism and apoA-I transcription is stimulated by thyroid hormone in the rat (Staels et al., 1990).

Insulin-mediated effect It is clear that a strong link exists between perturbations in insulin signal transduction and alterations in lipid and lipoprotein metabolism. Insulin and

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glucose are known to directly regulate lipid synthesis and secretion (Kok et al., 1996). Insulin controls, at the transcriptional level, hepatic sterol regulatory element binding protein (SREBP) expression which is a key transcription factor responsible for regulating fatty acid and cholesterol biosynthesis. SREBP can activate a cascade of enzymes involved in cholesterol biosynthetic pathways, such as HMG-CoA reductase and fatty acid synthase (Bennett et al., 1995; Brown and Goldstein, 1997). SREBP binds to sterol response elements (SRE) in the promoter/enhancer regions of target genes. Expression of SREBP is enhanced by insulin in liver, adipose tissue and skeletal muscle. Under normal conditions, insulin stimulates cellular uptake of glucose, as well as free fatty acids (FFA) esterification and storage. Equally, insulin suppresses the hormone sensitive lipase, the major regulator of FFA release from adipose tissue. At the same time, insulin and glucose are believed to stimulate LPL activity from adipose tissue, thus regulating the first step of FFA delivery to this tissue for storage and TG plasma removal (Goldberg and Merkel, 2001). In the insulin resistant state, there is an increased release of FFA from adipose tissue concomitant with a decrease in TG-derived FFA uptake by muscle tissue. Since only visceral fat is linked to the liver through the portal vein, this may result in an increased influx of FFA into the liver (Arner, 2002). This may result in a vicious cycle which results in typical dyslipidemia of insulin resistance characterized by increased TG level, decreased HDL and compositional changes in LDL. The elevated FFA and glucose levels regulate the VLDL output from the liver and elevated TG in the liver inhibit apoB degradation and result in an increased assembly and secretion of VLDL particles. Furthermore, LPL levels and VLDL clearance are decreased (Howard, 1999; Avramoglu et al., 2006). Possible mechanisms of decreased HDL in insulin resistance include impaired VLDL lipolysis, which impairs HDL maturation by decreased transfer of TGRL remnants, increased activity of HL, alterations in hepatic function, which inhibit production of apoA-I and secretion of nascent HDL, enhanced CETP activity as a result of increased CE acceptors, i.e., VLDL (Howard, 1999).

INFLUENCE OF PHYSICAL ACTIVITY ON THE REVERSE CHOLESTEROL TRANSPORT PATHWAY Physical activity favorably affects a number of cardiovascular disease risk factors such as obesity, insulin resistance and blood pressure and it also has a beneficial effect on lipoprotein profile. The major effect of physical exercise is on HDL particle number and HDL cholesterol content. The exercise-induced changes in HDL-C level are the result of the interaction among exercise intensity, frequency, duration and length of the exercise period and could also be gender specific. Athletes had increased HDL-C and apoA-I concentrations, increased LCAT activity and preb-HDL levels as compared to sedentary controls (Olchawa et al., 2004). Furthermore, cholesterol efflux from

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macrophages to total plasma was increased in athletes as a result of increased HDL particle number. Fitness increases HDL-C but this increase is not correlated to very high level physical activity, thus indicating an upper threshold value of the HDL-C increase with physical activity. HDL-C was increased, mainly in the HDL2 subfraction, in a group of soccer players as compared to sedentary controls (Brites et al., 2004). However, no relevant differences were observed for all the other HDL-related parameters, thus suggesting that increased HDL-C was due to enrichment of the particles in cholesterol and not to increased particle number. Increase in HDL level could be due to either increased synthesis or catabolism. It is not clear whether increased HDL in fit subjects is due to one or the other reason. Interestingly, ABCA1 expression in humans is associated with physical activity. Mechanisms underlying the effect of exercise on HDL are multiple. Free cholesterol and PL are transferred to HDL during the catabolism of TGRL by LPL. Furthermore, muscles are TG depleted during exercise and it is possible that, in this situation, cholesterol is mobilized by muscle cells to its primary acceptor, HDL. As demonstrated by Sviridov et al. (2003), preb-HDL could be formed extrahepatically at the level of the muscle after exercise, thus contributing to the RCT process.

INFLUENCE OF DIET ON THE REVERSE CHOLESTEROL TRANSPORT PATHWAY The protective role of the consumption of monounsaturated fatty acids (MUFA) or polyunsaturated fatty acids (PUFA) against the development of atherosclerosis has been known for many years (Psota et al., 2006). Indeed, epidemiologic studies show that the Mediterranean diet, in which olive oil (enriched in oleic acid) predominates, is associated with a reduction in cardiovascular pathology. n-6 PUFA (linoleic and arachidonic acids) decrease plasma LDL-cholesterol concentrations while saturated fatty acids increase it. Moreover, n-3 PUFA (alpha-linolenic acid), reduce triglyceridemia and have anti-thrombotic and anti-atherogenic properties. Recently, a study conducted in normolipidemic subjects without cardiovascular disease showed that consumption of PUFA improves the anti-inflammatory properties of HDL particles while consumption of SFA reduces their anti-inflammatory activity and degrades endothelial function (Nicholls et al., 2006). These studies suggest that the functionality of HDL particles in patients presenting a high cardiovascular risk can be modulated by fatty acid consumption. Dietary fats induce physicochemical changes in HDL particles, which can modulate cellular cholesterol efflux. Contrastingly, when fat intake is limited to the NCEP (National cholesterol education program) (25e30% of total calories intake in the form of fat) and when the proportion of MUFA in the diet is fixed, differential percentages of PUFA, SFA or trans fatty acids (TFA) do not influence the capacity of HDL2 or HDL3 to promote cholesterol efflux capacity (Buonacorso et al., 2007). In monkeys,

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dietary fatty acids modified HDL-PL content without altering cholesterol efflux capacity from hepatoma cells (Gillotte et al., 1998). Consumption of MUFA increases the capacity of whole plasma from normolipidemic subjects to promote cellular free cholesterol efflux via the SR-BI pathway as a result of an enrichment in phospholipids (Sakr et al., 1996), a greatest fluidity, a higher cholesteryl ester content and a smaller HDL3 particle size (Sola et al., 1993). Changes in the FA composition of the diet can have several effects on the FA composition of membrane lipids. The fact that both n-6 and n-3 PUFA classes cannot be synthesized de novo in humans, suggests that the fatty acid composition of membrane lipids is strongly influenced by the relative abundance of these PUFA in the diet (Hulbert et al., 2005). Variations in membrane composition can alter processes through which cholesterol desorbs or absorbs on membranes such as free cholesterol efflux or cholesteryl esters uptake, thus influencing RCT. At the same time, when membranes are in contact with FA, these latter can act as modulators of cholesterol transporters at different levels. ABCA1 and ABCG1 are sterol transporters critical for cholesterol efflux and HDL formation, both at the liver and intestine level. Wang and Oram (2007) observed that incubation of mouse macrophages in an oleate-rich medium resulted in more than 50% reduction in cholesterol efflux to apoA-I even if cells were stimulated with 8-Br cAMP or LXR-RXR ligand to induce ABCA1 expression. The unsaturated fatty acids palmitoleate, oleate, linoleate and arachidonate decreased apoA-I mediated cholesterol and PL efflux and apoA-I binding as a result of an increase in degradation of ABCA1. Preincubation of cells in the presence of unsaturated fatty acids results in a reduction of cholesterol efflux via both the ABCA1 and ABCG1 pathways by a downregulation of ABC transporter gene expression (Wang and Oram, 2002; Wang et al., 2004; Uehara et al., 2002, 2007). In addition, enrichment of cellular membrane with n-3 PUFA enhanced HDL-mediated cholesterol efflux from smooth muscle cell (Dusserre et al., 1995) or from human fibroblast (Pal and Davis, 1990).

INFLUENCE OF ALCOHOL CONSUMPTION ON THE REVERSE CHOLESTEROL TRANSPORT PATHWAY Many lines of evidence support a link between moderate drinking and lower risk of CVD but definitive proof remains elusive. Alcohol consumption shifts HDL particle distribution towards large, lipid-rich HDL2b in the heavy alcohol drinkers (Lagrost et al., 1996; Makela et al., 2008). Several studies also document an increase in paraoxonase-1 (PON-1) activity with alcohol intake. In moderate alcohol users, CETP, PLTP and LCAT mass and activities are unchanged, while in heavy alcohol abusers CETP activity and mass are decreased and PLTP activity increased, thus increasing HDL-PL content (Hannuksela paraoxonase-1, 2004). Moderate alcohol intake with dinner affects the postprandial phase by increasing HDL-C, PL and TG (Hendriks

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et al., 1998; van Tol et al., 1998). In addition, fatty acid composition of PL and CE in HDL is affected by moderate alcohol intake. Alcohol effects on cholesterol efflux were evaluated in both moderate and heavy alcohol consumers. Senault et al. (2000) observed that serum from moderate red wine consumers showed increased capacity of efflux on Fu5AH cells as compared to non-consumers, and that this was correlated to HDL-C, HDL3-C and HDL-PL variations. In alcoholic abusers with or without liver disease, the capacity of HDL particles to mediate cholesterol efflux from macrophages was decreased approximately by 80% and HDLeCE liver uptake was decreased by 60% as compared to non-drinkers. Alcohol is responsible for apoE desialylation that leads to decreased association of apoE with HDL, thus possibly affecting HDL functionality (Rao et al., 2000). By contrast, Makela et al. (2008) recently showed that large PL-rich HDL2 are better acceptors for FC than controls. Furthermore, postprandial serum shows an increased capacity to mediate cellular free cholesterol efflux, independently of the type of alcoholic beverage as compared to fasting serum (van der Gaag et al., 2001). In addition to its action on cholesterol efflux, alcohol could increase another step in RCT such as LCAT-induced free cholesterol esterification or CETP-mediated cholesteryl ester transfer between plasma lipoproteins. Indeed, LCAT activity is increased during the postprandial phase and apoA-I levels increase with wine consumption. Recently, it has been suggested that alcohol may inhibit the glycosylation of CETP, which could affect the binding of CETP to lipoproteins in alcohol drinkers (Liinamaa et al., 2006). Given that the association of alcohol with HDL-C and possibly cardiovascular risk may be partly mediated by CETP activity, genetic variation in CETP may modulate these relationships. A lightto-moderate alcohol intake was associated with a lower risk of CHD among B2 carriers of the Taq1B polymorphism (Jensen et al., 2008) which has been reported to be associated with increased HDL cholesterol levels and decreased CETP activity and levels (Hannuksela et al., 1994).

INFLUENCE OF SMOKING ON THE REVERSE CHOLESTEROL TRANSPORT PATHWAY Cigarette smoking is associated with changes in the levels of plasma lipoproteins with a dose response relationship between the number of cigarettes smoked per day and the extent of lipid abnormalities. However, very little has been understood concerning the molecular mechanisms through which cigarette smoke could accelerate atherosclerosis leading to CHD. Smoking could accelerate atherosclerosis by promoting modifications of lipoproteins and thus influencing HDL functionality. Cigarette smokers have reduced concentrations of plasma HDL and LCAT compared to non-smokers and exposure of human plasma to cigarette smoke resulted in cross-linking of HDL, inhibition of LCAT activity, shift in HDL particle size and dissociation of CETP from particles (Bielicki et al., 1995). HDL that were exposed to whole cigarette smoke

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extracts showed a remarkable increase in the level of conjugated dienes as in oxidized HDL, that inversely correlated with cholesterol efflux capacity. Cigarette smoke extracts contain a variety of free radicals and active oxygens that could be involved in FA peroxidation of HDL (Ueyama et al., 1998). Acrolein is a major component of cigarette smoke and one of the aldehydes that could modify HDL structure and charge. It has been demonstrated that this toxic aldehyde is able to modify apoA-I by site specific modification of lys-226 on HDL and to decrease ABCA1 mediated cholesterol efflux (Shao et al., 2005).

INFLUENCE OF POSTPRANDIAL STATE ON THE REVERSE CHOLESTEROL TRANSPORT PATHWAY Postprandial lipoprotein metabolism is characterized by transient accumulation of intestinally-derived chylomicrons (CM) and hepatically-derived VLDL and their remnants. Hydrolysis of the triglyceride core of CM and VLDL by lipoprotein lipase facilitates the delivery of free fatty acids to muscle and adipose tissue, with formation of CM- and VLDL-remnants which are efficiently removed from the circulation by receptor-mediated pathways in the liver (Cooper, 1997). During lipolysis of postprandial triglyceride-rich lipoproteins, an excess of surface components containing apolipoproteins, unesterified cholesterol and phospholipids is generated and sequesters to HDL potentially via the action of hepatic lipase and PLTP, thereby increasing the total circulating HDL pool and enhancing the transformation of small HDL3 to large CE-rich HDL2 particles (Zilversmit, 1995). Equally, CETP mediates heterotransfer of cholesteryl esters and triglycerides between HDL on the one hand, and apoB-containing lipoproteins on the other; such transfer is accelerated under postprandial conditions with CE enrichment of triglyceride-rich lipoprotein particles, and transient transformation of CE-enriched HDL into TG-enriched particles which become a substrate for hepatic lipase (Castro and Fielding, 1985; Tall et al., 1986; Contacos et al., 1998). In this way, HDL particle chemical composition and size are modulated during the postprandial phase. Elevated postprandial TG is associated with adverse events such as the formation of atherogenic CM remnants, small dense LDL particles and reduction of HDL-C. Fasting plasma TG levels account in part for the amplitude of the postprandial phase and elevated fasting TG is indicative of abnormal postprandial lipoprotein metabolism, for which delayed elimination of postprandial TGRL is associated with atherosclerosis. In patients with coronary artery disease (CAD), postprandial TG levels are elevated and remain so over a prolonged period in comparison with patients without CAD. In type IIb patients, plasma TG accumulation during the postprandial phase was increased as compared to normolipidemic subjects taking the same meal and the incremental area under the curve (AUC) for TG was elevated four-fold (Guerin et al., 2002). Since most people consume fat-containing meals or snack at regular

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4e5 h intervals, it is clear that the usual state of TG metabolism for most humans is a postprandial one (Lairon, 2007). The postprandial TG response refers to a series of metabolic events that occur after ingestion of a fatcontaining meal and the magnitude of this response is determined by several factors: it increases with fasting plasma TG concentration, age, sedentary life style, and is greater in males than females. Furthermore, the capacity of individuals to regulate circulating TG and clear TGRL is a reflection of their metabolic efficiency, which can be modulated by various gene polymorphisms for key proteins, such as apoE, apoB, apoA-IV and apoC-III, LDL receptor, microsomal transfer protein, fatty acid-binding protein, CETP, LPL and HL (Lairon et al., 2007). Efficient postprandial lipid metabolism with rapid clearance of CMs, promotes HDL formation and could stimulate RCT. After ingestion of dietary fat, an increase in plasma TG concentration is a normal metabolic consequence that is associated with CM and VLDL production and lipolysis. The small intestine delivers CM to the circulation packaged with a truncated form of apoB called apoB48. CM are very quickly transformed to CM remnants by the action of LPL with reduced TG content and particle size. The accumulation of these remnants in the circulation is thought to be one of the most detrimental consequences of the postprandial period, since they can accumulate in the plaque (Rapp et al., 1994) thereby promoting atherosclerosis. During the lipolytic process, surface components (FC, PL and apoE and apoA-I lipoproteins) of CM and VLDL are sequestered to HDL. In this state, CE transfer rate is modulated by changes in plasma lipoproteins and may increase as a result of an increase in CE acceptors and/or CETP concentrations (Tall et al., 1987). This process favors CE enrichment of TGRL and allows transformation of CE-rich HDL in TG-rich HDL that becomes a substrate for HL. On a quantitative basis, VLDL but not CM represent the preferential acceptors among TGRL during the postprandial phase (Lassel et al., 1998) and on a qualitative basis VLDL2 are the preferential ones. In NLS, LDL particles are the major acceptors of CE from HDL in both the fasting and postprandial state despite increases in TGRL (Lassel et al., 1999). However, LDL-CE are secondarily transferred to CM through CETP action, thus CE-enriched CM can be cleared by the liver and irreversibly removed from the body; in this case RCT in the postprandial state is enhanced, thus representing an anti-atherogenic process in normolipidemic subjects. PLTP could play an important role during the postprandial phase by transferring surface PL to the plasma HDL fraction and it is documented that CETP and PLTP activities are modulated by a large intravenous fat load in healthy men (Riemens et al., 1999). Syeda et al. (2003) observed that, during the late postprandial period (6e8 h after meal intake), CETP activation was accompanied by a large increase in HDL-PL but no change in PLTP activity. Furthermore, serum cholesterol efflux capacity was increased during the late postprandial phase in normolipidemic subjects. Guerin et al. (2002) observed that four hours after meal intake, the capacity of postprandial plasma from type IIb subjects to mediate FC efflux was increased

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as compared with the baseline value obtained before meal intake. However, efflux rates to IIb and control plasmas were similar at 2, 4, and 8 h postprandially.

CONCLUSION Interventional studies with lipid-lowering drugs have demonstrated a reduction of up to 50% in LDL-cholesterol levels with concomitant diminution in cardiovascular morbi-mortality of up to approximately 40% in dyslipidemic patients at high risk in both primary and secondary prevention. However, approximately 60% of CV risk, the residual risk, remains to be corrected by nutritional, pharmacological and behavioral approaches. A high proportion of patients exhibiting significant residual risk frequently present with a low HDLcholesterol level (1.063 kg/L) and a small size (5e17 nm). Regarding the basic composition, HDL particles have the highest proportion of protein relative to lipid compared to other plasma lipoproteins. The major proteins are apolipoprotein A-I (apoA-I) and apoA-II, with small amounts of apo C, E, A-IV, D and J. The principal lipid components are the phospholipids, with lesser amounts of cholesterol esters, unesterified cholesterol and triglycerides. HDL particles also carry several important enzymes: paraoxonase-1 (PON1), platelet-activating factor acetyl hydrolase (PAF-AH), glutathione selenoperoxidase (GSPx), lecithin:cholesterol acyltransferase (LCAT) and phospholipid transfer protein (PLTP).

HDL subclasses Variations in the qualitative and quantitative content of lipids and apolipoproteins result in the presence of various HDL subclasses, characterized by different shape, charge, density and size (Skinner, 1994). This remarkable heterogeneity arises from their complex synthesis and continuous intravascular remodeling processes. The understanding that structure and composition of each HDL subclass reflect its different metabolic function challenges the traditional approach of using only HDL-C level for the management of cardiovascular risk. To date, investigators have described several different approaches to separate HDL subclasses based on a variety of their physicochemical properties including particle density and size, charge and apolipoprotein composition. Here, we address the most commonly used techniques. Density gradient ultracentrifugation represents the original procedure from which subsequent methods have been calibrated and validated. When fractionated by ultracentrifugation, HDL particles are usually separated into two major subclasses; HDL2 (density range 1.063e1.125 kg/L) and HDL3 (density

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range 1.125e1.210 kg/L) (Chapman et al., 1981). However, during the ultracentrifugation process labile lipoproteins may be substantially altered by the high salt conditions and centrifugal forces which can lead to loss of loosely bound apolipoproteins and enzymes (Kunitake and Kane, 1982; Cheung and Wolf, 1988). In spite of numerous disadvantages, ultracentrifugation is still an irreplaceable preparative technique in numerous research laboratories. Electrophoretic methods separate HDL particles on the basis of their charge and size. In agarose gel electrophoresis, most HDL particles have a-mobility with the exception of a minor fraction that migrates in the pre-b region (Kunitake et al., 1985). Non-denaturing polyacrylamide gradient gel electrophoresis separates HDL into five distinct subpopulations ranging from smaller, denser HDL3c (7.2e7.8 nm), HDL3b (7.8e8.2 nm) and HDL3a (8.2e8.8 nm) to larger HDL2a (8.8e9.7 nm) and HDL2b (9.7e12 nm) subclasses (Blanceh et al., 1981). Finally, HDL subclasses are often classified based on their particular apolipoprotein composition into HDL particles that contain only apoA-I (LpAI) and those that contain both apoA-I and apoA-II (LpAI:AII) (Cheung and Albers, 1982). In our laboratory, we separate HDL subclasses using a modified gradient (3e31%) gel electrophoretic method originally developed by Rainwater et al. (1997). In addition to HDL, this method also provides the separation of LDL particles on the same gradient gel (Figure 5.1). From our own experience, this procedure is reasonably reproducible, relatively economical and therefore suitable for use in population and clinical studies (Vekic et al., 2007a). A key question remains to be answered. Do the various HDL subclasses equally contribute to the anti-atherogenic properties of HDL? The main controversy centers around the role of smaller, denser HDL particles. Numerous studies have investigated whether smaller, denser HDL particles are more or less protective than their larger counterparts. However, attempts to resolve the question have provided somewhat ambiguous results. It is worth mentioning that the use of different methodological approaches and, consequently, different nomenclature for the same class of particles may be the explanation of inconsistent and confusing results. Another reason could be the fact that HDL particles can undergo dramatic changes during inflammatory conditions which considerably alter or attenuate their anti-atherogenic effects (Ansell et al., 2005). Thus, smaller, denser HDL particles appear to have a dual role in atherogenesis; functional particles could be protective, whereas alterations in their structure could implicate pro-atherogenic properties.

Anti-atherogenic properties of HDL The protective role of HDL is well documented, but the precise mechanisms by which it exerts its anti-atherogenic effects are still being characterized. HDL particles prevent the development of atherosclerosis through multiple pathways including both reverse cholesterol transport (RCT) and cholesterol-independent

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FIGURE 5.1 Separation of LDL and HDL particles on a polyacrylamide gradient (3e31%) gel. Electrophoresis was performed at 8 C using Tris-boric acid-Na2EDTA buffer, pH 8.35 for 20 hours. Separated fractions were stained with Sudan Black. The relative distribution of each subfraction can be estimated by determining the areas under the peaks of the densitometric scan of the sample.

mechanisms. Although this chapter mostly concerns antioxidative properties of HDL particles, such a discussion would be incomplete without briefly considering their other significant roles.

Reverse cholesterol transport The process involves the unique ability of HDL particles to remove excess cholesterol from peripheral tissues to the liver. To start the process the liver and intestine synthesize lipid-poor apoA-I. Once in the bloodstream, circulating nascent HDL particles receive free cholesterol from tissues via their interactions with the ATP-binding membrane cassette transport proteins A1 (ABCA1) and G1/G4 (ABCAG1/G4) expressed on macrophages, scavenger receptor class B type 1 (SR-B1), caveolin and even via passive diffusion (Yancey et al., 2003). Subsequently, LCAT esterifies free cholesterol within nascent HDL and produces mature HDL3 (smaller, denser HDL) and HDL2 (larger, less dense HDL) particles (Jonas, 1991). Mature HDL has two metabolic pathways. In the direct pathway, cholesterol esters within HDL undergo selective uptake by hepatocytes via SR-B1 receptors or LDL receptors (if HDL contains apoE) and subsequent excretion into the bile. In the indirect pathway, cholesterol esters within HDL are exchanged for triglycerides in apolipoprotein B

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(apoB)-containing particles (LDL and VLDL) through the action of cholesterol ester transfer protein (CETP). Finally, apoB-rich lipoproteins deliver cholesterol esters to the liver via LDL receptors. After removal of cholesterol esters, triglyceride-rich HDL undergoes hydrolysis by hepatic lipase (HL) and endothelial lipase (EL). The remaining smaller, denser HDL releases apoA-I which then either participates in the next lipidation cycle or is catabolized in the kidneys (Lewis and Rader, 2005). Although the mechanism of the RCT process is still not fully elucidated, its efficiency is considered to be an important determinant of HDL’s anti-atherogenic potential.

Anti-inflammatory effects Some authors have suggested that HDL is capable of inhibiting cytokineinduced expression of adhesion molecules (vascular cell adhesion molecule (VCAM)-1, intracellular adhesion molecule (ICAM)-1 and E-selectin) thereby impeding monocyte adhesion on the endothelial surface (Cockerill et al., 2001). However, we failed to confirm the proposed association between low HDL-C concentrations and adhesion molecule overexpression in normolipidemic subjects (Bogavac-Stanojevic et al., 2005). Furthermore, there is evidence suggesting that the main protein in HDL (apoA-I) is able to bind to bacterial endotoxin thereby inhibiting endotoxin-induced cellular activation (Ma et al., 2004). In addition, the significant anti-inflammatory potential of HDL is a consequence of its antioxidative effects. Vasoprotective and anti-thrombotic effects HDL preserves the integrity of the endothelium via stimulation of endothelial cell migration and suppression of their apoptosis (Nofer et al., 2001). Incubation of cultured endothelial cells with HDL particles activates nitric oxide (NO) synthase (eNOS), with subsequent NO release and NO-dependent vasodilatation (Kuvin et al., 2002). Furthermore, by stimulating endothelial prostacyclin synthesis, HDL can further enhance vasorelaxation (Spector et al., 1985). The inhibitory action of HDL on platelet activation and aggregation can be accomplished directly (Lerch et al., 1998), or by modulating the production of the above-mentioned endothelial factors. In addition, HDL may prevent thrombogenesis via regulation of thrombomodulin synthesis and enhancement of protein C and protein S activities (Griffin et al., 1999; Nicholls et al., 2005). Antioxidative effects HDL is also believed to protect against atherosclerosis by inhibiting the oxidative modification of LDL thereby attenuating the biological activities of oxidized LDL (Ng et al., 2005). Such antioxidative properties have been attributed to various proteins associated with HDL, mainly a set of enzymes: LCAT, PAF-AH, PON1, proteinase (elastase-like activity) and phospholipase D but also due to albumin and apoA-I (Durrington et al., 2001). PON1 was found

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to be more effective than LCAT or apoA-I in protecting LDL against oxidation, although it was documented that the antioxidative effect of PON1 was slightly enhanced in the presence of LCAT and apoA-I (Arrol et al., 1996). The activity of PAF-AH is believed to be similar to that of PON1. However, studies using PON1-knockout mice have demonstrated that PAF-AH was unable to perform its function in the absence of PON1, indicating that PON1 is the primary enzyme responsible for LDL protection (Rodrigo et al., 2001). HDL is the most abundant lipoprotein in tissue fluid and the only lipoprotein in the central nervous system. Based on the evidence that PON1 is present not only in the circulation but also in other tissue fluids, some authors suspect that the antioxidative function of PON1 has evolved to protect against the development of atherosclerosis, a disease prevalent for less than a century (Durrington et al., 2001).

PARAOXONASE-1 (PON1) PON1 (EC 3.1.8.1, aryldialkylphosphatase) is specifically an HDL-associated serum enzyme (Durrington et al., 2001). Its primary function was first described as an organophosphate hydrolase, meaning that it could degrade commonly used insecticides and nerve gases. Recent reports have suggested that PON1 is largely responsible for the breakdown of lipid peroxides before they accumulate in LDL particles (Mackness et al., 1991a). In addition, PON1 also renders HDL resistant to oxidation (Durrington et al., 2001). According to Aviram et al. (2000) PON1 is able to hydrolyze lipid peroxides in human atherosclerotic lesions. Recent evidence has confirmed that PON1 is primarily a lactonase acting upon certain lactones/hydroxyl acids. Therefore, one of the physiological roles of PON1 is the metabolism of lipid species arising from the oxidation of polyunsaturated fatty acids (Draganov et al., 2005).

PON1 polymorphism Research regarding the PON1 gene sequence has demonstrated nearly 200 polymorphic sites (La Du, 2003). PON1 has two common coding polymorphisms: a methionine (M) to leucine (L) substitution at position 55 and a glutamine (Q) to arginine (R) substitution at position 192. In addition, PON1 has at least three structural variants in the same region. The polymorphic sites are also found in the non-coding regions of the PON1 gene: 7 are in the 5’-untranslated region, 171 are in intronic regions and 15 are in the 3’-untranslated region. However, the most prominent coding polymorphisms (Q192R and L55M) have particular pathophysiological significance. Numerous studies have indicated that both polymorphisms could be associated with CVD, stroke, familial hypercholesterolemia, type 2 diabetes mellitus and Parkinson’s disease (Ruiz et al., 1995; Schmidt et al., 1998; Akhmedova et al., 2001; Ng et al., 2005). Of particular note is the fact that PON1Q192R isoforms have

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substrate-dependent differences in their enzyme activity; the Q variant has a higher diazoxonase activity (DZOase) whereas paraoxonase activity (POase) predominates in the R variant (Serrato and Marian, 1995). By measuring the DZOase/POase activity ratio it is possible to discriminate between the PON1192QQ, PON1192QR and PON1192RR phenotypes.

PON1 status Possible involvement of the PON1Q192R polymorphism in vascular disease development has often been evaluated only by determination of PON1 genotype without considering PON1 concentration and/or activity (Richter et al., 2003). Richter and Furlong (1999) have characterized “PON1 status” as PON1 enzymatic activities towards two non-physiological organophosphate substrates e paraoxon and diazoxon in combination with PON1Q192R phenotype determination. By plotting the rates of PON1’s ability to hydrolyze diazoxon (DZOase PON1 activity) vs. paraoxon (POase PON1 activity), the study population can be divided into three distinct groups: individuals functionally homozygous for PON1192QQ, heterozygotes (PON1192QR) and individuals homozygous for PON1192RR. Therefore, PON1 status determination provides information not only on the PON1Q192R polymorphism but also on the two PON1 enzymatic activities. Richter et al. (2003) found that the PON1 status of individuals with different PON1Q192R genotypes can be quite similar, suggesting that PCR analysis was unable to discover inactive alleles. This finding indicates that PON1 status determination provides insight into the functional state of PON1 genomics in any given individual (Richter et al., 2003).

THE ASSOCIATION BETWEEN PON1 AND HDL The relationship between PON1 and HDL relies on a unique reciprocal arrangement. HDL facilitates the secretion of PON1 and offers a hydrophobic harbor for its anchoring. It also provides a hydrophobic environment that is essential for optimal PON1 activity (James and Deakin, 2004). In return, the enzyme prevents the oxidation of HDL and stimulates the cholesterol efflux from cells which enhances the capacity of RCT (Rosenblat et al., 2005). Therefore, the association between PON1 and HDL should be considered in both normal and pathological conditions.

PON1 and HDL in healthy subjects Variations in PON1 activity and phenotype distribution may be the cause of different predispositions to atherosclerosis within populations (Brophy et al., 2002). Such diversity in PON1 activities is largely genetically determined but it can also be modulated by various dietary (Shih et al., 1996; Kleemola et al.,

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2002) and lifestyle factors (Ferre´ et al., 2003). Recently, we studied the PON1Q192R polymorphism in Serbia (Kotur-Stevuljevic et al., 2006). We concluded that the frequencies of PON1 phenotypes in our healthy population were in Hardy-Weinberg equilibrium and comparable to those previously reported in other European populations (Table 5.1). A recent study by Blatter Garin et al. (2006) confirmed a significant positive association between PON1 activity and apoA-I and HDL-C concentrations and considerable influence of PON1 polymorphism on apoA-I and HDL-C levels. With the current appreciation of HDL variability, it has been postulated that PON1 is not equally distributed among HDL particles but, rather, it has a specific association with particular HDL subpopulations. Zech et al. (1993) suggested that the PON1-HDL association follows a normal remodeling process of HDL particles. Initially, PON1 is associated with smaller, denser HDL3 subfractions which will subsequently be converted into larger HDL2 particles. However, different studies have reported conflicting results. PON1 has been found to be associated with larger HDL particles isolated by gel filtration (Blatter et al., 1993) or with smaller HDL particles fractionated by ultracentrifugation (Kontush et al., 2003). On the other hand, it has been suggested that PON1 is preferentially associated with larger HDL particles in vivo and that it can be displaced to smaller HDL particles (Kontush et al., 2003; Bergmeier et al., 2004) or even accumulated in the lipoprotein-free plasma (Rosenblat et al., 2006) upon ultracentrifugation. Knowledge concerning the mechanisms of antioxidative protection by HDL is mainly derived from the results of basic science studies. Recently, we studied the association of oxidative stress and PON1 status and HDL particle size

TABLE 5.1

PON1Q192R allele frequencies in European populations PON1 alleles

Population

N

Q

R

Reference

Serbian

105

0.72

0.28

Kotur-Stevuljevic et al. (2006)

Italian

162

0.70

0.30

Ombres et al. (1998)

Austrian

144

0.75

0.25

Schmidt et al. (1998)

German

971

0.73

0.27

Cascorbi et al. (1999)

Swiss

273

0.73

0.27

James et al. (2000)

British

152

0.75

0.25

Mackness et al. (2000)

Spanish

310

0.70

0.30

Senti et al. (2000)

Russian

117

0.74

0.26

Akhmedova et al. (2001)

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heterogeneity in a clinical setting (Vekic et al., 2007b). All study participants were deemed clinically healthy and were middle-aged. We found that oxidative stress parameters positively correlated with the levels of smaller, denser HDL3b and HDL3c but negatively with larger HDL2a subclasses. In addition, PON1 activity exhibited a profound inverse relationship with HDL size. Our findings could represent in vivo evidence that oxidative stress could alter the maturation of HDL particles, perhaps due to impaired LCAT activity (Mertens et al., 2003) and also to attenuate their antioxidative potential by inactivation of PON1 (Mertens et al., 2003). Another interesting finding was significantly reduced HDL particle size among the carriers of the PON1192R allele (Vekic et al., 2007b). According to Mackness at al. (1998), the expression of the R allele is directly related to a lower capacity of PON1 to protect LDL from oxidation. Thus, we concluded that smaller, denser HDL particles could have a lower antioxidative potential. Furthermore, in a recent study, smaller HDL particles were found to be associated with elevated uric acid and markers of inflammation in apparently healthy subjects (Vekic et al., 2009). There is evidence to suggest that uric acid (UA) may function as a potent antioxidant by scavenging free radicals and also by stabilizing ascorbate in biological fluids. Therefore, elevated UA could also be considered to be a compensatory mechanism that counteracts oxidative stress related to the preponderance of smaller HDL particles.

PON1 and HDL in cardiovascular disease Reports that sought associations between the PON1192R allele (or PON1192RR genotype) and CVD are considered controversial. Some studies reported a positive association (Humbert et al., 1993; Zama et al., 1997; Sanghera et al., 1998), whereas others failed to show any relationship (Antikainen et al., 1996; Suehiro et al., 1996; Rice et al., 1997; Ombres et al., 1998). There is also evidence to suggest that low PON1 activity is associated with atherosclerosis irrespective of the PON1 phenotype (Mackness et al., 1991b, 2003; Jarvik et al., 2003). Jarvik et al. (2003) recently showed that PON1 activity was a better predictor of disease development than the PON1 genotype. In our own study, we found a significantly lower POase activity (24% reduced) in combination with reduced, but marginally significant, DZOase activity in a group of coronary heart disease (CHD) patients when compared with a healthy population (Kotur-Stevuljevic et al., 2006). Moreover, after the patients were separated according to the results of coronary angiography (CAD and CADþ), both groups had lower POase and DZOase activities compared with the controls (Figure 5.2). The distribution of the PON1Q192R phenotypes (estimation based on Figure 5.3) in the CHD patients was not significantly different from the control population. Moreover, the phenotype distribution within the CADþ and CAD groups did not differ between themselves nor from the control population.

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FIGURE 5.2 PON1 POase and DZOase activities in coronary heart disease (CHD) patients and controls. CHD patients were also separated according to coronary angiography results into CADþ and CAD groups (*PR genetic polymorphism. J Lipid Res, 40, 133e139. Chapuis, J., Bosher, M., Bensemain, F., Cottel, D., Amouyel, P., & Lambert, J. C. (2009). Association of the paraoxonase 1 gene with the risk of developing Alzheimer’s desease. Neurobiol Aging, 30, 152e156. Chen, N., Liu, Y., Greiner, C. D., & Holtzman, J. L. (2000). Physiologic concentrations of homocysteine inhibit the human plasma GSH peroxidase that reduces organic hydroperoxides. J Lab Clin Med, 136, 58e65. Cherki, M., Derouiche, A., Drissi, A., et al. (2005). Consumption of argan oil may have an antiatherogenic effect by improving paraoxonase activities and antioxidant status: Intervention study in healthy men. Nutr Metab Cardiovasc Dis, 15, 352e360. Ciftci, H., Yeni, E., Savas, M., Verit, A., & Celik, H. (2007). Paraoxonase activity in patients with erectile dysfunction. Int J Impot Res, 19, 517e520. Cle´ry-Barraud, C., Renault, F., Leva, J., El Bakdouri, N., Masson, P., & Rochu, D. (2009). Exploring the structural and functional stabilities of different paraoxonase-1 formulations through electrophoretic mobilities and enzyme activity parameters under hydrostatic pressure. Biochim Biophys Acta, 1794, 680e688. Cole, T. B., Jampsa, R. L., Walter, B. J., et al. (2003). Expression of human paraoxonase (PON1) during development. Pharmacogenetics, 13, 1e8.

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Teiber, J. F., Draganov, D. I., & La Du, B. N. (2004). Purified human serum PON1 does not protect LDL against oxidation in the in vitro assays initiated with copper or AAPH. J Lipid Res, 45, 2260e2268. Tomas, M., Senti, M., Garcia-Faria, F., et al. (2000). Effect of simvastatin therapy on paraoxonase activity and related lipoproteins in familial hypercholesterolemic patients. Arterioscler Thromb Vasc Biol, 20, 2113e2119. Tward, A., Xia, Y. R., Wang, X. P., et al. (2002). Decreased atherosclerotic lesion formation in human serum paraoxonase transgenic mice. Circulation, 106, 484e490. Ulevitch, R. J., Johnston, A. R., & Weinstein, D. B. (1979). New function for high density lipoproteins. Their participation in intravascular reactions of bacterial lipopolysaccharides. J Clin Invest, 64, 1516e1524. Uriel, J. (1961). Characterization of cholinesterase and other carboxylic esterases after electrophoresis and immunoelectrophoresis on agar. I. Application to the study of esterases of normal human serum. Ann Inst Pasteur, 101, 104e119. Uzun, H., Yanardag, H., Gelisgen, R., et al. (2008). Levels of paraoxonase, an index of antioxidant defense, in patients with active sarcoidosis. Curr Med Res Opin, 24, 1651e1657. Vaisar, T., Pennathur, S., Green, P. S., et al. (2007). Shotgun proteomics implicates protease inhibition and complement activation in the antiinflammatory properties of HDL. J Clin Invest, 117, 746e756. Van der Gaag, M. S., van Tol, A., Scheek, L. M., et al. (1999). Daily moderate alcohol consumption increases serum paraoxonase activity; a diet-controlled, randomised intervention study in middle-aged men. Atherosclerosis, 147, 405e410. van Himbergen, T. M., van Tits, L. J. H., Hectors, M. P. C., de Graaf, J., Roest, M., & Stalenhoef, A. F. H. (2005). Paraoxonase-1 and linoleic acid oxidation in familial hypercholesterolemia. Biochem Biophys Res Commun, 333, 787e793. Van Lenten, B. J., Hama, S. Y., de Beer, F. C., et al. (1995). Anti-inflammatory HDL becomes proinflammatory during the acute phase response. Loss of protective effect of HDL against LDL oxidation in aortic wall cell cocultures. J Clin Invest, 96, 2758e2767. Van Lenten, B. J., Wagner, A. C., Navab, M., & Fogelman, A. M. (2001). Oxidized phospholipids induce changes in hepatic paraoxonase and ApoJ but not monocyte chemoattractant protein-1 via interleukin-6. J Biol Chem, 276, 1923e1929. Wheeler, J. G., Keavney, B. D., Watkins, H., Collins, R., & Danesh, J. (2004). Four paraoxonase gene polymorphisms in 11212 cases of coronary heart disease and 12786 controls: metaanalysis of 43 studies. Lancet, 363, 689e695. Wu, A., Hinds, C. J., & Thiemermann, C. (2004). High-density lipoproteins in sepsis and septic shock: metabolism, actions, and therapeutic applications. Shock, 21, 210e221.

Chapter 7

Apolipoprotein A-I Mutations Akira Matsunaga, Yoshinari Uehara, Bo Zhang and Keijiro Saku Department of Laboratory Medicine, Department of Cardiology, Fukuoka University School of Medicine, Fukuoka, Japan

INTRODUCTION High density lipoprotein (HDL) is synthesized via a complex pathway. Although the underlying genetic defects in many cases of primary low HDL cholesterolemia are not clearly understood, mutations in three pivotal genes, namely apolipoprotein A-I (apoA-I), ATP binding cassette transporter A1 (ABCA1) and lecithin:cholesterol acyltransferase (LCAT), are associated with low plasma HDL cholesterol (HDL-C) levels (Miller et al., 2003). Some mutations of these genes are also associated with an increased risk of premature coronary artery disease (CAD). The most severe form of HDL deficiency is Tangier disease, which is a recessive disorder characterized by cholesteryl ester (CE) accumulation in macrophages, orange-yellow tonsils, hepatosplenomegaly, peripheral nerve neuropathy, corneal opacifications and premature CAD in 50% of cases (Hobbs and Rader, 1999). Tangier disease is caused by mutations of the gene encoding ABCA1, a 2261-amino acid peptide (Hobbs and Rader, 1999; Uehara et al., 2007). LCAT catalyzes the transfer of a preferentially unesterified fatty acid from phosphatidylcholine to free cholesterol, thereby producing a CE. The LCAT gene contains six exons and encodes a 440-amino acid protein including a 24-amino acid signal peptide (Kuivenhoven et al., 1997). Depending on the mutation, homozygous or compound heterozygous patients with LCAT mutations present with one of two clinical phenotypes, namely classical LCAT deficiency or fish-eye disease. Both disorders are characterized by the occurrence of corneal opacifications as hallmarks (Moriyama et al., 1995; Miida et al., 2004). However, premature CAD is uncommon, even in the presence of cardiovascular risk factors. ApoA-I is the major protein component of HDL, and plays essential roles in the biogenesis and functions of HDL. Approximately 70% of the HDL protein The HDL Handbook. ISBN: 978-0-12-382171-3 Copyright Ó 2010 Elsevier Inc. All rights of reproduction in any form reserved.

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mass is comprised of apoA-I with another 15e20% comprised of apoA-II (Mahley et al., 1984; Davidson and Thompson, 2007). HDL assembly initially involves cell surface ABCA1 transporter-mediated transfer of cellular phospholipids (PL) and unesterified cholesterol to extracellular lipid-poor apoA-I particles (preb-HDL particles). Overall, 61 mutations of the ApoA-I gene have been reported to date (45 missense, five nonsense and six frameshift mutations, and five in-frame deletions), and these are mostly located in the coding region. The nonsense and frameshift mutations, which affect the complete translation of apoA-I mRNA, are always associated with reduced levels of apoA-I and HDL-C. In contrast, only half of the missense mutations are associated with low levels of apoA-I and HDL-C. However, only a minority of these apoA-I mutations are associated with premature CAD. Most of the mutations of the ApoA-I gene have been found in heterozygous subjects. The wide allelic heterogeneity and condition of heterozygosity make it difficult to clearly define the phenotypic expression of ApoA-I gene mutations in heterozygotes with respect to the development of premature CAD. For these reasons, the identification of individuals homozygous for mutations of the ApoA-I gene, leading to severe apoA-I deficiency, is very useful for better understanding of the roles of apoA-I and HDL in protection against CAD. Some homozygous subjects with virtually no HDL or apoA-I have been reported (Funke et al., 1991; Matsunaga et al., 1991).

GENOMIC AND PROTEIN STRUCTURE OF APOA-I Mature apoA-I in plasma is a 243-amino acid peptide primarily synthesized in the liver and intestine. ApoA-I is a 28-kDa single polypeptide that lacks glycosylation or disulfide linkages. The ApoA-I gene resides on chromosome 11q23-q24, contains four exons and encodes a 267-amino acid peptide chain including an 18-amino acid prepeptide and a 6-amino acid propeptide (Fielding and Fielding, 1995). Exon 3 of the ApoA-I gene encodes the N-terminal region of mature apoA-I (residues 1-43) and the remaining regions are encoded by exon 4. Large parts of the mature protein form homologous repeats of amphipathic alpha-helices, each encompassing 11 or 22 amino acids (Nolte and Atkinson, 1992; Brouillette and Anantharamaiah, 1995; Borhani et al., 1997).

PHYSIOLOGICAL FUNCTIONS OF APOA-I As the quantitatively predominant component of HDL, apoA-I is crucial for HDL formation. It 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 ABCA1 or G1 (Gelissen et al., 2006; Zanis et al., 2006). In mice and rabbits, knockout of the ApoA-I gene causes HDL deficiency and, conversely, transgenic overexpression of apoA-I increases HDL-C in a gene-dose-dependent manner. In susceptible animals with an

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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 (Williamson et al., 1992).

APOA-I DEFICIENCIES WITH LARGE DELETION, NONSENSE OR FRAMESHIFT MUTATIONS In total, 13 types of ApoA-I deficiency caused by large deletion, nonsense or frameshift mutations have been reported to date (Table 7.1) (Norum et al., 1982; Schaefer et al., 1985; Funke et al., 1991; Matsunaga et al., 1991, 1999; Lackner et al., 1993; Nakata et al., 1993; Ng et al., 1994; Romling et al., 1994; Takata et al., 1995; Miccoli et al., 1996; Moriyama et al., 1996; Yokota et al., 2002; Ikewaki et al., 2004; Dastani et al., 2006). The probands of ApoA-I/C-III deficiency were two sisters aged 29 and 31 years, who had skin and tendon xanthomas, corneal opacities and severe CAD (Norum et al., 1982). The rearrangement in these probands consisted of a DNA inversion involving portions of the 30 ends of the ApoA-I and ApoC-III genes, including the DNA region between these genes (Karathanasis et al., 1987). Their plasma levels of HDL-C were 4 and 7 mg/dL (0.10 and 0.18 mmol/L, respectively), and only traces of apoA-I were detected in whole plasma. The proband of ApoA-I/C-III/A-IV deficiency was a 45-year-old white female (Schaefer et al., 1985). The defect in this proband was complete deletion of the ApoA-I, ApoC-III, ApoA-IV gene complex on chromosome 11 (Ordovas et al., 1989). She had severe CAD and corneal opacities, but no hepatosplenomegaly or xanthomas were detected. Her plasma level of HDL-C was 1 mg/dL. She died after coronary bypass surgery. The proband of Q-2X was a 34-year-old Caucasian female who had xanthelasma, Achilles xanthomas, bilateral cataracts, retinal detachments, mild midline cerebellar ataxia and asymmetric bilateral neurosensory hearing loss (Ng et al., 1994). Her plasma HDL-C was 2.3 mg/dL. This proband and four of her 11 siblings had a homozygous mutation of Q-2X, severe deficiency of HDL (TAG at codon-2

Homozygote

34

F

Canadian

þ

She had retinopathy Ng et al. (1994) cataracts, spinocerebellar ataxia and tendon xanthomas

4.Q5fsX36)

Insertion of C at codon 5

Homozygote Heterozygote

11 10

F F

Turkish Japanese

 

She had planar xanthomas

5. Q5fsX11)

Deletion of C at codon 5

Compound))

47

M

Italian

þ

Another was apoA-I L141RPisa. He had corneal opacities

6. W8XWakayama

TGG->TGA at codon 8

Homozygote

39

M

Japanese



He had corneal opacities

Takata et al. (1995)

7. Q32X

CAG->TAG at codon 32

Homozygote

31

F

Italian



She had perioirbital xanthelasmas

Romling et al. (1994)

Lackner et al. (1993) Nakata et al. (1993)

The HDL Handbook

She had planar xanthomas Another was a missense mutation in the TATA box

Matsunaga et al. (1991) Matsunaga et al. (1999)

French Canadian

þ

E136X mutations were found in 17 carriers

Dastani et al. (2006)

F

Japanese

þ

She had corneal opacities and planar xanthomas

Ikewaki et al. (2004)

50

F

Japanese



She had corneal opacities and skin and tendon xanthomas

Moriyama et al. (1996)

Homozygote

69

F

Japanese



She had corneal opacities and corneal rings

Yokota et al. (2002)

Homozygote

42

M

German



He had corneal opacities

Funke et al. (1991)

Homozygote Compound))

9. E136X

Deletion of C at codon 136

Heterozygote

10. A154fsX178 Shinbashi)

Deletion of GC at codon 154

Homozygote

51

11. H162fsX208 Sasebo

Insertion of 23 pb from codon 162

Homozygote

12. K184FSX200)

Deletion of C at codon 184

13. T202fsX200)

Deletion of G at codon 202

)

Frameshift mutation;

60 13

F M

Apolipoprotein A-I Mutations

þ 

CAG->TAG at codon 84

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Japanese Japanese

8. Q84X

))

Compound: compound heterozygote; CAD: coronary artery disease

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3 mg/dL. The patient of W8XWakayama was a 39-year-old Japanese male (Takata et al., 1995). He had corneal opacities, but no CAD or xanthomas. His plasma HDL-C and apoA-I levels were 6 and Asp). Biochim Biophys Acta, 1214, 272e278. Assmann, G., von Eckardstein, A., & Funke, H. (1993). High density lipoproteins, reverse transport of cholesterol, and coronary artery disease. Insights from mutations. Circulation, 87, III28e34. Baralle, M., & Baralle, F. E. (2000). Genetics and molecular biology. Curr Opin Lipidol, 11, 653e656. Bielicki, J. K., & Oda, M. N. (2002). Apolipoprotein A-I(Milano) and apolipoprotein A-I(Paris) exhibit an antioxidant activity distinct from that of wild-type apolipoprotein A-I. Biochemistry, 41, 2089e2096. Booth, D. R., Tan, S. Y., Booth, S. E., et al. (1995). A new apolipoprotein Al variant, Trp50Arg, causes hereditary amyloidosis. QJM, 88:695e702. Booth, D. R., Tan, S. Y., Booth, S. E., Tennent, G. A., Hutchinson, W. L., Hsuan, J. J., et al. (1996). Hereditary hepatic and systemic amyloidosis caused by a new deletion/insertion mutation in the apolipoprotein AI gene. J Clin Invest, 97, 2714e2721. Borhani, D. W., Rogers, D. P., Engler, J. A., & Brouillette, C. G. (1997). Crystal structure of truncated human apolipoprotein A-I suggests a lipid-bound conformation. Proc Natl Acad Sci USA, 94, 12291e12296. Breslow, J. L. (1988). Apolipoprotein genetic variation and human disease. Physiol Rev, 68, 85e132. Brewer, H. B., Jr. (2004). Focus on high-density lipoproteins in reducing cardiovascular risk. Am Heart J, 148, S14e18. Brouillette, C. G., & Anantharamaiah, G. M. (1995). Structural models of human apolipoprotein A-I. Biochim Biophys Acta, 1256, 103e129. Bruckert, E., von Eckardstein, A., Funke, H., Beucler, I., Wiebusch, H., Turpin, G., et al. (1997). The replacement of arginine by cysteine at residue 151 in apolipoprotein A-I produces a phenotype similar to that of apolipoprotein A-Imilano. Atherosclerosis, 128, 121e128. Calabresi, L., Vecchio, G., Frigerio, F., Vavassori, L., Sirtori, C. R., & Franceschini, G. (1997). Reconstituted high-density lipoproteins with a disulfide-linked apolipoprotein A-I dimer: evidence for restricted particle size heterogeneity. Biochemistry, 36, 12428e12433. Chiesa, G., & Sirtori, C. R. (2003). Apolipoprotein A-I(Milano): current perspectives. Curr Opin Lipidol, 14, 159e163. Chiesa, G., Monteggia, E., Marchesi, M., et al. (2002). Recombinant apolipoprotein A-I(Milano) infusion into rabbit carotid artery rapidly removes lipid from fatty streaks. Circ Res, 90, 974e980. Chroni, A., Duka, A., Kan, H. Y., Liu, T., & Zannis, V. I. (2005). Point mutations in apolipoprotein A-I mimic the phenotype observed in patients with classical lecithin:cholesterol acyltransferase deficiency. Biochemistry, 44, 14353e14366. Coriu, D., Dispenzieri, A., Stevens, F. J., et al. (2003). Hepatic amyloidosis resulting from deposition of the apolipoprotein A-I variant Leu75Pro. Amyloid, 10, 215e223. Dastani, Z., Dangoisse, C., Boucher, B., et al. (2006). A novel nonsense apolipoprotein A-I mutation (apoA-I(E136X)) causes low HDL cholesterol in French Canadians. Atherosclerosis, 185, 127e136. Daum, U., Langer, C., Duverger, N., et al. (1999a). Apolipoprotein A-I (R151C)Paris is defective in activation of lecithin: cholesterol acyltransferase but not in initial lipid binding, formation of reconstituted lipoproteins, or promotion of cholesterol efflux. J Mol Med, 77, 614e622.

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Daum, U., Leren, T. P., Langer, C., et al. (1999b). Multiple dysfunctions of two apolipoprotein A-I variants, apoA-I(R160L)Oslo and apoA-I(P165R), that are associated with hypoalphalipoproteinemia in heterozygous carriers. J Lipid Res, 40, 486e494. Davidson, W. S., & Thompson, T. B. (2007). The structure of apolipoprotein A-I in high density lipoproteins. J Biol Chem, 282, 22249e22253. de Sousa, M. M., Vital, C., Ostler, D., et al. (2000). Apolipoprotein AI and transthyretin as components of amyloid fibrils in a kindred with apoAI Leu178His amyloidosis. Am J Pathol, 156, 1911e1917. Deeb, S. S., Cheung, M. C., Peng, R. L., et al. (1991). A mutation in the human apolipoprotein A-I gene. Dominant effect on the level and characteristics of plasma high density lipoproteins. J Biol Chem, 266, 13654e13660. Fielding, C. J., & Fielding, P. E. (1995). Molecular physiology of reverse cholesterol transport. J Lipid Res, 36, 211e228. Funke, H., von Eckardstein, A., Pritchard, P. H., Karas, M., Albers, J. J., & Assmann, G. (1991). A frameshift mutation in the human apolipoprotein A-I gene causes high density lipoprotein deficiency, partial lecithin: cholesterol-acyltransferase deficiency, and corneal opacities. J Clin Invest, 87, 371e376. Gelissen, I. C., Harris, M., Rye, K. A., et al. (2006). ABCA1 and ABCG1 synergize to mediate cholesterol export to apoA-I. Arterioscler Thromb Vasc Biol, 26, 534e540. Gregorini, G., Izzi, C., Obici, L., et al. (2005). Renal apolipoprotein A-I amyloidosis: a rare and usually ignored cause of hereditary tubulointerstitial nephritis. J Am Soc Nephrol, 16, 3680e3686. Hamidi Asl, L., Liepnieks, J. J., Hamidi Asl, K., et al. (1999a). Hereditary amyloid cardiomyopathy caused by a variant apolipoprotein A1. Am J Pathol, 154, 221e227. Hamidi Asl, K., Liepnieks, J. J., Nakamura, M., Parker, F., & Benson, M. D. (1999b). A novel apolipoprotein A-1 variant, Arg173Pro, associated with cardiac and cutaneous amyloidosis. Biochem Biophys Res Commun, 257, 584e588. Han, H., Sasaki, J., Matsunaga, A., et al. (1999). A novel mutant, ApoA-I nichinan (Glu235e>0), is associated with low HDL cholesterol levels and decreased cholesterol efflux from cells. Arterioscler Thromb Vasc Biol, 19, 1447e1455. Hobbs, H. H., & Rader, D. J. (1999). ABC1: connecting yellow tonsils, neuropathy, and very low HDL. J Clin Invest, 104, 1015e1017. Holvoet, P., Zhao, Z., Vanloo, B., et al. (1995). Phospholipid binding and lecithin-cholesterol acyltransferase activation properties of apolipoprotein A-I mutants. Biochemistry, 34, 13334e13342. Hovingh, G. K., Brownlie, A., Bisoendial, R. J., et al. (2004). A novel apoA-I mutation (L178P) leads to endothelial dysfunction, increased arterial wall thickness, and premature coronary artery disease. J Am Coll Cardiol, 44, 1429e1435. Huang, W., Sasaki, J., Matsunaga, A., et al. (1998). A novel homozygous missense mutation in the apo A-I gene with apo A-I deficiency. Arterioscler Thromb Vasc Biol, 18, 389e396. Huang, W., Sasaki, J., Matsunaga, A., Han, H., Li, W., Koga, T., et al. (2000). A single amino acid deletion in the carboxy terminal of apolipoprotein A-I impairs lipid binding and cellular interaction. Arterioscler Thromb Vasc Biol, 20, 210e216. Ikewaki, K., Matsunaga, A., Han, H., et al. (2004). A novel two nucleotide deletion in the apolipoprotein A-I gene, apoA-I Shinbashi, associated with high density lipoprotein deficiency, corneal opacities, planar xanthomas, and premature coronary artery disease. Atherosclerosis, 172, 39e45.

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Ji, Y., & Jonas, A. (1995). Properties of an N-terminal proteolytic fragment of apolipoprotein AI in solution and in reconstituted high density lipoproteins. J Biol Chem, 270, 11290e11297. Karathanasis, S. K., Ferris, E., & Haddad, I. A. (1987). DNA inversion within the apolipoproteins AI/CIII/AIV-encoding gene cluster of certain patients with premature atherosclerosis. Proc Natl Acad Sci USA, 84, 7198e7202. Kaul, S., Coin, B., Hedayiti, A., et al. (2004). Rapid reversal of endothelial dysfunction in hypercholesterolemic apolipoprotein E-null mice by recombinant apolipoprotein A-I(Milano)phospholipid complex. J Am Coll Cardiol, 44, 1311e1319. Koukos, G., Chroni, A., Duka, A., Kardassis, D., & Zannis, V. I. (2007a). LCAT can rescue the abnormal phenotype produced by the natural ApoA-I mutations (Leu141Arg)(Pisa) and (Leu159Arg)(FIN). Biochemistry, 406, 167e174. Koukos, G., Chroni, A., Duka, A., Kardassis, D., & Zannis, V. I. (2007b). Naturally occurring and bioengineered apoA-I mutations that inhibit the conversion of discoidal to spherical HDL: the abnormal HDL phenotypes can be corrected by treatment with LCAT. Biochem J, 406, 167e174. Kuivenhoven, J. A., Pritchard, H., Hill, J., Frohlich, J., Assmann, G., & Kastelein, J. (1997). The molecular pathology of lecithin:cholesterol acyltransferase (LCAT) deficiency syndromes. J Lipid Res, 38, 191e205. Lachmann, H. J., Booth, D. R., Booth, S. E., et al. (2002). Misdiagnosis of hereditary amyloidosis as AL (primary) amyloidosis. N Engl J Med, 346, 1786e1791. Lackner, K. J., Dieplinger, H., Nowicka, G., & Schmitz, G. (1993). High density lipoprotein deficiency with xanthomas. A defect in reverse cholesterol transport caused by a point mutation in the apolipoprotein A-I gene. J Clin Invest, 92, 2262e2273. Ladias, J. A., Kwiterovich, P. O., Jr., Smith, H. H., Karathanasis, S. K., & Antonarakis, S. E. (1990). Apolipoprotein A1 Baltimore (Arg10e>Leu), a new apoA1 variant. Hum Genet, 84, 439e445. Lebherz, C., Sanmiguel, J., Wilson, J. M., & Rader, D. J. (2007). Gene transfer of wild-type apoAI and apoA-I Milano reduce atherosclerosis to a similar extent. Cardiovasc Diabetol, 6, 15. Leren, T. P., Bakken, K. S., Daum, U., et al. (1997). Heterozygosity for apolipoprotein A-I(R160L) Oslo is associated with low levels of high density lipoprotein cholesterol and HDL-subclass LpA-I/A-II but normal levels of HDL-subclass LpA-I. J Lipid Res, 38, 121e131. Lindholm, E. M., Bielicki, J. K., Curtiss, L. K., Rubin, E. M., & Forte, T. M. (1998). Deletion of amino acids Glu146e>Arg160 in human apolipoprotein A-I (ApoA-ISeattle) alters lecithin: cholesterol acyltransferase activity and recruitment of cell phospholipid. Biochemistry, 37, 4863e4868. Mahley, R. W., Innerarity, T. L., Rall, S. C., Jr., & Weisgraber, K. H. (1984). Plasma lipoproteins: apolipoprotein structure and function. J Lipid Res, 25, 1277e1294. Martin-Campos, J. M., Julve, J., Escola, J. C., et al. (2002). ApoA-I(MALLORCA) impairs LCAT activation and induces dominant familial hypoalphalipoproteinemia. J Lipid Res, 43, 115e123. Matsunaga, A., Sasaki, J., Han, H., et al. (1999). Compound heterozygosity for an apolipoprotein A1 gene promoter mutation and a structural nonsense mutation with apolipoprotein A1 deficiency. Arterioscler Thromb Vasc Biol, 19, 348e355. Matsunaga, T., Hiasa, Y., Yanagi, H., et al. (1991). Apolipoprotein A-I deficiency due to a codon 84 nonsense mutation of the apolipoprotein A-I gene. Proc Natl Acad Sci USA, 88, 2793e2797. McManus, D. C., Scott, B. R., Franklin, V., Sparks, D. L., & Marcel, Y. L. (2001). Proteolytic degradation and impaired secretion of an apolipoprotein A-I mutant associated with dominantly inherited hypoalphalipoproteinemia. J Biol Chem, 276, 21292e21302.

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Miccoli, R., Bertolotto, A., Navalesi, R., et al. (1996). Compound heterozygosity for a structural apolipoprotein A-I variant, apo A-I(L141R)Pisa, and an apolipoprotein A-I null allele in patients with absence of HDL cholesterol, corneal opacifications, and coronary heart disease. Circulation, 94, 1622e1628. Miccoli, R., Zhu, Y., Daum, U., et al. (1997). A natural apolipoprotein A-I variant, apoA-I (L141R) Pisa, interferes with the formation of alpha-high density lipoproteins (HDL) but not with the formation of pre beta 1-HDL and influences efflux of cholesterol into plasma. J Lipid Res, 38, 1242e1253. Miettinen, H. E., Gylling, H., Miettinen, T. A., Viikari, J., Paulin, L., & Kontula, K. (1997). Apolipoprotein A-IFin. Dominantly inherited hypoalphalipoproteinemia due to a single base substitution in the apolipoprotein A-I gene. Arterioscler Thromb Vasc Biol, 17, 83e90. Miida, T., Zhang, B., Obayashi, K., et al. (2004). T13M mutation of lecithin-cholesterol acyltransferase gene causes fish-eye disease. Clin Chim Acta, 343, 201e208. Miller, M., Aiello, D., Pritchard, H., Friel, G., & Zeller, K. (1998). Apolipoprotein A-I(Zavalla) (Leu159e>Pro): HDL cholesterol deficiency in a kindred associated with premature coronary artery disease. Arterioscler Thromb Vasc Biol, 18, 1242e1247. Miller, M., Rhyne, J., Hamlette, S., Birnbaum, J., & Rodriguez, A. (2003). Genetics of HDL regulation in humans. Curr Opin Lipidol, 14, 273e279. Minnich, A., Collet, X., Roghani, A., et al. (1992). Site-directed mutagenesis and structurefunction analysis of the human apolipoprotein A-I. Relation between lecithin-cholesterol acyltransferase activation and lipid binding. J Biol Chem, 267, 16553e16560. Moriyama, K., Sasaki, J., Arakawa, F., et al. (1995). Two novel point mutations in the lecithin: cholesterol acyltransferase (LCAT) gene resulting in LCAT deficiency: LCAT (G873 deletion) and LCAT (Gly344e>Ser). J Lipid Res, 36, 2329e2343. Moriyama, K., Sasaki, J., Matsunaga, A., & Arakawa, K. (1996a). Identification of two apolipoprotein variants, A-I Kaho (Asp 51e>Val) and A-I Lys 107 deletion. J Atheroscler Thromb, 3, 12e16. Moriyama, K., Sasaki, J., Matsunaga, A., Takada, Y., Kagimoto, M., & Arakawa, K. (1996b). Identification of two apolipoprotein variants, A-I Karatsu (Tyr 100e>His) and A-I Kurume (His 162e>Gln). Clin Genet, 49, 79e84. Moriyama, K., Sasaki, J., Takada, Y., et al. (1996). A cysteine-containing truncated apo A-I variant associated with HDL deficiency. Arterioscler Thromb Vasc Biol, 16, 1416e1423. Mucchiano, G. I., Haggqvist, B., Sletten, K., & Westermark, P. (2001). Apolipoprotein A-1derived amyloid in atherosclerotic plaques of the human aorta. J Pathol, 193, 270e275. Murphy, C. L., Wang, S., Weaver, K., Gertz, M. A., Weiss, D. T., & Solomon, A. (2004). Renal apolipoprotein A-I amyloidosis associated with a novel mutant Leu64Pro. Am J Kidney Dis, 44, 1103e1109. Nakata, K., Kobayashi, K., Yanagi, H., et al. (1993). Autosomal dominant hypoalphalipoproteinemia due to a completely defective apolipoprotein A-I gene. Biochem Biophys Res Commun, 196, 950e955. Newton, R. S., & Krause, B. R. (2002). HDL therapy for the acute treatment of atherosclerosis. Atheroscler Suppl, 3, 31e38. Ng, D. S., Leiter, L. A., Vezina, C., Connelly, P. W., & Hegele, R. A. (1994). Apolipoprotein A-I Q (-2)X causing isolated apolipoprotein A-I deficiency in a family with analphalipoproteinemia. J Clin Invest, 93, 223e229. Nichols, W. C., Dwulet, F. E., Liepnieks, J., & Benson, M. D. (1988). Variant apolipoprotein AI as a major constituent of a human hereditary amyloid. Biochem Biophys Res Commun, 156, 762e768.

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Nissen, S. E., Tsunoda, T., Tuzcu, E. M., et al. (2003). Effect of recombinant ApoA-I Milano on coronary atherosclerosis in patients with acute coronary syndromes: a randomized controlled trial. J Am Med Assoc, 290, 2292e2300. Nolte, R. T., & Atkinson, D. (1992). Conformational analysis of apolipoprotein A-I and E-3 based on primary sequence and circular dichroism. Biophys J, 63, 1221e1239. Norum, R. A., Lakier, J. B., Goldstein, S., et al. (1982). Familial deficiency of apolipoproteins A-I and C-III and precocious coronary-artery disease. N Engl J Med, 306, 1513e1519. Obici, L., Bellotti, V., Mangione, P., et al. (1999). The new apolipoprotein A-I variant leu(174) e> Ser causes hereditary cardiac amyloidosis, and the amyloid fibrils are constituted by the 93-residue N-terminal polypeptide. Am J Pathol, 155, 695e702. Obici, L., Palladini, G., Giorgetti, S., et al. (2004). Liver biopsy discloses a new apolipoprotein A-I hereditary amyloidosis in several unrelated Italian families. Gastroenterology, 126, 1416e1422. Ordovas, J. M., Cassidy, D. K., Civeira, F., Bisgaier, C. L., & Schaefer, E. J. (1989). Familial apolipoprotein A-I, C-III, and A-IV deficiency and premature atherosclerosis due to deletion of a gene complex on chromosome 11. J Biol Chem, 264, 16339e16342. Panzenbock, U., & Stocker, R. (2005). Formation of methionine sulfoxide-containing specific forms of oxidized high-density lipoproteins. Biochim Biophys Acta, 1703, 171e181. Parks, J. S., Li, H., Gebre, A. K., Smith, T. L., & Maeda, N. (1995). Effect of apolipoprotein A-I deficiency on lecithin:cholesterol acyltransferase activation in mouse plasma. J Lipid Res, 36, 349e355. Parolini, C., Chiesa, G., Gong, E., et al. (2005). Apolipoprotein A-I and the molecular variant apoA-I(Milano): evaluation of the antiatherogenic effects in knock-in mouse model. Atherosclerosis, 183, 222e229. Paszty, C., Maeda, N., Verstuyft, J., & Rubin, E. M. (1994). Apolipoprotein AI transgene corrects apolipoprotein E deficiency-induced atherosclerosis in mice. J Clin Invest, 94, 899e903. Persey, M. R., Booth, D. R., Booth, S. E., et al. (1998). Hereditary nephropathic systemic amyloidosis caused by a novel variant apolipoprotein A-I. Kidney Int, 53, 276e281. Plump, A. S., Scott, C. J., & Breslow, J. L. (1994). Human apolipoprotein A-I gene expression increases high density lipoprotein and suppresses atherosclerosis in the apolipoprotein E-deficient mouse. Proc Natl Acad Sci USA, 91, 9607e9611. Rall, S. C., Jr., Weisgraber, K. H., Mahley, R. W., et al. (1984). Abnormal lecithin:cholesterol acyltransferase activation by a human apolipoprotein A-I variant in which a single lysine residue is deleted. J Biol Chem, 259, 10063e10070. Recalde, D., Velez-Carrasco, W., Civeira, F., et al. (2001). Enhanced fractional catabolic rate of apo A-I and apo A-II in heterozygous subjects for apo A-I(Zaragoza) (L144R). Atherosclerosis, 154, 613e623. Romling, R., von Eckardstein, A., Funke, H., et al. (1994). A nonsense mutation in the apolipoprotein A-I gene is associated with high-density lipoprotein deficiency and periorbital xanthelasmas. Arterioscler Thromb, 14, 1915e1922. Rubin, E. M., Krauss, R. M., Spangler, E. A., Verstuyft, J. G., & Clift, S. M. (1991). Inhibition of early atherogenesis in transgenic mice by human apolipoprotein AI. Nature, 353, 265e267. Schaefer, E. J., Ordovas, J. M., Law, S. W., et al. (1985). Familial apolipoprotein A-I and C-III deficiency, variant II. J Lipid Res, 26, 1089e1101. Shah, P. K., Nilsson, J., Kaul, S., et al. (1998). Effects of recombinant apolipoprotein A-I(Milano) on aortic atherosclerosis in apolipoprotein E-deficient mice. Circulation, 97, 780e785. Shah, P. K., Yano, J., Reyes, O., Chyu, K. Y., Kaul, S., Bisgaier, C. L., et al. (2001). High-dose recombinant apolipoprotein A-I(milano) mobilizes tissue cholesterol and rapidly reduces plaque lipid and macrophage content in apolipoprotein e-deficient mice. Potential implications for acute plaque stabilization. Circulation, 103, 3047e3050.

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Sorci-Thomas, M. G., & Thomas, M. J. (2002). The effects of altered apolipoprotein A-I structure on plasma HDL concentration. Trends Cardiovasc Med, 12, 121e128. Sorci-Thomas, M., Kearns, M. W., & Lee, J. P. (1993). Apolipoprotein A-I domains involved in lecithin-cholesterol acyltransferase activation. Structure:function relationships. J Biol Chem, 268, 21403e21409. Sorci-Thomas, M. G., Thomas, M., Curtiss, L., & Landrum, M. (2000). Single repeat deletion in apoA-I blocks cholesterol esterification and results in rapid catabolism of delta6 and wild-type apoA-I in transgenic mice. J Biol Chem, 275, 12156e12163. Soutar, A. K., Hawkins, P. N., Vigushin, D. M., et al. (1992). Apolipoprotein AI mutation Arg-60 causes autosomal dominant amyloidosis. Proc Natl Acad Sci USA, 89, 7389e7393. Strobl, W., Jabs, H. U., Hayde, M., Holzinger, T., Assmann, G., & Widhalm, K. (1988). Apolipoprotein A-I (Glu 198e>Lys): a mutant of the major apolipoprotein of high-density lipoproteins occurring in a family with dyslipoproteinemia. Pediatr Res, 24, 222e228. Sviridov, D., Pyle, L. E., & Fidge, N. (1996). Efflux of cellular cholesterol and phospholipid to apolipoprotein A-I mutants. J Biol Chem, 271, 33277e33283. Takada, Y., Sasaki, J., Ogata, S., Nakanishi, T., Ikehara, Y., & Arakawa, K. (1990). Isolation and characterization of human apolipoprotein A-I Fukuoka (110 Glue>Lys). A novel apolipoprotein variant. Biochim Biophys Acta, 1043, 169e176. Takada, Y., Sasaki, J., Seki, M., Ogata, S., Teranishi, Y., & Arakawa, K. (1991). Characterization of a new human apolipoprotein A-I Yame by direct sequencing of polymerase chain reactionamplified DNA. J Lipid Res, 32, 1275e1280. Takata, K., Saku, K., Ohta, T., et al. (1995). A new case of apoA-I deficiency showing codon 8 nonsense mutation of the apoA-I gene without evidence of coronary heart disease. Arterioscler Thromb Vasc Biol, 15, 1866e1874. Tilly-Kiesi, M., Zhang, Q., Ehnholm, S., et al. (1995). ApoA-IHelsinki (Lys107e>0) associated with reduced HDL cholesterol and LpA-I:A-II deficiency. Arterioscler Thromb Vasc Biol, 15, 1294e1306. Uehara, Y., Tsuboi, Y., Zhang, B., et al. (2007). POPC/apoA-I discs as a potent lipoprotein modulator in Tangier disease. Atherosclerosis, 197, 283e289. Utermann, G., Haas, J., Steinmetz, A., et al. (1984). Apolipoprotein A-IGiessen (Pro143e>Arg). A mutant that is defective in activating lecithin:cholesterol acyltransferase. Eur J Biochem, 144, 325e331. Van Allen, M. W., Frohlich, J. A., & Davis, J. R. (1969). Inherited predisposition to generalized amyloidosis. Clinical and pathological study of a family with neuropathy, nephropathy, and peptic ulcer. Neurology, 19, 10e25. von Eckardstein, A., Funke, H., Henke, A., Altland, K., Benninghoven, A., & Assmann, G. (1989). Apolipoprotein A-I variants. Naturally occurring substitutions of proline residues affect plasma concentration of apolipoprotein A-I. J Clin Invest, 84, 1722e1730. von Eckardstein, A., Funke, H., Walter, M., Altland, K., Benninghoven, A., & Assmann, G. (1990). Structural analysis of human apolipoprotein A-I variants. Amino acid substitutions are nonrandomly distributed throughout the apolipoprotein A-I primary structure. J Biol Chem, 265, 8610e8617. von Eckardstein, A., Walter, M., Holz, H., Benninghoven, A., & Assmann, G. (1991). Site-specific methionine sulfoxide formation is the structural basis of chromatographic heterogeneity of apolipoproteins A-I, C-II, and C-III. J Lipid Res, 32, 1465e1476. Wang, L., Sharifi, B. G., Pan, T., Song, L., Yukht, A., & Shah, P. K. (2006). Bone marrow transplantation shows superior atheroprotective effects of gene therapy with apolipoprotein A-I Milano compared with wild-type apolipoprotein A-I in hyperlipidemic mice. J Am Coll Cardiol, 48, 1459e1468.

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Weisgraber, K. H., Bersot, T. P., Mahley, R. W., Franceschini, G., & Sirtori, C. R. (1980). A-Imilano apoprotein. Isolation and characterization of a cysteine-containing variant of the A-I apoprotein from human high density lipoproteins. J Clin Invest, 66, 901e907. Williamson, R., Lee, D., Hagaman, J., & Maeda, N. (1992). Marked reduction of high density lipoprotein cholesterol in mice genetically modified to lack apolipoprotein A-I. Proc Natl Acad Sci USA, 89, 7134e7138. Yamakawa-Kobayashi, K., Yanagi, H., Fukayama, H., et al. (1999). Frequent occurrence of hypoalphalipoproteinemia due to mutant apolipoprotein A-I gene in the population: a population-based survey. Hum Mol Genet, 8, 331e336. Yokota, H., Hashimoto, Y., Okubo, S., et al. (2002). Apolipoprotein A-I deficiency with accumulated risk for CHD but no symptoms of CHD. Atherosclerosis, 162, 399e407. Zannis, V. I., Chroni, A., & Krieger, M. (2006). Role of apoA-I, ABCA1, LCAT, and SR-BI in the biogenesis of HDL. J Mol Med, 84, 276e294.

Chapter 8

The Scavenger Receptor Class B Type I: An HDL Receptor Involved in Lipid Transport and HDL Dependent Signaling Aishah Al-Jarallah, Rachelle Brunet and Bernardo Trigatti Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada

OVERVIEW The scavenger receptor, class B, type I (SR-BI) is a palmitylated cell surface glycoprotein that binds high and low density lipoproteins (HDL and LDL) with high affinity. It was the first molecularly well defined HDL receptor that was shown to mediate the selective uptake of HDL lipids and HDL-dependent cholesterol efflux from cells. This chapter will begin with a discussion of the impact of studies from genetically engineered mice in revealing the significance of SR-BI in HDL metabolism, atherosclerosis and coronary heart disease. We will then discuss the cellular biology of SR-BI, its activities in lipid transfer between HDL and cells and more recent insights into its role in mediating the activation of cellular signaling pathways by HDL in various cell types, and their possible contribution to atheroprotection.

ROLE OF SR-BI IN HDL METABOLISM, ATHEROSCLEROSIS AND CORONARY HEART DISEASE e STUDIES FROM GENETICALLY ALTERED MICE The scavenger receptor, class B type I was first reported by Acton et al. (1996) to be a high affinity receptor for HDL that could mediate selective HDL lipid uptake into cells. Since then, studies of gene-targeted SR-BI knockout mice have shed light on its physiological role in HDL cholesterol clearance by the liver, driving reverse cholesterol transport, and its role in both liver and bone The HDL Handbook. ISBN: 978-0-12-382171-3 Copyright Ó 2010 Elsevier Inc. All rights of reproduction in any form reserved.

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marrow derived cells in protection against atherosclerosis and coronary heart disease. Mice that do not express SR-BI have increased plasma concentrations of cholesterol associated with enlarged HDL particles with increased unesterified cholesterol content and altered apolipoprotein composition (Rigotti et al., 1997; Trigatti et al., 1999; Miettinen et al., 2001; Braun et al., 2003; Van Eck et al., 2004). Hepatic concentrations of unesterified and esterified cholesterol are unchanged but biliary cholesterol is reduced (Trigatti et al., 1999; Mardones et al., 2001), consistent with studies demonstrating reduced hepatic clearance of HDL cholesterol from blood in mice with reduced hepatic SR-BI expression (Varban et al., 1998; Out et al., 2004; Brundert et al., 2005). In contrast, overexpression of SR-BI in livers of mice results in increased clearance of HDL cholesterol from blood and increased biliary secretion (Kozarsky et al., 1997; Wang et al., 1998; Ueda et al., 1999). Recent studies modeling reverse cholesterol transport from macrophages have demonstrated that hepatic SR-BI plays an important role in driving this process (Zhang et al., 2005b). Elimination of SR-BI expression also results in impaired cholesterol storage in steroidogenic cells in adrenals and ovaries (Rigotti et al., 1997; Trigatti et al., 1999) most likely due to the important role of SR-BI in HDL lipid uptake by steroidogenic cells (Temel et al., 1997). These studies and others demonstrated the important physiological role played by SR-BI in cellular uptake of HDL lipids including cholesterol ester, free cholesterol and bioactive lipids, such as a-tocopherol (vitamin E) (Mardones et al., 2002). Consistent with impaired reverse cholesterol transport (reduced hepatic HDL lipid uptake and secretion into bile), mice that are deficient in SR-BI are more susceptible to atherosclerosis. SR-BI KO mice exhibit increased development of early atherosclerotic plaques (fatty streaks) in the aortic sinus compared to wild type controls when fed atherogenic, high fat/high cholesterol diets (Van Eck et al., 2004; Zhang et al., 2005b). Similarly, mice deficient in both SR-BI and the LDL receptor (LDL-R) (SRBI/LDL-R double KO mice) develop increased levels of high fat diet induced atherosclerosis in the descending aorta compared to LDL-R single KO mice (Covey et al., 2003). SR-BI KO mice that are also deficient in apolipoprotein (apo) E (SR-BI/apoE dKO mice) develop greatly accelerated spontaneous aortic sinus atherosclerosis compared to apoE KO controls (Trigatti et al., 1999). Similar findings were reported in high fat diet fed SR-BI KO mice that contain a mutation (apoER61hypomorphic) that results in the reduced expression of a mutant form of apoE with impaired clearance (Raffai et al., 2001). The level of expression of the mutant form of apoE in these mice is only z5% of normal levels of apoE expression (Raffai et al., 2001; Raffai and Weisgraber, 2002). SR-BI/apoE dKO mice and high fat diet fed SR-BI KO/apoE-R61hypo mice also develop extensive, occlusive coronary artery disease, resulting in myocardial ischemia and infarction and which is associated with increased heart size, reduced heart function and death of mice within weeks of either birth (SR-BI/apoE dKO mice) or initiation of high fat diet feeding (SR-BI KO/apoE-R61hypo mice)

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(Braun et al., 2002, 2003; Zhang et al., 2005a; Karackattu et al., 2006). These findings suggest that SR-BI normally plays an important role in protection against the development of atherosclerosis in coronary arteries and that SR-BI KO/apoE mutant mice may represent novel models of human coronary heart disease. The important role of hepatic SR-BI in protection against atherosclerosis is illustrated by the ability of hepatic SR-BI overexpression to reduce atherosclerosis in homozygous and heterozygous LDL-R KO mice (Arai et al., 1999; Kozarsky et al., 2000). Moderate levels of hepatic SR-BI overexpression also protect against diet induced atherosclerosis in apoB transgenic mice (Arai et al., 1999; Kozarsky et al., 2000; Ueda et al., 2000). To test the role of SR-BI in bone marrow derived cells, including macrophages in atherosclerosis, we and others have used bone marrow transplantation to generate chimeric mice with specific disruption of the gene encoding SR-BI in bone marrow derived cells (Covey et al., 2003; Zhang et al., 2003; Van Eck et al., 2004). Replacement of bone marrow stem cells of LDL-R KO mice with those from SR-BI KO mice resulted in increased development of diet induced atherosclerosis compared to mice receiving wild type bone marrow (Covey et al., 2003; Van Eck et al., 2004). Similar results were obtained when wild type mice were used as recipients, although the atherosclerotic plaques were much smaller in size (Covey et al., 2003). Similarly, transplantation of bone marrow stem cells from SR-BI/apoE dKO mice into apoE single KO recipients resulted in increased levels of spontaneous aortic atherosclerosis compared to control mice transplanted with apoE KO mouse bone marrow stem cells (Zhang et al., 2003). These studies indicate that in addition to its role in the liver in driving reverse cholesterol transport, SR-BI plays an important atheroprotective role in bone marrow derived cells, possibly macrophages, in atherosclerotic plaques. Together, these studies suggest that SR-BI plays a key role in multiple tissues/cell types in normal HDL metabolism, and protection against atherosclerosis.

SR-BI MEDIATED HDL LIPID UPTAKE AND EFFLUX SR-BI mediates the selective uptake of cholesterol ester and free cholesterol from HDL in various tissues without the net internalization and degradation of the lipoprotein particle (Acton et al., 1996; Ji et al., 1999; Silver et al., 2001a; Brundert et al., 2005; Shetty et al., 2006; Sun et al., 2006). Selective uptake involves the movement of cholesterol from plasma HDL to various tissues, including the liver, without degradation of the lipoprotein particles (Glass et al., 1983; Stein et al., 1983). SR-BI mediated selective uptake requires lipoproteinreceptor binding and is followed by lipid transfer into the cell (Gu et al., 1998; Williams et al., 2000; Nieland et al., 2002; Temel et al., 2002). Although the mechanisms of lipid transfer remain largely unknown, recent studies suggest that it may occur on the cell surface and does not require internalization of the lipoprotein particles (Liu and Krieger, 2002; Nieland et al., 2005a; Eckhardt

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et al., 2006; Harder et al., 2007). The internalization of SR-BI may, on the other hand, represent a mechanism by which its activity can be regulated (Eckhardt et al., 2006; Zhang et al., 2007a,b). Overexpression of SR-BI in cultured cells also increases the efflux of cholesterol tracer to HDL or phospholipid vesicles (Ji et al., 1997; Jian et al., 1998). This has led to the idea that SR-BI can mediate the bidirectional flux of free cholesterol between cells and lipoprotein particles; the direction of net flux being determined by the concentration gradient of cholesterol between the cells and HDL particles (Yancey et al., 2003). In contrast to selective lipid uptake, cholesterol efflux appears to involve the prior internalization and re-secretion of the HDL particle (Stangl et al., 1998). The importance of SR-BI-mediated free cholesterol efflux from macrophages to HDL for macrophage reverse cholesterol transport and protection against atherosclerosis, however, is not clear. Macrophages from mice lacking SR-BI do not appear to have significantly reduced capacity to efflux cholesterol to HDL acceptors either when measured in vitro or in a reconstituted efflux assay in mice (Zhang et al., 2003; Van Eck et al., 2004; Wang et al., 2007). On the other hand, macrophages deficient in both SR-BI and apoE do exhibit altered cholesterol homeostasis: they have increased cellular levels of both free cholesterol and cholesteryl ester and accumulate free cholesterol further upon incubation with HDL (Yancey et al., 2007). Thus, SR-BI may be required for cholesterol efflux and proper cholesterol homeostasis in macrophages under certain physiological contexts, such as the absence of apoE.

CELLULAR BIOLOGY OF SR-BI: LOCALIZATION SR-BI is a 509 amino acid and 82 kDa protein made up of short amino- and carboxyterminal cytoplasmic domains, each situated next to a transmembrane domain, with a large, heavily glycosylated extracellular region in between (Acton et al., 1994; Babitt et al., 1997). It is expressed in a variety of cell types involved in metabolism and in the development of atherosclerosis. These include liver hepatocytes and intestinal enterocytes, adipocytes, macrophages, vascular smooth muscle cells, endothelial cells, and steroidogenic cells in the adrenal cortex, testes and ovaries (Acton et al., 1996; Landschulz et al., 1996; Hatzopoulos et al., 1998; Hirano et al., 1999; Yuhanna et al., 2001; Yeh et al., 2002; Nakagawa-Toyama et al., 2005). Immunohistochemistry and immunofluorescence analyses of macrophages from cultured human monocytes or in sections of human atherosclerotic plaques and Kupffer cells in sections of human liver demonstrated that SR-BI is distributed in a heterogeneous punctate pattern (Hirano et al., 1999; Nakagawa-Toyama et al., 2005). Immunofluorescent detection of SR-BI in ldlA7 cells (Chinese hamster ovary (CHO) cells deficient in the LDL-R) stably expressing murine SR-BI (mSR-BI), and in Y1-BS1 murine adrenocortical cells also shows a heterogeneous, punctate as well as a cell peripheral distribution of the receptor (Babitt et al., 1997). Fluorescence microscopy images of CHO cells transfected with enhanced

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green fluorescent protein (EGFP) tagged mSR-BI reveal a similar localization pattern (Eckhardt et al., 2004; Nieland et al., 2005b; Zhang et al., 2007a). However, immunofluorescence and immunohistochemical staining of sections of rat and human adrenal glands revealed a cell peripheral distribution of SR-BI (Landschulz et al., 1996; Nakagawa-Toyama et al., 2005). In 3T3-L1 adipocytes, SR-BI immunoreactivity was distributed in a punctate pattern throughout the cytoplasm and scarce cell surface labeling was reported (Tondu et al., 2005). Similarly, immunoelectronmicroscopy analyses of SR-BI in smooth muscle cells of rat aortic sections revealed that, although some cell-surface localization was observed, SR-BI was largely found in the cytoplasm (Yeh et al., 2002). Research conducted on human intestinal Caco 2 cells showed that SR-BI immunoreactivity was localized to the apical and basolateral surfaces of cells (Cai et al., 2001). In addition, immunofluorescence analyses of sections of intestine from mice revealed scarce SR-BI localization in the apical and basolateral membranes of the ileum, distribution of the receptor to both the apical and basolateral membranes in the jejunum, and strong SR-BI immunoreactivity in the apical membrane of the brush border of the duodenum (Cai et al., 2001). SR-BI in transfected CHO-derived ldlA7cells and in human microvascular endothelial cells co-fractionates and/or co-immunoprecipitates with caveolin-1, indicating that the receptor is localized to plasma membrane caveolae (Babitt et al., 1997; Uittenbogaard et al., 2000). Furthermore, in endothelial cells of rat aortic sections, immuno-electron microscopy analyses showed that SR-BI was distributed on the luminal and adluminal surfaces of the cell, but was not localized to the cytoplasm (Yeh et al., 2002). The localization of SR-BI has also been studied in rat, murine and human hepatocytes. Isolation of apical and basolateral membranes from rat livers, followed by immunoblotting for SR-BI revealed that it was localized to both the apical and basolateral surfaces of the cell (Harder et al., 2007). Immunofluorescence and immunohistochemical analyses of mouse liver sections have suggested apical or basolateral distributions in different studies (Sehayek et al., 2003; Yesilaltay et al., 2006). Analyses of primary murine hepatocyte couplets by immunofluorescence microscopy for SR-BI suggested it was localized to the apical membrane and co-localized with transferrin in the endosomal recycling compartment (ERC) (Silver et al., 2001b). In WIF-B cells, a model of polarized hepatocytes, yellow fluorescent protein (YFP)-tagged recombinant human SRBI localized to the apical and the basolateral membranes (Harder et al., 2007). Some of these apparent inconsistencies in the subcellular localization of SR-BI in different tissues and/or different studies may be explained by the dynamics of its localization. Research in both polarized Madin-Darby canine kidney (MDCK) cells and polarized hepatocytes revealed that SR-BI on the basolateral cell surface is continually internalized and recycled (Silver et al., 2001b; Burgos et al., 2004; Harder et al., 2007). Furthermore, recycling back

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to the basolateral membrane versus trafficking to the apical cell surface appears to be dependent on the cellular cholesterol content and regulated by protein kinase A (Burgos et al., 2004; Harder et al., 2007). Depletion of cellular cholesterol with cyclodextrin (CD) in polarized MDCK cells has been reported to divert the trafficking of internalized basolateral SR-BI from recycling to transcytosis to the apical cell surface via a pathway involving protein kinase A (PKA) (Burgos et al., 2004). On the other hand, cholesterol depletion in WIF-B cells resulted in the redistribution of SR-BI away from the apical (canalicular) membrane to an entirely basolateral localization (Harder et al., 2007). A similar process may take place in non-polarized cells such as adipocytes and transfected CHO-derived cells (Tondu et al., 2005; Zhang et al., 2007a), where SR-BI appears to be continually internalized and recycled back to the cell surface. In these cells, and in hepatocytes, SR-BI’s recycling back to the cell surface and possibly also its internalization appear to be regulated by insulin, angiotensin II (Ang II) and/or HDL via the phosphatidyl inositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway (Tondu et al., 2005; Shetty et al., 2006; Zhang et al., 2007a). PI3K plays a major role in endosomal membrane trafficking which involves the recruitment of regulatory proteins to the plasma membrane and the endocytic uptake and recycling of receptors (Backer, 2000; Covera, 2001). The involvement of PI3K in regulating the recruitment of SR-BI to the plasma membrane in adipocytes and hepatocytes was demonstrated using wortmannin, a relatively specific PI3K inhibitor. This abolished Ang II and insulin induced SR-BI recruitment to the cell surface in adipocytes and hepatocytes as well as the HDL-dependent cell surface localization of SR-BI in hepatocytes and CHOderived cells (Tondu et al., 2005; Shetty et al., 2006; Zhang et al., 2007a). These alterations in the levels of cell surface SR-BI in wortmannin-treated cells were associated with reduced SR-BI activity, suggesting a physiological role for this pathway in regulating selective HDL lipid uptake and reverse cholesterol transport (Tondu et al., 2005; Shetty et al., 2006; Yvan-Charvet et al., 2007; Zhang et al., 2007a). Therefore, SR-BI internalization and resecretion appear to present opportunities for regulation of SR-BI activity. SR-BII, a splice variant of SR-BI with identical extracellular regions, N and C-terminal transmembrane segments and N-terminal cytoplasmic region but a different C-terminal cytoplasmic region, exhibits lower lipid uptake activity, related to its lower steady state level on the cell surface (Webb et al., 1998). SR-BII contains a di-Leu endocytic motif in its C-terminal cytoplasmic region and is efficiently endocytosed (Webb et al., 1998; Eckhardt et al., 2006). Transfer of the di-Leu motif from SR-BII’s cytoplasmic tail to SR-BI, which contains no identifiable endocytic motif (Zhang et al., 2007b) converts SR-BI into an endocytic receptor, resulting in a decrease in its selective uptake activity (Eckhardt et al., 2004, 2006). Indeed, unlike mouse SR-BI, disruption of endocytosis in cells expressing human

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SR-BI appears to alter its selective uptake activity, possibly by altering the steady state level of SR-BI on the cell surface (Zhang at al., 2007b). Therefore, regulation of the recycling of SR-BI may be an important means of regulating cellular HDL lipid uptake and/or efflux.

SR-BI-MEDIATED ENDOCYTOSIS OF HDL In addition to selective HDL lipid uptake, SR-BI also mediates the endocytic uptake of HDL (Silver et al., 2001b; Burgos et al., 2004; Rhode et al., 2004; Wu¨stner et al., 2004; Nieland et al., 2005a; Pagler et al., 2006; Sun et al., 2006). The endocytosis of HDL is not blocked by inhibitors of SR-BI-mediated selective lipid uptake, such as blockers of lipid transport-1 and 4 (BLT-1 and BLT-4) or glyburide (Nieland et al., 2005a; Pagler et al., 2006; Sun et al., 2006). However, SR-BI mediated internalization of HDL is reduced by general inhibitors of endocytosis, such as hypertonic sucrose, potassium depletion or disruption of the actin cytoskeleton (Silver et al., 2001b; Nieland et al., 2005a; Harder et al., 2006; Zhang et al., 2007a). HDL endocytosis in CHO cells expressing SR-BI is not inhibited by the expression of either a dominant negative dynamin-1 mutant, which inhibits formation of clathrin-coated vesicles, or a dominant negative caveolin-1 mutant that disrupts caveolae-dependent endocytosis (Silver et al., 2001b). These results suggest that SR-BImediated endocytosis of HDL initially bound at the plasma membrane may occur by a pathway that is independent of either clathrin coated pits or caveolae. HDL particles and SR-BI are internalized and accumulate in perinuclear vesicles, that also accumulate transferrin, suggesting they comprise the ERC (Silver et al., 2001b; Rhode et al., 2004; Nieland et al., 2005a; Sun et al., 2006). In addition, fluorescently labeled SR-BI and HDL particles colocalize to the cell surface, and endocytosis of SR-BI occurs along a similar route and with similar velocity as that of HDL (Pagler et al., 2006). HDL particles internalized by endocytosis in an SR-BI dependent manner are resecreted from both polarized and non-polarized cells (Silver et al., 2001b; Rhode et al., 2004; Pagler et al., 2006; Sun et al., 2006). In polarized MDCK cells, cholesteryl ester and free cholesterol components of HDL, internalized via SR-BI from the basolateral membrane, are selectively sorted away from the HDL particle and secreted at the apical membrane, while the remaining HDL particle is re-secreted at the basolateral membrane (Silver et al., 2001). In contrast, in CHO-derived cells and in THP-1 macrophages, there is evidence that HDL is internalized, loaded with cholesterol during recycling, and then re-secreted, and that HDL internalization and re-secretion are required for SR-BI-mediated cholesterol efflux to HDL from cells (Pagler et al., 2006). These data suggest that SR-BI does mediate the internalization of HDL and possibly its shuttling, in both non-polarized and polarized cells.

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MULTIMERIZATION OF SR-BI SR-BI appears to exist as a monomer in cells and also forms dimers and possibly tetramers (Reaven et al., 2004; Sahoo et al., 2006, 2007). This was shown by co-expressing recombinant SR-BI fused to two different epitope tags in the same cells followed by immunoprecipitation of both tagged versions of SR-BI with an antibody specific to only one or the other epitope tag. To ensure that SR-BI molecules multimerize within the cells themselves, Sahoo and coworkers (2007) used fluorescence resonance energy transfer (FRET) to detect SR-BI dimerization in live cells in vivo. For these experiments, recombinant SR-BI tagged with either yellow or cyan fluorescent proteins (YFP or CFP) on their amino or carboxy termini were co-expressed in Cos-7 cells (Sahoo et al., 2007). Live cell FRET signals were detected only when both constructs encoded SR-BI tagged on the C-termini, indicating that the C-termini of SR-BI monomers were within 10 nm of one another (Sahoo et al., 2007). Since it has previously been shown that mutant SR-BI lacking its C-terminal tail can form oligomers, it has been proposed that oligomerization of the protein is mediated by the carboxy-terminal transmembrane domain or a portion of the extracellular loop in close proximity to this domain (Sahoo et al., 2006, 2007). The importance of SR-BI’s homo-oligomerization for its function is not currently known.

PDZK1, AN ADAPTOR PROTEIN THAT BINDS TO SR-BI’S CARBOXY TERMINAL CYTOPLASMIC TAIL PDZK1 is a 519 amino acid, 63 kDa adapter protein that contains four PDZ (postsynaptic density protein (PSD-95)/Drosophila discs-large (dlg)/tightjunction protein (ZO1)) proteineprotein interaction domains (Kocher et al., 1998; Silver, 2002; Yesilaltay et al., 2005). SR-BI interacts with PDZK1 through the last three amino acids, Ala-Lys-Leu, of its C-terminal tail (Ikemoto et al., 2000; Silver, 2002). Deletion of these three amino acids, or even the C-terminal amino acid, Leu, prevents the interaction between SR-BI and PDZK1 (Silver, 2002). Transgenic mice overexpressing a mutant SR-BI unable to interact with PDZK1, exhibited striking increases in SR-BI mRNA levels but only modest increases in SR-BI protein in the liver and no changes in plasma total cholesterol concentrations (Silver, 2002). PDZK1 KO mice, like SR-BI KO mice, exhibit increased plasma total cholesterol levels associated with HDL particles of increased size (Kocher et al., 2003; Yesilaltay et al., 2006). This is because these mice exhibit a 95% reduction in the level of SR-BI protein in the liver (Yesilaltay et al., 2006). They also exhibit a 50% reduction in SR-BI protein in small intestine but normal levels of SR-BI protein in steroidogenic tissues (Yesilaltay et al., 2006). Overexpression of wild-type SR-BI in livers of PDZK1 KO mice leads to the production of functional cell surface-localized SR-BI, and restores normal steady state HDL cholesterol levels and HDL

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composition, despite the PDZK1 deficiency (Yesilaltay et al., 2006). These studies indicate that PDZK1 stabilizes SR-BI protein, protecting it from degradation, but does not affect its function or cell surface localization in liver hepatocytes (Yesilaltay et al., 2006). The degradation of SR-BI in mouse liver hepatocytes is stimulated by fibrates, dependent on PPARa and requires neither lysosomal proteases, calpain nor the proteasome (Mardones et al., 2003; Lan and Silver, 2005). Fibrates also stimulate the turnover of PDZK1 but this does not appear to be the trigger for SRBI turnover (Mardones et al., 2003; Lan and Silver, 2005). Together, these studies suggest that PDZK1 protects SR-BI from degradation in the liver. Regulation of PDZK1 may therefore represent another step at which SR-BI and reverse cholesterol transport can be regulated (Namgaladze and Brune, 2006). PDZK1 is also expressed in endothelial cells. Unlike liver hepatocytes, reduction of PDZK1 levels in endothelial cells does not increase the degradation of SR-BI (Kimura et al., 2006). Instead, SR-BI-mediated HDL induction of intracellular signaling appears to be impaired in the absence of PDZK1 in endothelial cells (Kimura et al., 2006). This will be discussed further below.

REGULATION OF SR-BI SR-BI expression is regulated by a variety of hormonal, metabolic, dietary, and pharmacological stimuli. The hormones estrogen (Stangl et al., 2002; Srivastava, 2003), adrenocorticotrophic hormone (Sun et al., 1999), Ang II (Yvan-Charvet et al., 2007; Yu et al., 2007) and insulin (Sporstol et al., 2007; Yvan-Charvet et al., 2007) have been shown to alter expression of SR-BI transcripts. Dietary cholesterol (Fluiter et al., 1998; Mardones et al., 2001), polyunsaturated fatty acids (Spady et al., 1999), saturated fatty acids (Loison et al., 2002), and vitamins A (Jeyakumar et al., 2006) and E (Witt et al., 2000) were also shown to either up- or downregulate SR-BI transcript and/or protein levels. The regulation of SR-BI gene expression in response to proinflammatory mediators such as lipopolyscaccharides (LPS), tumor necrosis factor (TNF)-a and inferon-g were also reported in various cell types (Buechler et al., 1999; Khovidhunkit et al., 2001). SR-BI gene regulation has been recently reviewed elsewhere (Rigotti et al., 2003; Rhainds and Brissette, 2004).

MOLECULAR MECHANISMS OF HDL-MEDIATED SIGNALING In addition to its role in cholesterol metabolism, HDL initiates signaling events in various cell types relevant to atherogenesis such as endothelial and vascular smooth muscle cells (VSMC), monocytes and platelets (Nofer et al., 2002; Grewal et al., 2003). These signaling events include the activation of phospholipases C and D (Walter et al., 1995), protein kinase C (PKC) (Mendez et al., 1991; Nofer et al., 1998; Grewal et al., 2003), mitogen

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activated protein kinases (MAPKs) (Deeg et al., 1997; Han et al., 2001) and PI3K/Akt (Li et al., 2002a; Mineo et al., 2003; Cao et al., 2004), the release of intracellular calcium (Porn et al., 1991), the phosphorylation of cellular proteins, the production of cyclic AMP (cAMP), ceramide and nitric oxide (NO) (Li et al., 2002a). HDL signaling has been most extensively studied in endothelial cells (Kimura et al., 2003; Mineo and Shaul, 2003; Mineo et al., 2003; Li et al., 2005). Based on their extensive research in this field, Shaul and his colleagues proposed a model for HDL-induced signaling in endothelial cells (Mineo et al., 2006). They have suggested that cholesterol flux is required for HDL-induced signal initiation and that SR-BI acts as a cholesterol sensor in the plasma membrane. This was based on a number of observations: Cholesterol depletion with CD induced endothelial NO synthase (eNOS) activation in bovine aortic endothelial cells (BAECs) while cholesterol loaded CD did not. An SR-BI neutralizing antibody that prevents SR-BI-mediated cholesterol efflux inhibited signaling by native HDL. Enrichment of HDL with phosphatidylcholine, which increases the efflux rate, resulted in enhanced signaling (Assanasen et al., 2005). It also has been shown that the C-terminal transmembrane and the Cterminal PDZK1 interacting domains of SR-BI are both required for HDL signaling and it has been suggested that perturbations in cholesterol binding to SR-BI results in conformational change(s) that modify its association with PDZK1 or other proteins critical for downstream signaling (Assanasen et al., 2005). The involvement of PDZK1 was demonstrated both by the inability of a mutant form of SR-BI, incapable of interacting with PDZK1, to mediate eNOS activation by HDL, and by knocking down PDZK1 using RNAi, which disrupted HDL-dependent eNOS activation (Assanasen et al., 2005; Kimura et al., 2006), reviewed in Mineo et al. (2006). Although HDL-dependent signaling in some instances has been linked to HDL-associated bioactive lipids, including ceramide (Li et al., 2002b; Nofer et al., 2004), estrogen (Gong et al., 2003), sphingosine-1-phosphate (Kimura et al., 2003, 2006; Nofer et al., 2004; Argraves and Argraves, 2007), and lysophospholipids (Nofer et al., 2004), it is also clear that they are not necessary since signaling can be triggered by reconstituted HDL consisting only of apoA1, phospholipids and cholesterol (Assanasen et al., 2005), reviewed in Mineo et al. (2006). This is consistent with the idea that HDL-dependent signaling may be the result of alterations in membrane cholesterol (Assanasen et al., 2005), reviewed in Mineo et al. (2006).

PROTEIN KINASE C (PKC) PATHWAY The PKC enzyme family regulates a variety of cellular responses, including cell proliferation, differentiation, apoptosis and cell metabolism, reviewed in Dempsey et al. (2000). There are three categories of PKC isoforms that are grouped based on their co-factor requirements. Classical PKCs (a, b and g)

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require both diacylgylerol (DAG) and Ca2þ ions for their complete activation; novel PKCs (d, 3, h and q) require only DAG; atypical PKCs (z and i/l) are activated independently of either DAG or Ca2þ (Farhadi et al., 2006). HDL induces the activation of PKC which stimulates cholesterol mobilization in human skin fibroblasts (Walter et al., 1995; Francis et al., 1996). HDL induces the hydrolysis of phosphatidylcholine and phosphatidylinositol bisphosphate by phospholipases C and D generating DAG, phosphatidic acid and inositol phosphates (Walter et al., 1995). Mendenz et al. (1991) showed that the binding of HDL3 to cholesterol loaded fibroblasts stimulated the translocation of PKC to the cell membrane and that downregulation of PKC with sphingosine, a PKC inhibitor, or the long-term treatment with phorbol ester, inhibited HDL-induced cholesterol translocation and efflux. Furthermore, PKC activation, in rat VSMCs, has been shown to be a prerequisite for mobilization of intracellular cholesterol to a pool readily available for apolipoprotein-mediated efflux (Li and Yokoyama, 1995). HDL has been shown to increase intracellular calcium concentrations in CHO cells (Grewal et al., 2005), human skin fibroblasts (Porn et al., 1991; Nofer et al., 2000), smooth muscle cells (Bochkov et al., 1992) and endothelial cells (Honda et al., 1999; Nofer et al., 2004). Elevation of intracellular calcium upon HDL stimulation should result in inducible targeting of PKCa to the plasma membrane (Rentero et al., 2006). This may partially explain the PKCdependent activation of Raf-1/MEK/MAPK upon HDL-SR-BI interaction in annexin A6 overexpressing CHO cells (Rentero et al., 2006). Pilon et al. (2003) reported that SR-BI expression is increased in human NCI-H295R adrenocortical cell line in response to PKC activators, resulting in higher lipoprotein binding and specific cholesteryl ester uptake utilized for steroidogenesis. HDLdependent activation of PKC, mediated by HDL binding to SR-BI, in transfected CHO-derived cells, may also directly modulate the activity of SR-BI. This is based on the finding that a phorbol ester activator of PKC increased, while a PKC inhibitor, Ro31-8220, reduced SR-BI-dependent selective HDL lipid uptake, without affecting either the subcellular distribution or amount of SR-BI protein (Zhang et al., 2007a). Thus, SR-BI may be both a mediator and target of HDL-dependent PKC activation.

MITOGEN ACTIVATED PROTEIN KINASE (MAPK) PATHWAY HDL stimulates the proliferation of several different cell types, including VSMCs (Nofer et al., 2001), adrenocortical cells (Murao et al., 2006), endothelial cells (Nofer et al., 2002) and human lymphocytes (Cuthbert and Lipsky, 1989). HDL’s proliferative effects may be mediated by supply of nutrients such as cholesterol and fatty acids (reviewed in Von Eckardstein and Assmann, 2000) or by induction of specific signaling cascades. The mitogenic effects of HDL in the absence of growth factors such as insulin, fibroblast growth factor or fetal bovine serum were clearly demonstrated in VSMCs (Nofer et al., 2001).

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In these cells, HDL increased the proportion of cells in S and M phases of the cell cycle and decreased the number of cells in G0/G1 phase; it also induced the phosphorylation of Rb and the sequential expression of cyclins D1, A and E (Nofer et al., 2001). HDL itself is a strong mitogen and is capable of inducing cell proliferation via the activation of Raf-1, MEK1, and ERK1/2 components of the MAPK cascade (Nofer et al., 2001). Additionally, HDL increased the expression of c-fos, the downstream target of ERK1/2 in mitogen induced-cell proliferation. Although PKC can phosphorylate and activate Raf-1 (Kolch et al., 1993), it seems that HDL-dependent activation of MAPK and subsequent cell proliferation of VSMC and fibroblasts occurs in a PKC-independent manner and involves a pertussis toxin (PTX)-sensitive G-protein (Deeg et al., 1997; Nofer et al., 2001; Grewal et al., 2003). The contribution of p21ras (Ras), one of the best characterized activators of the Raf-1/MAPK pathway, to HDLinduced activation of the MAPK pathway was reported in CHO cells (Grewal et al., 2003). It was shown that the binding of HDL to SR-BI activates Ras in a PKC-independent manner with consequent activation of the MAPK cascade (Grewal et al., 2003). Han and colleagues (2002) have shown that HDL stimulates the phosphorylation of p42 and p44 components of the MAPK cascade in RAW264.7 cells and the incubation of J774 macrophages with CD resulted in the phosphorylation of these isoforms suggesting that the removal of cholesterol, not the addition of lipids or lipoproteins, is the key element in HDLmediated signaling. The rapid activation of ERK1/2 and p38 components of the MAPK pathway was also demonstrated in cells overexpressing human SR-BI after incubation with HDL (Baranova et al., 2005; Zhang et al., 2007a). The activation of p38 MAPK has been reported to be involved in the reorganization of the actin cytoskeleton and filamentous actin polymerization (Schafer et al., 1998; Wang and Doerschuk, 2001). This in turn could suggest the involvement of SR-BI in intracellular actin trafficking upon HDL binding (Baranova et al., 2005). Norata et al. (2004) have shown that HDL3 induces the expression of cyclooxygenase-2 and the subsequent production of prostacyclin in endothelial cells in a p38 MAPK/CREB-dependent manner. Finally, increased expression of c-fos, c-myc and erg-1, the ultimate nuclear effectors of the cascade, was detected in smooth muscle cells upon HDL stimulation in the absence of serum (Nofer et al., 2001). Reports identifying the mitogenic components of HDL are conflicting. Nofer et al. (2001) showed that neither apoA-I nor apoA-II alone are capable of inducing DNA synthesis or cell proliferation in VSMCs. Still, others showed that apoA-I and apoC-I components of HDL induce the growth of endothelial, lymphoblastic and adenocarcinoma cells (Favre et al., 1993). In contrast, others reported apoA-I and apoE inhibition of growth factor-induced proliferation and cell cycle progression in VSMCs and lymphocytes (Ishigami et al., 1998). On the other hand, sphingosylphosphorylcholine and lysosulfatide, bioactive lysosphingolipids present in HDL, were shown to exert strong mitogenic activities, activate phosphoinositide-specific phospholipase C and liberate

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intracellular calcium (Nofer et al., 2000). These suggest that the lipid components of HDL may account for its mitogenic properties (Guha and Mackman, 2002).

PI3K/AKT PATHWAY PI3K is a ubiquitous lipid kinase that regulates the activity of various signaling cascades (Guha and Mackman, 2002). PI3K is a heterodimeric protein that consists of a regulatory, p85, and a catalytic, p110 subunit, which comprise Class IA isoforms of PI3K (Jones, 2000). The p110a and p110b are widely distributed in mammalian tissues; however, the p110d is usually restricted to leukocytes (Jones, 2000). A single Class IB variant containing the p110g catalytic subunit complexed with a 101-kDa regulatory subunit is thought to interact with G-proteins in leukocytes (Jones, 2000). In contrast, however, Class IA PI3Ks are activated by phosphorylation of tyrosine residues on the Src homology 2 domain of the regulatory subunit (Guha and Mackman, 2002). Phosphorylation stimulates docking of PI3K to the plasma membrane and results in allosteric modifications that increase its catalytic activity (Scheid and Woodgett, 2002). Activated PI3K phosphorylates membrane phosopholipids producing phosphatidylinositol-3,4,5-trisphosphate (PIP3) and phosphatidylinositol-3,4-bisphosphate (PIP2) in the plasma membrane. These membrane lipids stimulate the recruitment of protein kinase B/Akt, phosphatidylinositoldependent kinase 1 (PDK1) and other kinases which contain pleckstrin homology (PH) domains that mediate their binding to PIP2 and PIP3 (Klippel et al., 1997). PDK1 and other kinases phosphorylate Akt’s critical serine residues (Ser473 in Akt1) in their regulatory domains and threonine residues (Thr308 in Akt1) in their catalytic domains (Osaki et al., 2004), activating the Akt kinase (Franke et al., 1997). Like the MAPK pathway, HDL stimulation of PI3K/Akt has been extensively studied in endothelial cells. HDLeSR-BI interaction has been shown to activate the PI3K/Akt pathway and regulates endothelial cell apoptosis, proliferation and migration (Seetharam et al., 2006) (reviewed in Mineo et al., 2006). HDL has been shown to stimulate NO production via the activation of PI3K (Mineo and Shaul, 2003). Both receptor and non-receptor tyrosine kinases (Src) are involved in the activation of PI3K (reviewed in Mineo et al., 2006). Active PI3K induces the independent activation of both Akt and MAPK pathways (Mineo and Shaul, 2003). Akt phosphorylates eNOS at Ser1179, however, the molecular basis of MAPK mediated activation of eNOS is currently unclear (Mineo and Shaul, 2003). Unlike the phosphorylation at Ser1179, the phosphorylation at Thr497 attenuates enzyme activity and it has been reported that the effects of MAPK on eNOS can either be positive or negative (reviewed in Mineo et al., 2006). Evidence of HDL activation of these signaling pathways and the subsequent production of NO came from the use of dominant negative constructs, blocking antibodies and pharmacological antagonists that inhibit the activity of key signaling

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molecules. Dominant-negative PI3K and Akt inhibited both HDL-mediated phosphorylation and activation of the enzyme (Mineo et al., 2003). In addition, MEK inhibition by PD98059 blocked eNOS activation by HDL without affecting Akt or Ser-1179 phosphorylation (Mineo et al., 2003). These results suggest that HDL stimulates eNOS through a common upstream mediator, Src or PI3K, which leads to a parallel activation of Akt and MAPK and an independent modulation of the enzyme (Mineo et al., 2003). Deletion of the PDZK1 interaction domain on SR-BI or knocking down PDZK1 in endothelial cells prevented HDL-dependent eNOS activation suggesting that PDZK1 is essential for SR-BI-mediated signaling, possibly by acting as a scaffold linking the receptor to the downstream kinases required for the activation of eNOS by HDL (Assanasen et al., 2005; Kimura et al., 2006). Studies that investigated HDL components involved in the activation of eNOS revealed that apoA-I is necessary but not sufficient for eNOS stimulation (Mineo and Shaul, 2003). Evidence came from experiments in which anti-apoA-I antibodies inhibited HDL-induced eNOS activation, however, lipid free apoA-I did not activate eNOS (Yu et al., 2007). Recently, Yu et al. (2007) investigated the effects of Ang II on SR-BI expression in endothelial cells. Ang II decreased SR-BI protein and mRNA expression in human umbilical cord endothelial cells (HUVECs) (Yu et al., 2007). In addition, Ang II inhibited the activity of the human SR-BI promoter in a dose-dependent manner that was sensitive to the PI3K inhibitors, wortmannin and LY294002 (Yu et al., 2007). Therefore, PI3K appears to mediate Ang II-induced inhibition of SR-BI expression. The involvement of PI3K/Akt pathway was further confirmed by examining the phosphorylation state of Akt and by examining the response to Ang II treatment in the context of expression of either a dominant negative (DN) or a constitutively active form of Akt. Ag-II induced a time dependent phosporylation of Akt that was evident within 5 min and peaked at 15 min (Yu et al., 2007). The constitutively active Akt suppressed human SR-BI promoter activity in HUVECs, while the DN-Akt abolished the actions of Ang II (Yu et al., 2007). The involvement of FoxO1, a downstream target of Akt, was also examined. FoxO is a subfamily of forkhead transcription factors that when phosphorylated by Akt are excluded from the nucleus resulting in the inhibition of the transcription of their target genes (Anderson et al., 1998; Brunet et al., 1999). Ang II induced a time dependent phosphorylation of FoxO1. Furthermore, the mutation of the FoxO1 binding site blocked the effect of Ang II on the human SR-BI promoter activity, confirming the involvement of the PI3K/Akt/FoxO1 pathway in Ang II-mediated suppression of SR-BI gene expression (Yu et al., 2007). As discussed earlier, Ang II, insulin and HDL all stimulate the recruitment of SR-BI to the cell surface in a PI3K/ Akt dependent manner in hepatocytes, adipocytes and CHO cells (Tondu et al., 2005; Shetty et al., 2006; Zhang et al., 2007a). If this also occurs in endothelial cells, then SR-BI may be subject to differential short- and long-term regulation. On the other hand, Ang II results in upregulation of SR-BI gene expression in

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adipocytes, contrasting with the downregulation in endothelial cells (Yu et al., 2007; Yvan-Charvet et al., 2007). Whether the differential cell type specific regulation of SR-BI gene expression in response to the same hormonal stimuli is also reflected in differential regulation of its subcellular distribution remains to be tested. It has also been shown that HDL signaling through SR-BI promotes endothelial cell migration by a pathway that is independent of eNOS activation (Seetharam et al., 2006). Seetharam et al. (2006) showed that HDL, via SR-BI, causes the rapid activation of Rac GTPase in concert with lamellipodia formation. HDL binding to SR-BI activates Src kinases, PI3K/Akt and p44/p42 MAPK cascades which independently activate Rac GTPase (Seetharam et al., 2006). Moreover, inhibition of PI3K or MAPK prevented HDL-induced Rac activation, however; these modulations did not alter lamellipodia formation suggesting the existence of alternative, Src-independent, pathway(s) that mediate initial lamellipodia formation (Seetharam et al., 2006). This process can also be mediated by lipoprotein particles reconstituted with different ratios of lipid free apoA-I, phospholipids and cholesterol (Seetharam et al., 2006). Cell apoptosis is a key regulator in the development of atherosclerotic lesions. Macrophage apoptosis occurs at all stages of atherosclerosis. In atherosclerotic lesions, macrophage foam cells undergo apoptosis induced by several factors (reviewed in Tobas, 2005). Examples include accumulation of large amounts of intracellular cholesterol and high concentrations of oxidized LDL, oxysterols and TNF-a (reviewed in Tabas, 2005). In early lesions, apoptotic foam cells are efficiently cleared by phagocytic macrophages and this is associated with diminished lesion cellularity and decreased lesion progression (Arai et al., 2005). In late lesions, macrophages also undergo apoptosis but tend to accumulate, suggesting a defect in the phagocytic clearance (Schrijvers et al., 2005). Defective phagocytosis would lead to secondary macrophage necrosis and enhanced inflammatory responses (Hegyi et al., 1996). Thus, the consequence of macrophage apoptosis on lesion development is dependent on the efficiency of apoptotic cell clearance by subendothelial phagocytes which are mostly macrophages. The activation of PI3K/Akt pathway has been shown to protect against cell death in response to a variety of apoptotic stimuli in endothelial cells (Piro et al., 2008), VSMCs (Wang et al., 2007) and in peritoneal macrophages, and macrophages from atherosclerotic lesions from high fat diet fed apoE KO and LDL-R KO mice (Boullier et al., 2006). On the other hand, inhibition of the PI3K/Akt pathway resulted in apoptosis in murine peritoneal macrophages (Koh et al., 1998) and primary human monocyte derived macrophages (Liu et al., 2001). Mechanisms by which Akt inhibition results in apoptosis involve the loss of mitochondrial transmembrane potential, the activation of caspases-9 and -3 and decreased expression of Mcl-1, suggesting the essential role of PI3K/Akt in macrophage survival (Liu et al., 2001). Moreover, activation of Scr/PI3K/Akt and Ras/Raf/MEK/ERK MAPK signaling cascades has been

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reported in granulocyte-macrophage colony stimulating factor (GM-CSF)induced microglial proliferation (Suh et al., 2005). More recently, Namgaladze and Brune (2006) have shown that phospholipase-A2 modified LDL promotes human and THP-1 monocytic cell survival via the activation of PI3K/Akt pathway. This suggests that HDL, via SR-BI, may activate Akt in macrophages as it does in endothelial cells, possibly involving a similar mechanism, triggering an anti-apoptotic response, potentially reducing the formation of a necrotic core. This has yet to be tested.

CONCLUSIONS The discovery of SR-BI by Acton et al. (1996) and further characterization of cellular biology of the receptor provided new insights into HDL metabolism and cellular responses to HDL. Studies from genetically engineered mice shed light onto the role of SR-BI in reverse cholesterol transport, in the protection against atherosclerosis and coronary heart disease. In addition, the more recently discovered interaction of SR-BI with the adaptor protein, PDZK1, (Ikemoto et al., 2000) clarified a complex picture of the cellular mechanisms regulating HDL metabolism and SR-BI induced signaling in various cell types. Studies on the molecular mechanisms of HDL/SR-BI induced signaling revealed the involvement of numerous pathways that mediate the atheroprotective actions of HDL in cells relevant to atherogenesis such as macrophages, endothelial cells and vascular smooth muscle cells.

ACKNOWLEDGMENTS Research in our laboratory is supported by grants NA6310 from the Heart and Stroke Foundation of Ontario and MOP74753 from the Canadian Institutions of Health Research, Institute of Genetics and a Program Grant (PRG 6502) from the Heart and Stroke Foundation of Ontario. BT has been a New Investigator of the Heart and Stroke Foundation of Canada. AA is supported by a graduate scholarship from Kuwait University. RB was supported by a graduate scholarship from the Heart and Stroke Foundation of Ontario.

REFERENCES Acton, S., Rigotti, A., Landschulz, K. T., Xu, S., Hobbs, H. H., & Krieger, M. (1996). Identification of scavenger receptor SR-BI as a high density lipoprotein receptor. Science, 271, 518e520. Acton, S. L., Scherer, P. E., Lodish, H. F., & Krieger, M. (1994). Expression cloning of SR-BI, a CD36-related class B scavenger receptor. J Biol Chem, 269, 21003e21009. Anderson, M., Viars, C. S., Czekay, S., Cavenee, W. K., & Arden, K. C. (1998). Cloning and characterization of three human forkhead genes that comprise an FKHR-like gene subfamily. Genomics, 47, 187e199. Arai, T., Wang, N., Bezouevski, M., Welch, C., & Tall, A. R. (1999). Decreased atherosclerosis in heterozygous low density lipoprotein receptor-deficient mice expressing the scavenger receptor BI transgene. J Biol Chem, 274, 2366e2371.

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Arai, S., Shelton, J. M., Chen, M., et al. (2005). A role for the apoptosis inhibitory factor AIM/ Spalpha/Api6 in atherosclerosis development. Cell Metab, 1, 201e213. Argraves, K. M., & Argraves, W. S. (2007). HDL serves as an S1P signaling platform mediating a multitude of cardiovascular effects. J Lipid Res, 48, 2325e2333. Assanasen, C., Mineo, C., Seetharam, D., et al. (2005). Cholesterol binding, efflux, and a PDZinteracting domain of scavenger receptor-BI mediate HDL-initiated signaling. J Clin Invest, 115, 969e977. Babitt, J., Trigatti, B., Rigotti, A., et al. (1997). Murine SR-BI, a high density lipoprotein receptor that mediates selective lipid uptake, is N-glycosylated and fatty acylated and colocalizes with plasma membrane caveolae. J Biol Chem, 272, 13242e13249. Backer, J. (2000). Phosphoinositide 3-kinases and the regulation of vesicular trafficking. Mol Cell Biol Res Commun, 3, 193e204. Baranova, I., Vishnyakova, T. G., Bocharov, A. V., et al. (2005). Serum amyloid A binding to CLA-1 (CD36 and LIMPII analogous-1) mediates serum amyloid A protein-induced activation of ERK1/2 and p38 mitogen-activated protein kinases. J Biol Chem, 280, 8031e8040. Bochkov, V., Tkachuk, V., Buhler, F., & Resink, T. (1992). Phosphoinositide and calcium signalling responses in smooth muscle cells: comparison between lipoproteins, Ang II, and PDGF. Biochem Biophys Res Commun, 188, 1295e1304. Boullier, A., Li, Y., Quehenberger, O., et al. (2006). Minimally oxidized LDL offsets the apoptotic effects of extensively oxidized LDL and free cholesterol in macrophages. Arterioscler Thromb Vasc Biol, 26, 1169e1176. Braun, A., Trigatti, B. L., Post, M. J., et al. (2002). Loss of SR-BI expression leads to the early onset of occlusive atherosclerotic coronary artery disease, spontaneous myocardial infarctions, severe cardiac dysfunction, and premature death in apolipoprotein E-deficient mice. Circ Res, 90, 270e276. Braun, A., Zhang, S., Miettinen, H. E., et al. (2003). Probucol prevents early coronary heart disease and death in the high-density lipoprotein receptor SR-BI/apolipoprotein E double knockout mouse. Proc Natl Acad Sci USA, 100, 7283e7288. Brundert, M., Ewert, A., Heeren, J., et al. (2005). Scavenger receptor class B type I mediates the selective uptake of high-density lipoprotein-associated cholesteryl ester by the liver in mice. Arterioscler Thromb Vasc Biol., 25, 143e148. Brunet, A., Bonni, A., Zigmond, M. J., et al. (1999). Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell, 96, 857e868. Buechler, C., Ritter, M., Quoc, C. D., Agildere, A., & Schmitz, G. (1999). Lipopolysaccharide inhibits the expression of the scavenger receptor Cla-1 in human monocytes and macrophages. Biochem Biophys Res Commun, 262, 251e254. Burgos, P., Klattenhoff, C., de la Fuente, E., Rigotti, A., & Gonza´lez, A. (2004). Cholesterol depletion induces PKA-mediated basolateral-to-apical transcytosis of the scavenger receptor class B type I in MDCK cells. Proc Natl Acad Sci USA, 101, 3845e3850. Cai, S., Kirby, R. J., Howles, P. N., & Hui, D. Y. (2001). Differentiation-dependent expression and localization of the class B type I scavenger receptor in intestine. J Lipid Res, 42, 902e909. Cao, W., Murao, K., Imachi, H., et al. (2004). A mutant high-density lipoprotein receptor inhibits proliferation of human breast cancer cells. Cancer Res, 64, 1515e1521. Covera, S. (2001). Phosphatidylinositol 3-kinase and the control of endosome dynamics: new players defined by structural motifs. Traffic, 2, 859e866. Covey, S., Krieger, M., Wang, W., Penman, M., & Trigatti, B. L. (2003). Scavenger receptor class B type I-mediated protection against atherosclerosis in LDL receptor-negative mice involves its expression in bone marrow-derived cells. Arterioscler Thromb Vasc Biol., 23, 1589e1594.

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Wang, X., Collins, H. L., Ranalletta, M., et al. (2007). Macrophage ABCA1 and ABCG1, but not SR-BI, promote macrophage reverse cholesterol transport in vivo. J Clin Invest, 117, 2216e2224. Webb, N., Connell, P. M., Graf, G. A., et al. (1998). SR-BII, an isoform of the scavenger receptor BI containing an alternate cytoplasmic tail, mediates lipid transfer between high density lipoprotein and cells. J Biol Chem, 273, 15241e15248. Williams, D. L., de La Llera-Moya, M., Thuahnai, S. T., et al. (2000). Binding and cross-linking studies show that scavenger receptor BI interacts with multiple sites in apolipoprotein A-I and identify the class A amphipathic alpha-helix as a recognition motif. J Biol Chem, 275, 18897e18904. Witt, W., Kolleck, I., Fechner, H., Sinha, P., & Rustow, B. (2000). Regulation by vitamin E of the scavenger receptor BI in rat liver and HepG2 cells. J Lipid Res, 41, 2009e2016. Wu¨stner, D., Mondal, M., Huang, A., & Maxfield, F. R. (2004). Different transport routes for high density lipoprotein and its associated free sterol in polarized hepatic cells. J Lipid Res, 45, 427e437. Yancey, P. G., Bortnick, A. E., Kellner-Weibel, G., de la Llera-Moya, M., Phillips, M. C., & Rothblat, G. H. (2003). Importance of different pathways of cellular cholesterol efflux. Arterioscler Thromb Vasc Biol., 23, 712e719. Yancey, P., Jerome, W. G., Yu, H., et al. (2007). Severely altered cholesterol homeostasis in macrophages lacking apoE and SR-BI. J Lipid Res, 48, 1140e1149. Yeh, Y. C., Hwang, G. Y., Liu, I. P., & Yang, V. C. (2002). Identification and expression of scavenger receptor SR-BI in endothelial cells and smooth muscle cells of rat aorta in vitro and in vivo. Atherosclerosis, 161, 95e103. Yesilaltay, A., Kocher, O., Pal, R., et al. (2006). PDZK1 is required for maintaining hepatic scavenger receptor, class B, type I (SR-BI) steady state levels but not its surface localization or function. J Biol Chem, 281, 28975e28980. Yesilaltay, A., Kocher, O., Rigotti, A., & Krieger, M. (2005). Regulation of SR-BI-mediated highdensity lipoprotein metabolism by the tissue-specific adaptor protein PDZK1. Curr Opin Lipidol, 16, 147e152. Yesilaltay, A., Morales, M. G., Amigo, L. M., et al. (2006). Effects of hepatic expression of the high-density lipoprotein receptor SR-BI on lipoprotein metabolism and female fertility. Endocrinology, 147, 1577e1588. Yu, X., Murao, K., Imachi, H., et al. (2007). Regulation of scavenger receptor class BI gene expression by angiotensin II in vascular endothelial cells. Hypertension, 49, 1378e1384. Yuhanna, I., Zhu, Y., Cox, B. E., et al. (2001). High-density lipoprotein binding to scavenger receptor-BI activates endothelial nitric oxide synthase. Nat. Med, 7, 853e857. Yvan-Charvet, L., Bobard, A., Bossard, P., et al. (2007). In vivo evidence for a role of adipose tissue SR-BI in the nutritional and hormonal regulation of adiposity and cholesterol homeostasis. Arterioscler Thromb Vasc Biol, 27, 1340e1345. Zhang, W., Yancey, P. G., Su, Y. R., et al. (2003). Inactivation of macrophage scavenger receptor class B type I promotes atherosclerotic lesion development in apolipoprotein E-deficient mice. Circulation, 108, 2258e2263. Zhang, S., Picard, M. H., Vasile, E., et al. (2005a). Diet-induced occlusive coronary atherosclerosis, myocardial infarction, cardiac dysfunction, and premature death in scavenger receptor class B type I-deficient, hypomorphic apolipoprotein ER61 mice. Circulation, 111, 457e464. Zhang, Y., Da Silva, J. R., Reilly, M., Billheimer, J. T., Rothblat, G. H., & Rader, D. J. (2005b). Hepatic expression of scavenger receptor class B type I (SR-BI) is a positive regulator of macrophage reverse cholesterol transport in vivo. J Clin Invest, 115, 2870e2874.

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Zhang, Y., Ahmed, A. M., McFarlane, N., et al. (2007a). Regulation of SR-BI-mediated selective lipid uptake in Chinese hamster ovary-derived cells by protein kinase signaling pathways. J Lip Res, 48, 405e416. Zhang, Y., Ahmed, A. M., Tran, T., et al. (2007b). The inhibition of endocytosis affects HDL-lipid uptake mediated by the human scavenger receptor class B type I. Mol Membr Biol, 24, 442e454.

Chapter 9

HDL Mimetic Peptides: Novel Therapeutic Strategies for the Treatment of Inflammatory Vascular Disease C. Roger White 1, G.M. Anantharamaiah 1, 2 and Geeta Datta 1 1 Departments of Medicine, and 2 Biochemistry, and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL, USA

INTRODUCTION Despite numerous therapeutic advances, cardiovascular disease (CVD) remains the leading cause of death in the Western world. The Framingham Heart Study demonstrated that CVD risk directly correlates with circulating levels of low density lipoprotein (LDL) (Gordon et al., 1981). Statin (HMGCoA reductase inhibitors) therapy has been successful in lowering LDL and the risk for coronary events (Cannon et al., 2004; Brousseau, 2005). However, even high doses of statins are unable to completely eliminate CVD risk and events. Several studies have established that a reduction in high density lipoprotein (HDL) is associated with increased severity of coronary artery disease (CAD) and is an independent risk factor for CVD (Castelli, 1988). Epidemiologic studies have shown that each 1mg/dL decrease in plasma HDL cholesterol (HDL-C) enhances CVD risk by 2e3%, while an equivalent increase in HDL-C reduces risk by 6%, independent of LDL-C levels (Gordon and Rifkind, 1989). An increase in HDL also reduces vascular complications associated with sepsis, diabetes and atherosclerosis, conditions that occur with greater frequency in aging populations (Rosenson, 2005; Kontush and Chapman, 2008). Despite these findings, analysis of published data from the Framingham Heart Study indicates that a significant number of events occur in subjects with normal LDL and HDL levels (Ansell et al., 2003). More recently, studies in highrisk patients suggest that inflammatory/anti-inflammatory properties of HDL The HDL Handbook. ISBN: 978-0-12-382171-3 Copyright Ó 2010 Elsevier Inc. All rights of reproduction in any form reserved.

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may be a better indicator of risk than HDL-C levels per se (Ansell et al., 2003). Therefore, therapeutic strategies aimed at increasing HDL and improving its functional properties are currently under development (LinselNitschke and Tall, 2005). Current approaches for raising HDL include both non-pharmacological (life-style) and pharmacological strategies (Singh et al., 2007). Exercise, weight loss, alcohol consumption and cessation of tobacco use are life-style modifications that are associated with a 5e10% increase in HDL-C (Roberts et al., 2006). HDL therapy represents an emerging field directed toward the development of new treatments that increase HDL-C levels and/or improve its anti-atherogenic properties. Pharmacological agents that increase HDL levels comprise a number of different drug classes. These drugs, however, may induce adverse reactions and/or side effects that may preclude their use. In this chapter, we will focus on agents that improve the function of HDL, specifically, apoA-I mimetic peptides.

HETEROGENEITY OF HDL HDL represents a class of heterogeneous particles that are characterized by high density (d > 1.06 g/mL) and low buoyancy. Subclasses of HDL also vary in their content of lipids, apolipoproteins, antioxidant enzymes, and lipid transfer proteins. Resulting differences in HDL size, density and composition ultimately influence particle function. The apolipoprotein composition of HDL determines, to a large extent, its function. Apolipoprotein A-I (apoA-I) accounts for 80e95% of the protein component of a-HDL, a fraction with a-mobility on agarose gels. In contrast, apoA-I comprises only 5e15% of the protein associated with lipid-poor preb-HDL. ApoA-I and apoE (a protein component of very low density lipoprotein (VLDL) and HDL) possess anti-inflammatory and atheroprotective properties. ApoA-II is another HDL-associated apolipoprotein that exerts antiatherogenic effects in numerous animal models (Meyers and Kashyap, 2004). The HDL-associated enzymes paraoxonase (PON) and platelet activating factor-acetyl hydrolase (PAF-AH) hydrolyze oxidized phospholipids and reduce lipid hydroperoxide levels in LDL and VLDL particles and thus render them non-atherogenic (Mackness et al., 2002; Oda et al., 2002). The relative concentration of these proteins per HDL particle will, in large part, reflect HDL function. In a recent study, the HDL proteome has been characterized and has revealed the presence of up to 48 proteins in human HDL (Vaisar et al., 2007). In addition to aforementioned proteins that regulate lipid transfer and metabolism, additional novel proteins that play a role in complement activation and proteolysis were identified (Vaisar et al., 2007). These observations underscore the complexity of HDL and suggest an additional important role for the particle in the regulation of immune function.

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PHYSIOLOGICAL MECHANISMS OF PROTECTIVE ACTION OF HDL AND APOA-I Anti-atherogenic effects of HDL and apoA-I may be explained by their functional properties. While a principal function of HDL is to mediate cholesterol efflux from peripheral cells, it also possesses anti-inflammatory and antioxidant properties which inhibit the initiation and progression of vascular disease.

Reverse Cholesterol Transport (RCT) RCT is the process by which excess cholesterol from non-hepatic tissues (especially cholesterol-laden, resident macrophages) is transferred to the liver for metabolism and excretion into the bile. Lipid-poor preb-HDL particles, produced in the liver or the intestine, initiate the efflux of cholesterol and phospholipids from cell membranes via interaction with the adenosine triphosphate-binding cassette transporter A1 (ABCA1). Subsequent action of lecithin-cholesterol acyl transferase (LCAT) esterifies cholesterol in preb-HDL particles and converts them to mature a-HDL particles. These particles can take up more cholesterol via the adenosine triphosphate-binding cassette transporter G1 (ABCG1). Mature HDL can deliver cholesterol to the liver either directly via the scavenger receptor type B1 (SR-B1) or indirectly by exchange of cholesteryl esters to apoB-containing particles for triglycerides (TG). In the latter pathway, cholesteryl esters can be exchanged for triglycerides in apoBrich particles (LDL and VLDL) by cholesteryl ester transfer protein (CETP). The uptake of apoB-rich particles via hepatic LDL receptors enables the delivery of cholesterol to the liver (approximately 50% of RCT). The lypolysis of TG in TG-rich HDL by hepatic lipase and endothelial lipase leads to a smaller HDL which re-enters the RCT cycle. Through this cycle, HDL mediates the delivery of cholesterol to the liver where it is metabolized and excreted into bile (Singh et al., 2007). Impairment of RCT due to dysfunctional or reduced HDL has been observed, among others, in the elderly and subjects with CAD, diabetes and Alzheimer’s disease (Clee et al., 2000; Singh-Manoux et al., 2008).

Endothelial function A decrease in nitric oxide (NO) bioavailability accompanied by an increase in leukocyte adhesion to the blood vessel wall are components of endothelial dysfunction. These represent critical early events in atherogenesis. In vitro studies show that HDL protects the endothelium by inhibiting the expression of adhesion molecules (VCAM-1, ICAM-1) and by inducing endothelial nitric oxide synthase (eNOS) activity and NO bioavailability (Assmann and Gotto, 2004). Beneficial effects of HDL have also been observed in vivo (Spiekeret al., 2002; Assmann and Nofer, 2003).

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Endothelial function in hypercholesterolemic patients is characterized by impairment of NO-dependent vasodilation compared to normolipemic subjects and can be restored by an infusion of cholesterol-free, reconstituted HDL (Nofer et al., 2002). However, this protective effect was not observed with free apoA-I, suggesting that either the lipid components or antioxidant enzymes of HDL contribute to the improvement in endothelial function. Several lines of evidence implicate bioactive sphingolipids (Nofer et al., 2002) in the initiation of signaling events leading to NO release and vasorelaxation.

Antioxidant properties of HDL Oxidized lipids and lipid hydroperoxides are associated with aging, CAD, diabetes and inflammation. Protective effects of HDL can be ascribed, in part, to its ability to inhibit LDL oxidation. Both metal ion- and 12-lipooxygenase-induced oxidation are inhibited by HDL (Nofer et al., 2002). Products of 12-lipooxygenase action, hydroperoxyeicosatetraenoic acid (HPETE) and hydroperoxyoctadecadienoic acid (HPODE), are “seeding molecules” that initiate the non-enzymatic oxidation of lipoprotein phospholipids (Assmann and Nofer, 2003). ApoA-I itself reduces HPETE and HPODE levels (Navab et al., 2000a,b, 2001). Oxidized phospholipids present in LDL are known to stimulate the production of cytokines and chemokines that induce monocyte adhesion to endothelial cells. PON1 and PAF-AH, present on HDL, effectively catalyze the degradation of oxidized phospholipids. This is further supported by transgenic animal studies. PON1 transgenic mice produce HDL that is resistant to oxidation, while PON1 deficient mice are more susceptible to atherosclerosis (Shih et al., 1998, 2000; Oda et al., 2002). Thus, HDL can scavenge oxidized lipids and lipid hydroperoxides and reduce LDL-associated inflammatory responses and atherogenicity (Navab et al., 2002).

Anti-inflammatory properties Inflammation is known to initiate atherosclerosis, diabetes, sepsis and is known to play a major role in aging (Chapman, 2007). Endothelial cells synthesize adhesion molecules (VCAM-1, ICAM-1) and release chemokines (eg., MCP-1) when induced by an inflammatory stimulus. HDL, apoA-I and reconstituted HDL (rHDL) have been shown to inhibit the upregulation of adhesion molecules and the transmigration of monocytes in vitro (Barter et al., 2004). The ability of HDL to modulate adhesion molecule expression in vivo was demonstrated by Dimayuga et al. (1999). Infusion of rHDL to apoE/ mice caused a 40% reduction in the expression of VCAM-1 within 1 week and significantly reduced monocyte infiltration and neointimal hyperplasia within 3 weeks.

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PRO-INFLAMMATORY HDL Despite the aforementioned protective properties of HDL, the concept that HDL can also be pro-inflammatory and dysfunctional has emerged (Vaisar et al., 2007). Alterations in the composition of HDL subclasses would impair the functional capacity of HDL to mediate cholesterol efflux from peripheral tissues and result in an increase in lipoprotein oxidation (Van Lenten et al., 1995; Navab et al., 2005a) and stimulation of inflammatory responses. Changes in HDL-associated proteins during inflammation induce the formation of pro-inflammatory HDL. Increased incorporation of ceruloplasmin, serum amyloid A (SAA), secretory phospholipase A2 (sPLA2), and apoJ in HDL reduces the capacity of the particle to inhibit LDL-induced chemokine production, monocyte adhesion and lipid hydroperoxide formation. A concomitant decrease in apoA-I, PON1 and PAF-AH further enhances the proinflammatory nature of HDL (Navab et al., 2006; White et al., 2008). Under inflammatory conditions, the neutrophilic enzyme myeloperoxidase (MPO) catalyzes the formation of the potent oxidant hypochlorous acid (HOCl) (Marcinkiewicz, 1997; Eiserich et al., 1998). Recent studies show that MPOderived HOCl oxidizes apoA-I and attenuates the ability of HDL to mediate RCT. Pro-inflammatory HDL has been observed in human subjects with dyslipidemia, hyperglycemia and hypertriglyceridemia (Kontush and Chapman, 2006, 2008). Results from The Framingham Heart Study indicated that 40% of CAD events were observed in subjects with “normal” or “high” HDL suggesting that HDL function, rather than HLD-C levels, is a better indicator of CVD risk. HDL function may be quantitatively assessed using the HDL inflammatory index (HII) (White et al., 2008). HII is the ratio of the LDL-induced monocyte chemotaxis in the presence or absence of test HDL. An increase in LDLinduced chemotaxis by the test HDL (HII > 1.0) indicates pro-inflammatory HDL and a decrease in chemotaxis by the test HDL (HII < 1.0) indicates antiinflammatory HDL (Navab et al., 2006; White et al., 2008). A study of 26 patients with CHD showed that HDL from patients prior to statin therapy had a high HII value which was reduced after treatment (Ansell et al., 2003; Navab et al., 2006). The pro-inflammatory nature of HDL was also noted in another study with 20 subjects who had CHD despite elevated HDL levels (Ansell et al., 2003). HII was found to be significantly elevated in these subjects. While measurement of the HII is useful in assessing functional properties of HDL, the identification of a readily measurable surrogate marker for pro-inflammatory HDL would be ideal. Identification of such a marker, to date, has been elusive.

HDL THERAPY Raising HDL-C levels and/or improving its anti-atherogenic function are principal goals of HDL therapy. The HDL-raising effects of several commonly

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prescribed medications have been tested and have shown mixed effects. Specifically, the efficacy of niacin, fibrates, and CETP inhibitors in raising serum HDL has been extensively reviewed (Assmann and Nofer, 2003; Singh et al., 2007; White et al., 2008) and will not be addressed by this chapter. The first demonstration that HDL administration served a protective function in experimental animals was reported by Badimon et al. (1990) who showed that administration of HDL to cholesterol-fed rabbits prevented the development of atherogenic lesions. HDL per se is not suitable as a drug since it is a heterogeneous mixture of proteins and lipids that is not easily duplicated by pharmaceutical preparations. In contrast, apoA-Ielipid complexes of defined composition (recombinant HDL or reconstituted HDL (rHDL)) have been tested in various dyslipidemic animal models (Chiesa et al., 2008) and shown prominent anti-atherogenic effects. Clinical studies with rHDL have been performed in small patient cohorts. The first study demonstrated that infusion of rHDL in four hypoalphalipoproteinemic (low HDL) patients induced an increase in plasma HDL-C and elimination of RCT-derived cholesterol degradation products (Eriksson et al., 1999; Chiesa et al., 2008). Patients with familial hypoalphalipo-proteinemia, such as seen in loss of functional mutations in ABCA1, have low HDL with normal levels of LDL and VLDL. Four hours after a single infusion of rHDL in such patients, forearm blood flow was restored, as measured by venous plethysmography. Hypercholesterolemia also causes endothelial dysfunction. In hypercholesterolemic patients, a single infusion of rHDL was able to increase flow-mediated dilatation in these patients and normalize endothelial dependent vasodilation by increasing NO bioavailability. In another small study, patients with established coronary disease received apoA-IMilano, a mutant of apoA-I that possesses enhanced antioxidant activity compared to wild type apoA-I (Bielicki and Oda, 2002). Five weekly treatments of apoAIMilano-phospholipid complexes (45 mg/kg each) reduced coronary atheroma burden as measured by intravascular ultrasound (IVUS). These studies underscore the efficacy of rHDL administration. Even though this treatment appears to have regressed the lesion, the large amount of the protein:lipid complexes required for this effect makes the approach impractical. In the recent ERASE study (Tardif et al., 2007), patients with acute coronary syndrome were given 40 mg or 80 mg/kg of rHDL. The changes in atheroma volume were measured by IVUS. In the 40 mg/kg group, an absolute reduction in atheroma volume of 5.34 mm3 was observed compared to baseline. At this dose, mild to moderate side effects were observed, while, at 80 mg/kg, hepatic dysfunction was significantly increased (Tardif et al., 2007). Reconstituted HDL infusion has also been shown to restore endothelial function in type 2 diabetes patients (Nieuwdorp et al., 2008). Although purified apoA-I appears to have many protective effects, the high concentrations of protein and lipid that are required to observe beneficial effects are financially and physically impractical. A new line of drugs, apolipoprotein mimetic peptides, whose

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design is based on the structure of apoA-I, represent a promising alternative to apoA-I.

STRUCTURAL CHARACTERISTICS OF APOA-I Exchangeable apolipoproteins possess lipid associating amphipathic helical domains that bind to lipids. This interaction is facilitated by their unique structure which consists of amphipathic helices arranged in tandem (Segrest et al., 1994). An amphipathic helix is defined as an alpha helix with opposing polar and non-polar faces oriented along its long axis. The sidedness of this structural motif imparts a structure complementary to that of phospholipids, thus promoting the interaction between proteins and lipids. Amphipathic helixes are grouped into seven different classes: A, H, L, G, K, C and M (Segrest et al., 1994), each with a distinct charge distribution and density. The most commonly found amphipathic helix in apolipoproteins is the Class A amphipathic a-helix, a zwitterionic helix characterized by a positively charged amino acid cluster at the polarenon-polar interface and the negatively charged residues at the center of the polar face. X-ray and molecular modeling studies of structures of D43-apo A-I (in which 1e43 residues of apo A-I are deleted) and full length apoA-I suggest a lipid bound structure in which two molecules of apoA-I form a belt-like structure in anti-parallel orientation with lipids in the center. The non-polar face of the protein is orientated toward the lipid acyl chains (Borhani et al., 1997; Li et al., 2004). Studies using other techniques such as mass spectroscopy have yielded similar structures, with some variations, depending on the protein/lipid ratios (Davidson and Thompson, 2007; Wu et al., 2007).

APOA-I MIMETIC PEPTIDES In 1985, an 18-residue Class A amphipathic peptide (DWLKAFYDKVAEKLKEAF), whose structure was based on helical repeating domains present in apoA-I, was designed in our laboratory (Anantharamaiah et al., 1985). This peptide was designated 18A and was found to mimic many of the properties of apoA-I. Similar to apoA-I, 18A interacted with lipids to form discoidal HDL-like structures; clarified suspensions of Dimyristoyl Phosphatidylcholine (DMPC) (Anantharamaiah et al., 1985); mediated cholesterol efflux from mouse fibroblasts and macrophages when complexed with DMPC (Mendez et al., 1994); and activated plasma enzyme lecithin cholesterol acyltransferase (LCAT) (Epand et al., 1987). 18A was subsequently modified by blocking the amino terminus with an acetyl group and the carboxy terminus with an amide group. These changes increased the helicity of the resulting peptide (Ac-18A-NH205 or 2F) and significantly enhanced its cholesterol efflux and LCAT activating properties compared to native 18A (Venkatachalapathi et al., 1993). Recently, high resolution NMR experiments on the structure of

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lipid-bound 2F demonstrated that the Class A peptide orients in a head to tail fashion around the edge of the lipids with the non-polar face toward the lipid (Mishra et al., 2006), in a manner similar to apoA-I (the belt model). Detailed structureefunction studies of Class A peptides were carried out by designing a family of peptides that differed in the content and position of aromatic amino acid residues (Datta et al., 2001). These peptides were generated by systematically increasing the hydrophobicity and lipid affinity of the peptide Ac-18A-NH2. This was achieved by replacing non-polar amino acids with phenylalanine (F) residues. Since Ac-18A-NH2 contains two F residues, it has been alternately designated 2F. The peptides 3F, 4F, 5F, 6F and 7F are additional analogs, all of which possess high lipid binding affinity. However, the increased hydrophobicity of 5F, 6F and 7F also leads to a decrease in solubility. This series of peptides mimicked many properties of apoA-I including the ability to mediate cholesterol efflux, LCAT activating ability and inhibition of monocyte chemotaxis. Although the physical properties of these peptides correlated well with their hydrophobicity, their biological properties were not directly related to hydrophobicity; 4F, 5F and 6F were equipotent in their ability to inhibit LDL-induced monocyte chemotaxis, while 5F had the highest LCAT activating property (Datta et al., 2001). Since all these peptides formed HDL-like structures when complexed to lipids and mimicked many properties of apoA-I, they have been classified as HDLmimetic or apoA-I mimetics. Studies with analogs of bioactive mimetics suggested that the position of the aromatic residues on the non-polar face plays an important role in conferring “biological activity” to these peptides (Data et al., 2004).

In vivo effects of apoA-I mimetic peptides Atheroprotective effects of apoA-I mimetic peptides have been demonstrated in dyslipidemic animal models. Several studies also show that they improve endothelial function, insulin sensitivity and attenuate weight gain in mouse models (Peterson et al., 2008). The peptide 18A protects rats from LPS-induced inflammatory responses and increases survival (Levine et al., 1993). The apoA-I mimetic peptide, 4F also inhibits inflammatory responses and improves survival in septic rats (Zhang et al., 2009). This was associated with an increase in HDL cholesterol and improvement in cardiac output. Recent studies also show an attenuation of vascular remodeling by apoA-I mimetics in transplantation models. These peptides protect against a wide range of inflammatory and vascular disorders.

ApoA-I mimetic peptides inhibit the formation of aortic lesions Since this class of peptides possessed many properties of apoA-I, we investigated their ability to inhibit aortic lesions in dyslipidemic mouse

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models. Although the peptide 2F possessed many of the properties of apoA-I, a significant inhibition in aortic lesion formation was not observed in C57BL/6J mice on a high fat diet (Datta et al., 2001). The more hydrophobic peptide, 5F, was the first Class A peptide that was shown to have atheroprotective activity. In vivo administration of 5F to C57BL/6J mice fed an atherogenic diet was shown to reduce aortic lesion area compared to mice treated with saline vehicle (Garber et al., 2001). No differences, however, were noted in total plasma cholesterol levels between groups, suggesting that reduction in fatty lesions observed in 5F-treated mice occurred by a mechanism that was independent of cholesterol levels. Examination of functional properties of HDL isolated from the plasma of 5F-treated mice revealed that HDL inhibited LDL-induced monocyte chemotaxis and LDL-associated lipid hydroperoxide levels, while the HDL from the vehicle treated mice did not (Garber et al., 2001). These results suggested that the protective effects of the peptide 5F were not due to changes in plasma HDL levels per se. Rather, it was suggested that antioxidative and anti-inflammatory properties of the peptide were related to an improvement in the functional properties of HDL. Although 5F had greater lipid affinity than 4F, they were both equipotent in inhibiting LDL-induced monocyte chemotaxis. Since 4F is more water soluble, subsequent studies have focused on the effects of 4F on atherosclerosis in apoE/ and LDLR/ mice. Oral administration of the peptide was made possible by the synthesis of 4F using D-amino acids resulting in a peptide (D-4F) that is resistant to the action of proteases. The presence of D-4F in the plasma of LDLR/ mice could be demonstrated 4h after administration by gavage (Navab et al., 2002). Chronic treatment with oral D-4F in LDLR/ mice fed a Western diet showed a 79% reduction in lesions. Similar to 5F treatment, there was no change in the plasma cholesterol or lipoprotein profiles compared to saline controls. However, HDL isolated from these mice showed greater inhibition of LDL-induced monocyte chemotaxis (Navab et al., 2002). Oral treatment with D-4F in apoE/ mice also significantly reduced aortic lesion formation (75% reduction compared to saline-treated controls) (Navab et al., 2004). It was shown that oral treatment with D-4F resulted in the rapid appearance (20 min) of small cholesterol containing particles in the plasma (Navab et al., 2004). These particles contained apoA-I, paraoxonase and had preb-HDL mobility on agarose gels. A decrease in plasma lipid hydroperoxides and an increase in cholesterol efflux were also noted in these experiments, suggesting that D-4F acts by improving HDL function (Navab et al., 2004). More interestingly, in older apoE/ mice, administration of low doses of D-4F in combination with a sub-therapeutic dose of pravastatin induced the formation of anti-inflammatory HDL and regression of aortic lesions in apoE/ mice and cynomologus monkeys (Navab et al., 2005b).

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ApoA-I mimetic peptides improve endothelial cell function Endothelial dysfunction and impaired vasodilation are early events in atherogenesis. Under hypercholesterolemic conditions, LDL impairs vasodilation by uncoupling eNOS, thus generating superoxide anion (O.2 ) instead of nitric oxide (NO). Using bovine aortic endothelial cells (BAECs), Ou et al. (2003a) showed that 4F, like HDL, improves endothelial vascular function by maintaining coupled eNOS activity to generate NO. Such protection by 4F has also been observed in vivo in LDLR/ (Ou et al., 2003b). Treatment of LDLR/ mice with 4F improved vasodilation in arterioles of these mice compared to vehicle controls. As in the cell culture studies, this improvement was associated with the inhibition of O.2  formation by 4F. The mechanism of O.2  generation in sickle cell disease (SCD) mice is different from the LDL-dependent mechanism observed in LDLR/ mice. In SCD mice, xanthine oxidase (XO) generates O.2  by its action on substrate xanthine and reduces NO activity (White et al., 1996). 4F treatment improved the vasodilation in SCD mice by preventing the binding of XO to endothelial cell surfaces and thus increasing NO bioavailability (Ou et al., 2003b). Recent studies with LDLR/ and LDLR//apoA-I/ double knockout mice demonstrated that 4F restores vascular endothelial cell eNOS function in both types of mice but reduces vessel wall thickness only in the LDLR/ mice suggesting that HDL containing apoA-I is required for 4F to reduce vessel wall thickness (Ou et al., 2005). The effects of 4F on vascular dilation were further corroborated by subsequent studies on tight-skin mice (Weihrauch et al., 2007). The tight-skin mouse (Tsk/þ) is a commonly used model for systemic sclerosis (SSc), an autoimmune disorder characterized by increased oxidant stress and impaired vascular function. Treatment with 4F improved vascular function and decreased myocardial inflammation and pro-inflammatory HDL.

ApoA-I mimetic peptides reduce complications associated with diabetes Oxidative stress plays an important role in diabetes and CVD. In the streptozotocin (STZ)-treated diabetic rat model, an increase in vascular O. 2 formation is associated with endothelial dysfunction and impaired relaxation (Peterson et al., 2007). The increase in superoxide anion and impaired vasodilation in diabetic rats were both rectified by the administration of 4F. These changes were associated with a 4F-induced increase in the antioxidant enzymes heme oxygenase-1 (HO-1) and extracellular superoxide dismutase (EC-SOD) and endothelial progenitor cells (EPC) (Peterson et al., 2007). In diabetic ob/ob mice, administration of 4F improved insulin sensitivity and glucose tolerance (Peterson et al., 2008). This was accompanied by an increase in plasma adiponectin levels and a decrease in inflammatory cytokines, IL-6 and IL-1b. Most

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interestingly, it also reduced visceral and subcutaneous fat content and restricted weight gain (Peterson et al., 2008).

ApoA-I mimetic peptides improve cognitive function Recent studies (Buga et al., 2006 and Handattu et al., 2009) have shown that administration of D-4F to LDLR null mice anf D-4F with pravastatin to APPSwe-PS1DE9 mice (a murine model of Alzheimer’s disease) significantly improved cognitive function. The treatment also reduced the amyloid burden in the APPSwe-PS1DE9 mice. These studies demonstrate that 4F (similar to HDL) improves symptoms of Alzheimer’s disease.

ApoA-I mimetic peptides inhibit rejection of organ transplantation Cardiac allograft vasculopathy (CAV) is characterized by lymphocyte infiltration and intimal lesion formation and is one of the main causes for the rejection of transplanted hearts. Inflammation and oxidative stress are implicated in the development of CAV. Since HDL and 4F have anti-inflammatory and antioxidant properties and have atheroprotective properties, the effect of 4F on CAV was studied (Weis and von Scheidt, 1997). Donor hearts transplanted in C57BL/ 6 mice treated with 4F or saline were examined. Treatment with 4F significantly reduced the severity of intimal lesions and lymphocyte infiltration after 24 days. These protective effects were, in part, due to the upregulation of HO-1. On the other hand, in vitro studies showed that 4F inhibited T-lymphocyte proliferation and cytokine production by an HO-1 independent pathway. These studies suggest that the protective effects of 4F on CAV are partly dependent on HO-1 but other HO-1 independent pathways are also invoked. Earlier studies showed that 4F administration inhibited lesion formation in vein grafts transplanted into apoE/ (Li et al., 2004). Daily administration of 4F for 4 weeks to mice in which a segment of the inferior vena cava was grafted into the right carotid artery significantly reduced lesions in the graft with a decrease in lipid content and inflammation (Li et al., 2004). However, it had no effect on established lesions in the aortic sinus. These results suggest that the time of 4F administration plays an important role. Most of the protective effects of 4F appear to be due to its anti-inflammatory and antioxidant properties (Figure 9.1). Since oxidized lipids initiate a number of inflammatory responses, the binding properties of 4F to oxidized lipids may be associated with these functions. A recent study examined the binding of 4F and apoA-I to oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine (PAPC) by surface plasma resonance (Van Lenten et al., 2008). The binding constant for 4F was four to six orders of magnitude higher than that of apoA-I. These data suggest that perhaps 4F, with its greater affinity for oxidized lipids, can bind oxidized lipids and inhibit their inflammatory activity.

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FIGURE 9.1 Under conditions of stress, acute phase proteins can displace apoA-I and PON (antiinflammatory proteins) from HDL converting it to dysfunctional HDL. However, apo-mimetic peptides can reverse this and restore HDL functions.

APOE MIMETIC PEPTIDES Another group of apo-mimetic peptides are the apoE mimetics. ApoE, a protein component of VLDL and HDL, plays a key role in lipoprotein metabolism and atherosclerosis. It facilitated the hepatic uptake of atherogenic lipoproteins and analogous to apoA-I, mediated cholesterol efflux, and has anti-inflammatory and antioxidant properties. Although the anti-atherosclerotic properties of apoE can be attributed, in part, to its role in regulating cholesterol homeostasis, its vascular protective role has been shown to be independent of its cholesterol transport function (Hui, 2004). Ac-hE18A-NH2, designed to structurally mimic apoE, is a unique, dual-domain peptide with the receptor binding domain of apoE (residues 141e150) covalently bound to a lipid associating domain (the Class A amphipathic peptide, 18A) and has the sequence AcLRKLRKRLLRDWLKAFYDKVAEKLKEAF-NH2 (Datta et al., 2000, 2001). We have shown that it binds LDL and VLDL and enhances their uptake by HepG2 cells via the heparan sulfate proteoglycan (HSPG) pathway. This peptide dramatically lowered plasma cholesterol in apoE/ and Watanabe rabbits and improved endothelial function (Garber et al., 2003; Gupta et al., 2005). Ac-hE18A-NH2 also reduced superoxide formation and LPS-induced VCAM-1 expression and has been shown to recycle (Datta et al., 2010). Another apoE mimetic peptide that has shown suppression of systemic and brain inflammation is the peptide corresponding to the 133e149 receptor binding region of apoE (Lynch et al., 2003). Co-administration of this peptide with LPS to wild type C57BL6/J mice significantly reduced serum IL-6 and TNF-a. To enhance transmembrane permeability, this peptide was fused to

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a protein transduction domain to create COG112 (Li et al., 2006). This peptide inhibited bacterially induced expression of iNOS and the activation of NF-kB in colonic epithelial cells. These results suggest that apoE mimetic peptides with cholesterol reducing and potent anti-inflammatory properties, have significant potential in treating vascular and other inflammation-induced diseases.

OTHER APO-MIMETIC PEPTIDES ApoJ is an apolipoprotein present on HDL which also plays a protective role against inflammation (Navab et al., 1997). Similar to apoA-I, apoJ also inhibits LDL-induced lipid hydroperoxide formation and monocyte chemotaxis (Navab et al., 1997). Screening of short amphipathic peptide sequences from apoJ yielded a class G* amphipathic helix (corresponding to amino acids at positions 113e122 of apoJ) which exerts anti-atherogenic and antiinflammatory effects in mice and monkeys by reducing lipoprotein-associated lipid hydroperoxides and by enhancing the activity of PON (Navab et al., 2005c). Small tetrapeptides (KRES and FREL) that are too small to form helices but have amphipathic properties have also been shown to interact with HDL (Navab et al., 2005d). The four amino acid peptides KRES and FREL exert similar effects on HDL quality but also increase HDL levels in apoE null mice (Navab et al., 2005d). Despite these effects on HDL, KRES does not induce preb-HDL formation or directly stimulate cholesterol efflux from macrophages (Navab et al., 2005d). This is in contrast to the known effects of apoA-I mimetic peptides such as 4F. Ongoing studies are defining mechanisms of small peptide action.

CONCLUSION Peptide mimetics of apolipoproteins A-I, E and J exert anti-inflammatory and atheroprotective effects in dyslipidemic animal models (summarized in Table 9.1). These effects are ascribed, in large part, to their abilities to improve the functional properties of HDL and to remove seeding peroxides from atherogenic lipoproteins. Peptide mimetics increase circulating levels of lipid-poor preb-HDL particles that are effective mediators of cholesterol efflux from macrophages. They also increase the activity of the HDL-associated enzyme PON-1 which reduces LDL atherogenicity by hydrolyzing oxidized lipids. Vasoprotective effects of apolipoprotein mimetics also include an increase in the expression of the antioxidants HO-1 and EC-SOD (Peterson et al., 2007). These enzymes decrease oxidant stress and the formation of pro-inflammatory lipid peroxides, while improving NO bioavailability and endothelial cell function. Whether the induction of HO-1 and EC-SOD is a direct effect of 4F or is related to improvement in HDL function is currently unknown. The apoA-I mimetic 4F is currently undergoing clinical evaluation and has been shown to

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TABLE 9.1

Properties of apomimetic peptides Peptide

Effect

2F

ApoA-I mimetic

Inhibits LPS-associated toxicity in mice (Levine et al., 1993)

4F

ApoA-I mimetic

Inhibits aortic lesion formation in dyslipidemic animal models (Navab et al., 2002, 2004, 2005b) Sepsis-induced inflammatory responses (Zhang et al., 2009) Organ transplant (Weis and von Scheidt, 1997) Reduces insulin sensitivity (Peterson et al., 2007) Improves cognitive functions (Bulga et al., 2006 and Hattandu et al., 2009) Improves endothelial function (Ou et al., 2003a)

5F

ApoA-I mimetic

Inhibits aortic lesion formation in dyslipidemic mouse models (Garber et al., 2001)

Ac-hE18A-NH2

ApoE mimetic

Inhibits aortic lesion formation in dyslipidemic animal models (Garber et al., 2003; Gupta et al., 2005) Inhibits LPS-induced inflammatory responses (Datta et al., 2010) Recycles from macrophages and hepatocytes (Datta et al., 2010)

ApoJ peptide

ApoJ mimetic

Anti-atherogenic in mice and monkeys (Navah et al., 2005c)

KRES and FREL

Short peptides

Anti-atherogenic (Navab et al., 2005d)

be safe and well tolerated in phase I studies (Bloedon et al., 2008). Most importantly, it reduces the HII in high-risk subjects. These exciting results underscore the therapeutic potential of apolipoprotein mimetic peptides in the treatment of cardiovascular diseases.

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Assmann, G., & Gotto, A. M., Jr. (2004). HDL cholesterol and protective factors in atherosclerosis. Circulation, 109(23 Suppl. 1), III8eII14. Assmann, G., & Nofer, J. R. (2003). Atheroprotective effects of high-density lipoproteins. Annu Rev Med, 54, 321e341. Badimon, J. J., Badimon, L., & Fuster, V. (1990). Regression of atherosclerotic lesions by high density lipoprotein plasma fraction in the cholesterol-fed rabbit. J Clin Invest, 85(4), 1234e1241. Barter, P. J., Nicholls, S., Rye, K. A., Anantharamaiah, G. M., Navab, M., & Fogelman, A. M. (2004). Antiinflammatory properties of HDL. Circ Res, 95(8), 764e772. Bielicki, J. K., & Oda, M. N. (2002). Apolipoprotein A-I(Milano) and apolipoprotein A-I(Paris) exhibit an antioxidant activity distinct from that of wild-type apolipoprotein A-I. Biochemistry, 41(6), 2089e2096. Bloedon, L. T., Dunbar, R., Duffy, D., et al. (2008). Safety, pharmacokinetics, and pharmacodynamics of oral apoA-I mimetic peptide D-4F in high-risk cardiovascular patients. J Lipid Res, 49(6), 1344e1352. Borhani, D. W., Rogers, D. P., Engler, J. A., & Brouillette, C. G. (1997). Crystal structure of truncated human apolipoprotein A-I suggests a lipid-bound conformation. Proc Natl Acad Sci USA, 94(23), 12291e12296. Brousseau, M. E. (2005). Emerging role of high-density lipoprotein in the prevention of cardiovascular disease. Drug Discov Today, 10(16), 1095e1101. Buga, G. M., Frank, J. S., Mottino, G. A., et al. (2006). D-4F decreases brain arteriole inflammation and improves cognitive performance in LDL receptor-null mice on a Western diet. J. Lipid Res, 47, 2148e2160. Cannon, C. P., Braunwald, E., McCabe, C. H., et al. (2004). Intensive versus moderate lipid lowering with statins after acute coronary syndromes. N Engl J Med, 350(15), 1495e1504. Castelli, W. P. (1988). Cholesterol and lipids in the risk of coronary artery disease e the Framingham Heart Study. Can J Cardiol, 4(Suppl A), 5Ae10A. Chapman, M. J. (2007). Metabolic syndrome and type 2 diabetes: lipid and physiological consequences. Diab Vasc Dis Res, 4(Suppl 3), S5eS8. Chiesa, G., Parolini, C., & Sirtori, C. R. (2008). Acute effects of high-density lipoproteins: biochemical basis and clinical findings. Curr Opin Cardiol, 23(4), 379e385. Clee, S. M., Kastelein, J. J., van Dam, M., et al. (2000). Age and residual cholesterol efflux affect HDL cholesterol levels and coronary artery disease in ABCA1 heterozygotes. J Clin Invest, 106(10), 1263e1270. Datta, G., Chaddha, M., Garber, D. W., et al. (2000). The receptor binding domain of apolipoprotein E, linked to a model class A amphipathic helix, enhances internalization and degradation of LDL by fibroblasts. Biochemistry, 39(1), 213e220. Datta, G., Chaddha, M., Hama, S., et al. (2001). Effects of increasing hydrophobicity on the physical-chemical and biological properties of a class A amphipathic helical peptide. J Lipid Res, 42(7), 1096e1104. Datta, G., Garber, D. W., Chung, B. H., et al. (2001). Cationic domain 141-150 of apoE covalently linked to a class A amphipathic helix enhances atherogenic lipoprotein metabolism in vitro and in vivo. J Lipid Res, 42(6), 959e966. Datta, G., Epand, R. F., Epand, R. M., et al. (2004). Aromatic residue position on the nonpolar face of class A amphipathic helical peptides determines biological activity. J. Biol Chem, 279(24), 26509e26517. Datta, G., White, C. R., Dashti, N., et al. (2010). Anti-inflammatory and recycling properties of an apolipoprotein mimetic peptide, Ac-hE18A-NH(2). Atherosclerosis, 208, 134e141.

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Davidson, W. S., & Thompson, T. B. (2007). The structure of apolipoprotein A-I in high density lipoproteins. J Biol Chem, 282(31), 22249e22253. Dimayuga, P., Zhu, J., Oguchi, S., et al. (1999). Reconstituted HDL containing human apolipoprotein A-1 reduces VCAM-1 expression and neointima formation following periadventitial cuff-induced carotid injury in apoE null mice. Biochem Biophys Res Commun, 264(2), 465e468. Eiserich, J. P., Hristova, M., Cross, C. E., et al. (1998). Formation of nitric oxide-derived inflammatory oxidants by myeloperoxidase in neutrophils. Nature, 391(6665), 393e397. Epand, R. M., Gawish, A., Iqbal, M., et al. (1987). Studies of synthetic peptide analogs of the amphipathic helix. Effect of charge distribution, hydrophobicity, and secondary structure on lipid association and lecithin:cholesterol acyltransferase activation. J Biol Chem, 262(19), 9389e9396. Eriksson, M., Carlson, L. A., Miettinen, T. A., & Angelin, B. (1999). Stimulation of fecal steroid excretion after infusion of recombinant proapolipoprotein A-I. Potential reverse cholesterol transport in humans. Circulation, 100(6), 594e598. Garber, D. W., Datta, G., Chaddha, M., et al. (2001). A new synthetic class A amphipathic peptide analogue protects mice from diet-induced atherosclerosis. J Lipid Res, 42(4), 545e552. Garber, D. W., Handattu, S., Aslan, I., Datta, G., Chaddha, M., & Anantharamaiah, G. M. (2003). Effect of an arginine-rich amphipathic helical peptide on plasma cholesterol in dyslipidemic mice. Atherosclerosis, 168(2), 229e237. Gordon, D. J., & Rifkind, B. M. (1989). High-density lipoprotein e the clinical implications of recent studies. N Engl J Med, 321(19), 1311e1316. Gordon, T., Kannel, W. B., Castelli, W. P., & Dawber, T. R. (1981). Lipoproteins, cardiovascular disease, and death. Arch Intern Med, 141(9), 1128e1131, The Framingham study. Gupta, H., White, C. R., Handattu, S., et al. (2005). Apolipoprotein E mimetic peptide dramatically lowers plasma cholesterol and restores endothelial function in watanabe heritable hyperlipidemic rabbits. Circulation, 111(23), 3112e3118. Handattu, S. P., Garber, D. W., Monroe, C. E., et al. (2009). Oral apolipoprotein A-I mimetic peptide improves cognitive function and reduces amyloid burden in a mouse model of Alzheimer’s disease. Neurobiol Dis, 34(3), 525e534. Hui, D. Y. (2004). Apolipoprotein E-induced cell signaling in the vessel wall. Rev Endocr Metab Disord, 5(4), 335e341. Kontush, A., & Chapman, M. J. (2006). Functionally defective high-density lipoprotein: a new therapeutic target at the crossroads of dyslipidemia, inflammation, and atherosclerosis. Pharmacol Rev, 58(3), 342e374. Kontush, A., & Chapman, M. J. (2008). Why is HDL functionally deficient in type 2 diabetes? Curr Diab Rep, 8(1), 51e59. Levine, D. M., Parker, T. S., Donnelly, T. M., Walsh, A., & Rubin, A. L. (1993). In vivo protection against endotoxin by plasma high density lipoprotein. Proc Natl Acad Sci USA, 90(24), 12040e12044. Li, L., Chen, J., Mishra, V. K., et al. (2004). Double belt structure of discoidal high density lipoproteins: molecular basis for size heterogeneity. J Mol Biol, 343(5), 1293e1311. Li, X., Chyu, K. Y., Faria Neto, J. R., et al. (2004). Differential effects of apolipoprotein AI-mimetic peptide on evolving and established atherosclerosis in apolipoprotein E-null mice. Circulation, 110(12), 1701e1705. Li, F. Q., Sempowski, G. D., McKenna, S. E., Laskowitz, D. T., Colton, C. A., & Vitek, M. P. (2006). Apolipoprotein E-derived peptides ameliorate clinical disability and inflammatory infiltrates into the spinal cord in a murine model of multiple sclerosis. J Pharmacol Exp Ther, 318(3), 956e965.

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Linsel-Nitschke, P., & Tall, A. R. (2005). HDL as a target in the treatment of atherosclerotic cardiovascular disease. Nat Rev Drug Discov, 4(3), 193e205. Lynch, J. R., Tang, W., Wang, H., et al. (2003). APOE genotype and an ApoE-mimetic peptide modify the systemic and central nervous system inflammatory response. J Biol Chem, 278(49), 48529e48533. Mackness, B., Durrington, P. N., Boulton, A. J., Hine, D., & Mackness, M. I. (2002). Serum paraoxonase activity in patients with type 1 diabetes compared to healthy controls. Eur J Clin Invest, 32(4), 259e264. Marcinkiewicz, J. (1997). Nitric oxide and antimicrobial activity of reactive oxygen intermediates. Immunopharmacology, 37(1), 35e41. Mendez, A. J., Anantharamaiah, G. M., Segrest, J. P., & Oram, J. F. (1994). Synthetic amphipathic helical peptides that mimic apolipoprotein A-I in clearing cellular cholesterol. J Clin Invest, 94(4), 1698e1705. Meyers, C. D., & Kashyap, M. L. (2004). Pharmacologic elevation of high-density lipoproteins: recent insights on mechanism of action and atherosclerosis protection. Curr Opin Cardiol, 19 (4), 366e373. Mishra, V. K., Anantharamaiah, G. M., Segrest, J. P., et al. (2006). Association of a model class A (apolipoprotein) amphipathic alpha helical peptide with lipid: high resolution NMR studies of peptide.lipid discoidal complexes. J Biol Chem, 281(10), 6511e6519. Navab, M., Anantharamaiah, G. M., & Fogelman, A. M. (2005a). The role of high-density lipoprotein in inflammation. Trends Cardiovasc Med, 15(4), 158e161. Navab, M., Anantharamaiah, G. M., Hama, S., et al. (2002). Oral administration of an Apo A-I mimetic peptide synthesized from D-amino acids dramatically reduces atherosclerosis in mice independent of plasma cholesterol. Circulation, 105(3), 290e292. Navab, M., Anantharamaiah, G. M., Reddy, S. T., et al. (2004). Oral D-4F causes formation of prebeta high-density lipoprotein and improves high-density lipoprotein-mediated cholesterol efflux and reverse cholesterol transport from macrophages in apolipoprotein E-null mice. Circulation, 109(25), 3215e3220. Navab, M., Anantharamaiah, G. M., Hama, S., et al. (2005b). D-4F and statins synergize to render HDL antiinflammatory in mice and monkeys and cause lesion regression in old apolipoprotein E-null mice. Arterioscler Thromb Vasc Biol, 25(7), 1426e1432. Navab, M., Anantharamaiah, G. M., Reddy, S. T., et al. (2005c). An oral apoJ peptide renders HDL antiinflammatory in mice and monkeys and dramatically reduces atherosclerosis in apolipoprotein E-null mice. Arterioscler Thromb Vasc Biol, 25(9), 1932e1937. Navab, M., Anantharamaiah, G. M., Reddy, S., et al. (2005d). Oral small peptides render HDL antiinflammatory in mice and monkeys and reduce atherosclerosis in ApoE null mice. Circ Res, 97(6), 524e532. Navab, M., Berliner, J. A., Subbanagounder, G., et al. (2001). HDL and the inflammatory response induced by LDL-derived oxidized phospholipids. Arterioscler Thromb Vasc Biol, 21(4), 481e488. Navab, M., Hama, S. Y., Anantharamaiah, G. M., et al. (2000a). Normal high density lipoprotein inhibits three steps in the formation of mildly oxidized low density lipoprotein: steps 2 and 3. J Lipid Res, 41(9), 1495e1508. Navab, M., Hama, S. Y., Cooke, C. J., et al. (2000b). Normal high density lipoprotein inhibits three steps in the formation of mildly oxidized low density lipoprotein: step 1. J Lipid Res, 41(9), 1481e1494. Navab, M., Hama-Levy, S., Van Lenten, B. J., et al. (1997). Mildly oxidized LDL induces an increased apolipoprotein J/paraoxonase ratio. J Clin Invest, 99(8), 2005e2019.

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Navab, M., Anantharamaiah, G. M., Reddy, S. T., Van Lenten, B. J., Ansell, B. J., & Fogelman, A. M. (2006). Mechanisms of disease: proatherogenic HDL e an evolving field. Nat Clin Pract Endocrinol Metab, 2(9), 504e511. Nieuwdorp, M., Vergeer, M., Bisoendial, R. J., et al. (2008). Reconstituted HDL infusion restores endothelial function in patients with type 2 diabetes mellitus. Diabetologia, 51(6), 1081e1084. Nofer, J. R., Kehrel, B., Fobker, M., Levkau, B., Assmann, G., & von Eckardstein, A. (2002). HDL and arteriosclerosis: beyond reverse cholesterol transport. Atherosclerosis, 161(1), 1e16. Oda, M. N., Bielicki, J. K., Ho, T. T., Berger, T., Rubin, E. M., & Forte, T. M. (2002). Paraoxonase 1 overexpression in mice and its effect on high-density lipoproteins. Biochem Biophys Res Commun, 290(3), 921e927. Ou, J., Ou, Z., Jones, D. W., et al. (2003b). L-4F, an apolipoprotein A-1 mimetic, dramatically improves vasodilation in hypercholesterolemia and sickle cell disease. Circulation, 107(18), 2337e2341. Ou, J., Wang, J., Xu, H., et al. (2005). Effects of D-4F on vasodilation and vessel wall thickness in hypercholesterolemic LDL receptor-null and LDL receptor/apolipoprotein A-I doubleknockout mice on Western diet. Circ Res, 97(11), 1190e1197. Ou, Z., Ou, J., Ackerman, A. W., Oldham, K. T., & Pritchard, K. A., Jr. (2003a). L-4F, an apolipoprotein A-1 mimetic, restores nitric oxide and superoxide anion balance in low-density lipoprotein-treated endothelial cells. Circulation, 107(11), 1520e1524. Peterson, S. J., Drummond, G., Kim, D. H., et al. (2008). L-4F treatment reduces adiposity, increases adiponectin levels, and improves insulin sensitivity in obese mice. J Lipid Res, 49(8), 1658e1669. Peterson, S. J., Husney, D., Kruger, A. L., et al. (2007). Long-term treatment with the apolipoprotein A1 mimetic peptide increases antioxidants and vascular repair in type I diabetic rats. J Pharmacol Exp Ther, 322(2), 514e520. Roberts, C. K., Ng, C., Hama, S., Eliseo, A. J., & Barnard, R. J. (2006). Effect of a short-term diet and exercise intervention on inflammatory/anti-inflammatory properties of HDL in overweight/obese men with cardiovascular risk factors. J Appl Physiol, 101(6), 1727e1732. Rosenson, R. S. (2005). HDL-C and the diabetic patient: target for therapeutic intervention? Diabetes Res Clin Pract, 68(Suppl 2), S36eS42. Segrest, J. P., Garber, D. W., Brouillette, C. G., Harvey, S. C., & Anantharamaiah, G. M. (1994). The amphipathic alpha helix: a multifunctional structural motif in plasma apolipoproteins. Adv Protein Chem, 45, 303e369. Shih, D. M., Gu, L., Xia, Y. R., et al. (1998). Mice lacking serum paraoxonase are susceptible to organophosphate toxicity and atherosclerosis. Nature, 394(6690), 284e287. Shih, D. M., Xia, Y. R., Wang, X. P., et al. (2000). Combined serum paraoxonase knockout/ apolipoprotein E knockout mice exhibit increased lipoprotein oxidation and atherosclerosis. J Biol Chem, 275(23), 17527e17535. Singh, I. M., Shishehbor, M. H., & Ansell, B. J. (2007). High-density lipoprotein as a therapeutic target: a systematic review. J Am Med Assoc, 298(7), 786e798. Singh-Manoux, A., Gimeno, D., Kivimaki, M., Brunner, E., & Marmot, M. G. (2008). Low HDL cholesterol is a risk factor for deficit and decline in memory in midlife: the Whitehall II study. Arterioscler Thromb Vasc Biol, 28(8), 1556e1562. Spieker, L. E., Sudano, I., Hurlimann, D., et al. (2002). High-density lipoprotein restores endothelial function in hypercholesterolemic men. Circulation, 105(12), 1399e1402. Tardif, J. C., Gregoire, J., L’Allier, P. L., et al. (2007). Effects of reconstituted high-density lipoprotein infusions on coronary atherosclerosis: a randomized controlled trial. J Am Med Assoc, 297(15), 1675e1682.

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Chapter 10

Sterol Efflux by ABCA1 and ABCG1 Naoki Terasaka Biological Research Laboratories, Daiichi Sankyo Co., Ltd, Tokyo, Japan

INTRODUCTION Plasma high density lipid (HDL) levels are inversely related to the risk of atherosclerotic cardiovascular disease (Rhoads et al., 1976; Castelli et al., 1986). HDL has anti-inflammatory, antioxidant, antithrombotic and vasodilatory properties that may be relevant to this relationship (Assmann and Gotto, 2004; Barter et al., 2004). One of the most important atheroprotective roles of HDL is reverse cholesterol transport (RCT), where excess cholesterol in macrophage foam cells undergoes efflux and then is transported to the liver for excretion in the bile (Rader, 2006). In recent years, there has been substantial progress in understanding the biology of HDL and its interaction with cells, and the RCT hypothesis has been modified and amplified. Progress has been emphasized by the discovery of new molecules involved in the different steps of RCT or other aspects of HDL biology, development of transgenic mouse models and the beginning of human clinical trials targeting HDL. A remarkable feature of the RCT pathway is that each of the individual steps involves molecules that are directly regulated by liver X receptors (LXRs), including ATP-binding cassette transporters ABCA1 and ABCG1 in macrophages (Tall et al., 2002; Tontonoz and Mangelsdorf, 2003). In the absence of ABCA1 and ABCG1, macrophages massively accumulate cholesteryl esters (CEs), highlighting the physiological importance of cholesterol efflux in the pathogenesis of atherosclerosis (Out et al., 2007a; Yvan-Charvet et al., 2007a). This chapter will summarize recent advances in the basic science of HDL and ABCA1 and ABCG1.

ABCA1-MEDIATED CHOLESTEROL EFFLUX The ability of HDL and its major apolipoprotein, apoA-I, to stimulate efflux of cholesterol from macrophage foam cells in atherosclerotic lesions is thought to be central to its anti-atherogenic mechanism, and to represent the first step in an The HDL Handbook. ISBN: 978-0-12-382171-3 Copyright Ó 2010 Elsevier Inc. All rights of reproduction in any form reserved.

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overall process of RCT. A major breakthrough in the HDL field was the discovery that mutations in the ABCA1 are responsible for Tangier disease (Bodzioch et al., 1999; Brooks-Wilson et al., 1999; Rust et al., 1999), a condition characterized by an almost complete deficiency of plasma HDL, macrophage foam cell accumulation in various tissues, peripheral neuropathy and an apparent moderate increase in atherosclerosis. More than twenty different mutations in ABCA1 have been shown to cause Tangier disease. Moreover, about 10% of individuals with very low HDL have a variety of different mutations in ABCA1 (Cohen et al., 2004; Frikke-Schmidt et al., 2004). The human ABCA1 gene has been mapped to chromosome 9q31 and is composed of 50 exons, which encode 2261-amino acid residues (SantamarinaFojo et al., 2000). The ABCA1 protein is a full-size ABC transporter containing two transmembrane domains of six alpha helices and two intracellular nucleotide binding domains (Santamarina-Fojo et al., 2000). Like Tangier disease patients, ABCA1 knockout mice exhibit HDL deficiency and reduced cellular cholesterol efflux activity (Fitzgerald et al., 2001). Both systemic and selective hepatic overexpression of ABCA1 in mice results in an increase of HDL (Vaisman et al., 2001; Singaraja et al., 2002). In mice with a liverspecific deletion of ABCA1, apoA-I and HDL plasma levels are dramatically reduced (Timmins et al., 2005). By contrast, the selective inactivation or expression of ABCA1 in macrophages had little or no effect on the plasma concentration of HDL (Haghpassand et al., 2001). Therefore, hepatic ABCA1 expression is a rate-limiting step for plasma HDL production, whereas macrophages do not contribute significantly to the formation of HDL. However, the selective knockout of ABCA1 in macrophages of either ApoE
/
or Ldlr
/
mice significantly enhanced the development of atherosclerosis (Francis et al., 1995). Thus, although ABCA1 in macrophages has little influence on HDL plasma levels, it is a crucial factor in the prevention of excessive cholesterol accumulation in macrophages of the arterial wall, independently of plasma HDL levels. ABCA1 gene expression is induced by LXRa or LXRb and the retinoid-Xreceptor (RXR) which form heterodimers and are activated by oxysterols and retinoids, respectively (Costet et al., 2000). The most likely physiological ligand for LXRa and LXRb is 27-hydroxycholesterol, which is produced by the cytochrome P450 enzyme 27-cholesterol hydroxylase (CYP27). The levels of 27-hydroxycholesterol correlate with cellular cholesterol levels highlighting the regulation of ABCA1-mediated cholesterol efflux by oxysterols. Indeed, increasing levels of cellular cholesterol lead to the formation of 27hydroxycholesterol, which binds to LXRs inducing ABCA1 and thereby cholesterol efflux (Fu et al., 2001). Conversely, unsaturated fatty acids inhibit ABCA1 expression presumably because they compete with oxysterols for the binding to LXRa and LXRb (Ou et al., 2001). cAMP not only stimulates the expression of ABCA1 in macrophages but also its phosphorylation (Oram et al., 2000; Haidar et al., 2002, 2004). This involves protein kinase A, occurs at serine residues 1042 and 2054 and

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increases the cholesterol efflux activity of ABCA1. Interestingly, apoA-I activates cAMP signaling and thereby the phosphorylation of ABCA1, its abundance on the cell surface, and cholesterol efflux (Haidar et al., 2004). Janus kinase 2 (JAK2) also phosphorylates ABCA1, a process that increases apoA-I binding and cholesterol efflux (Roosbeek et al., 2004; Tang et al., 2004). However, in the absence of apolipoproteins, phosphorylation of threonine residues 1286 and 1305 within the ABCA1 PEST motif is a signal for calpainmediated degradation of ABCA1 (Martinez et al., 2003). Other signals leading to ABCA1 degradation are exerted by unsaturated fatty acids via a phospholipase D2-dependent pathway (Wang and Oram, 2002, 2005) and by excess free cholesterol (FC), which induces proteasomal degradation. The intracellular targeting of ABCA1 is also regulated by various adapter proteins. a1syntrophin (Munehira et al., 2004), b1-syntrophin (Okuhira et al., 2005), b2syntrophin (Buechler et al., 2002), and Cdc42 (Tsukamoto et al., 2001), which interact with the carboxy-terminal domain of ABCA1.

ABCG1-MEDIATED CHOLESTEROL EFFLUX Whereas ABCA1 promotes cholesterol efflux to lipid-poor apoA-I, it only modestly stimulates lipid efflux to smaller HDL3 particles and does not promote cholesterol efflux to the larger HDL2 fraction (Francis et al., 1995; Wang et al., 2000). This suggested the possibility that there might be another cholesterol efflux pathway promoting lipid efflux to HDL particles which led to the discovery that ABCG1 promotes cholesterol efflux from transfected cells to HDL particles but not to lipid-poor apoA-I (Figure 10.1) (Wang et al., 2004). ApoE and lecithin:cholesterol acyltransferase (LCAT) in the HDL particles have an important role in promoting cholesterol efflux via ABCG1, especially in CETP deficiency states (Matsuura et al., 2006). ABCG1 is a half-transporter that is likely to act as homo-dimers. ABCG1 is highly expressed in macrophages and promotes cholesterol efflux from macrophage foam cells to HDL particles (Wang et al., 2006). The human ABCG1 gene has been mapped to chromosome 21q22.3. It is composed of 23 exons and has multiple transcripts (Kennedy et al., 2001). ABCG1 mRNA levels are highly increased when macrophages are incubated with LXR agonists (Nakamura et al., 2004). Its silencing by RNA interference results in reduced efflux of cholesterol and phospholipid to HDL. Conversely, its overexpression causes an increase in HDL-mediated cholesterol efflux and a reduction in cellular cholesterol mass (Nakamura et al., 2004; Wang et al., 2004; Vaughan and Oram, 2005). These results were confirmed in knockout and transgenic mice. The disruption of ABCG1 in mice on a high-fat and cholesterol-rich diet causes the accumulation of neutral lipids and phospholipids in hepatocytes and macrophages, although not affecting plasma lipids (Kennedy et al., 2005). In contrast, overexpression of ABCG1 is protective against lipid accumulation (Kennedy et al., 2005).

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HDL ABCG1

FC

LCAT ABCA1

HL, EL PLTP Free ApoAI

FIGURE 10.1 Role of ABCA1 and ABCG1 transporters in cholesterol efflux in macrophages. Lipid-free or lipid-poor apoA-I can interact with ABCA1 in macrophage foam cells in atherosclerotic lesions, promoting efflux of free cholesterol (FC). This results in the formation of nascent HDL particles that are further modified by lecithin:cholesterol acyltransferase (LCAT) generating cholesterol esters (CEs) and forming mature HDL. HDL is also formed by ABCA1 in the liver and intestine (not shown). Mature HDL particles can serve as acceptors for ABCG1-mediated cholesterol efflux. In the sub-endothelial space, mature HDL particles are remodeled in the arterial wall by hepatic lipases (HL), endothelial lipase (EL) and phospholipid transfer protein (PLTP) secreted by macrophages. In addition, large HDL may promote efflux of cholesterol and oxysterols from endothelial cells, which express abundant ABCG1.

CELLULAR MECHANISMS OF CHOLESTEROL EFFLUX VIA ABCA1 AND ABCG1 ABCA1 and ABCG1 are ATPases that promote unidirectional, net cholesterol efflux to lipid-poor helical apolipoproteins and lipoprotein particles, respectively (Wang et al., 2006). In transfected cells and in macrophages, ABCA1 and ABCG1 can act in a sequential fashion, with ABCA1 generating nascent HDL particles which then promote cholesterol efflux via ABCG1 (Gelissen et al., 2006). Genetic knock-down studies suggest that ABCA1 and ABCG1 together account for about 60e70% of the net cholesterol efflux to HDL or serum from cholesterol-loaded LXR-activated macrophages (Yvan-Charvet et al., 2007b). Even though scavenger receptor class BI (SR-BI) can promote the bidirectional exchange of FC between cells and HDL, SR-BI knockout macrophages have no change in net cholesterol efflux to HDL, suggesting it does not make a significant contribution to this process. ABCA1 promotes efflux of phospholipids and cholesterol to lipid-poor apoA-I in a process that involves the direct binding of apoA-I to the transporter (Oram et al., 2000; Wang et al., 2000). Most likely ABCA1 translocates phospholipids from the inner to the outer membrane leaflet of the plasma membrane, perhaps creating outward curvature and packing defects in the membrane; this may allow interpolation of amphipathic helices of apoA-I into the membrane and formation of nascent HDL particles (Vedhachalam et al.,

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A

B HDL

HDL

FC, 7-KC

PL, FC, 7-KC III

ABCG1

ABCG1

I

II

IV

FC, 7-KC

ER

unsaturated phospholipid

FC, 7-KC

ER

saturated phospholipid/sphingomyelin

FIGURE 10.2 Potential mechanisms of sterol efflux mediated by ABCG1. Two different models of ABCG1- mediated sterol efflux have been proposed. (A) One model suggests that ABCG transporters accomplish free cholesterol (FC) and 7-ketocholesterol (7-KC) transfer by helping sterol molecules to overcome the energy barrier for entry into the hydrophilic water layer perhaps by utilizing ATP to promote protrusion of the cholesterol molecule into water, followed by a transient collision with acceptor, in this case HDL. (B) The ability of ABCG1 to promote phospholipid (PL) efflux points to a function as a phospholipid flippase mediating the transfer of PL from the inner- to the outer-leaflet (I). This could lead to such an extensive change in the equilibrium of membrane components that the outer-leaflet becomes more attractive to sterol (e.g., an increased content of sphingomyelin (SM) or saturated phosphatidyl choline (PC)), followed by transbilayer diffuson of cholesterol molecules towards the outer leaflet (II) where they can dissociate onto HDL particles perhaps following non-specific binding of HDL to the plasma membrane (III). In both models, the movement of cholesterol from the inner to the outer membrane is followed by carrier-facilitated diffusion from cellular organelles notably the endoplasmic reticulum (IV) leading to altered sterol-mediated ER regulation of cholesterol homeostasis.

2007). Cholesterol and phospholipid efflux via ABCA1 appear to occur simultaneously (Smith et al., 2004) and it is possible that the transporter also translocates cholesterol onto the forming HDL particle (Gillotte-Taylor et al., 2002). In contrast, ABCG1 promotes efflux of cholesterol onto a variety of lipoprotein particles, including HDL, low density lipoprotein (LDL), phospholipid vesicles and cyclodextrin, but ABCG1 does not appear to bind lipoprotein particles (Wang et al., 2004, 2006). Overexpression of ABCG1 also promotes efflux of choline-containing phospholipids onto HDL (Wang et al., 2006), likely including both sphingomyelin (SM) and phosphatidyl choline (PC) (Kobayashi et al., 2006). Two potential mechanisms of sterol efflux by ABCG transporters have been proposed (Figure 10.2): 1. Small (2003) has suggested that ABCG5/8, heterodimeric transporters that promote secretion of cholesterol and plant sterols into bile, mediate protrusion of the hydrophobic sterol molecule into the aqueous phase, followed by

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collision with a micelle. By analogy ABCG1 could promote protrusion of cholesterol from the plasma membrane followed by transient collision with an HDL particle (see Figure 10.2A) 2. Alternatively, ABCG1 could change the organization of phospholipids in the plasma membrane such that the membrane more readily releases sterol to lipoprotein acceptors (see Figure 10.2B). Cholesterol efflux would then be facilitated by diffusion or collision of lipoprotein particles with the plasma membrane. This model is consistent with the non-specific nature and lack of binding of lipoprotein acceptors by ABCG1. It is notable that ABCG1 appears to promote movement of cholesterol from the endoplasmic reticulum (ER) to the plasma membrane, manifested as decreased cellular ACAT activity and decreased activity of SREBP-2 target genes, even in the absence of extracellular lipoprotein acceptors (Wang et al., 2006). Thus, ABCG1 appears to increase the affinity of the plasma membrane for ERderived sterols even while releasing sterols more readily to extracellular lipoprotein acceptors (Wang et al., 2006). This could be achieved by promoting the movement of sterol from the inner to the outer leaflet of the plasma membrane. Since sterol can readily flip-flop across bilayer membranes (Lange et al., 1981), these findings imply a change in phospholipid organization that results in a change in the bilayer distribution of sterol, such that the sterol content of the outer leaflet of the plasma membrane is increased.

THE ROLE OF ABCA1- AND ABCG1-MEDIATED CHOLESTEROL EFFLUX PATHWAYS IN ATHEROSCLEROSIS Several different potential cholesterol efflux pathways from macrophages to HDL have been documented: passive or diffusional efflux; efflux associated with macrophage apoE secretion; SR-BI-mediated cholesterol efflux; and active cholesterol efflux mediated by ABCA1 and ABCG1 (Yancey et al., 2003; Tall, 2003). Recent studies indicate that, together, ABCA1 and ABCG1 have the major role in mediating net cholesterol efflux from macrophages to HDL or serum (Adorni et al., 2007; Out et al., 2007a; Yvan-Charvet et al., 2007a, 2008). When one transporter is deficient, the other is induced as a result of sterol accumulation and LXR activation, resulting in mutual compensation in the activities of the two transporters (Ranalletta et al., 2006; Yvan-Charvet et al., 2007b). Macrophages with combined deficiency of ABCA1 and ABCG1 have major defects in cholesterol efflux to apoA-I, HDL and serum (Out et al., 2007a; Yvan-Charvet et al., 2007a). In one study, combined deficiency of ABCA1 and ABCG1 resulted in a 60% decrease in macrophage net cholesterol efflux to HDL (Yvan-Charvet et al., 2007a), while in another study, the decrease in cholesterol efflux was reported as 100% (Out et al., 2007a). A third set of studies suggested major roles of ABCA1 and ABCG1 in cholesterolloaded macrophages and a relatively larger role of passive cholesterol efflux

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from cells to HDL especially when macrophages are not loaded with cholesterol (Adorni et al., 2007). Abca1
/
Abcg1
/
mice showed much greater accumulation of cholesterol in peritoneal macrophages on either chow or high cholesterol diets compared to either Abca1
/
or Abcg1
/
mice (Out et al., 2007a,b; Yvan-Charvet et al., 2007a). Together these studies have confirmed that ABCA1 and ABCG1 account for the major portion of the net cholesterol efflux from cholesterol-loaded macrophages to plasma lipoproteins in vivo. The atherosclerosis studies provide a close parallel with the cholesterol efflux data and indicate that combined deficiency of ABCA1 and ABCG1 in bone marrow-derived cells results in a dramatic deterioration of atherosclerosis. Transplantation of Abca1
/
bone marrow into Ldlr
/
or ApoE
/
mice results in a modest increase in atherosclerosis (Aiello et al., 2002; van Eck et al., 2002). In contrast, transplantation of Abcg1
/
bone marrow into Ldlr
/
or apoE
/
mice resulted in either no change or a decrease in atherosclerosis (Baldan et al., 2006; Ranalletta et al., 2006; Out et al., 2007b). The decrease in atherosclerosis was attributed to either compensatory upregulation of ABCA1 in Abcg1
/
macrophages (Ranalletta et al., 2006), or to enhanced apoptosis of Abcg1
/
macrophage (Baldan et al., 2006). Importantly, transplantation of Abca1
/
Abcg1
/
bone marrow into Ldlrþ/
mice, followed by feeding a high cholesterol diet led to markedly increased atherosclerosis compared to mice receiving bone marrow with single deficiency of ABCA1 or ABCG1 (Yvan-Charvet et al., 2007a). Moreover, pharmacological studies with synthetic LXR agonists demonstrated the increased expression of ABCA1 and ABCG1 in the arterial wall, and consequent reduction of atherosclerosis in both ApoE
/
and Ldlr
/
mice (Joseph et al., 2002; Terasaka et al., 2003).

A POTENTIAL ROLE OF HDL, ABCA1 AND ABCG1 IN MACROPHAGE APOPTOSIS AND INFLAMMATION IN PLAQUES The death of macrophages in atherosclerotic lesions by apoptosis or postapoptotic necrosis is thought to contribute to inflammation, necrotic core formation and destabilization of plaques (Tabas, 2005). HDL was found to protect macrophages from apoptosis induced by oxidized LDL, or by loading with FC (Cui et al., 2007; Terasaka et al., 2007). In the case of oxidized LDLinduced apoptosis, the protective effect of HDL was abolished in Abcg1
/
macrophages (Terasaka et al., 2007). In contrast, for FC-induced apoptosis, deficiency of both ABCG1 and ABCA1 was required to see an abolition of the protective effect of HDL (Yvan-Charvet et al., 2007b). ABCG1 has a specific role in promoting cellular efflux of sterols modified at the 7-position such as 7-ketocholesterol (7-KC) (Terasaka et al., 2007). 7-KC is a spontaneously formed cholesterol oxidation product that is present in high cholesterol diets, and is the most abundant oxysterol in oxidized LDL and in human atherosclerotic plaques (Brown et al., 1996; Vine et al., 1998; Brown and Jessup,

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1999). Dietary 7-KC is normally absorbed on chylomicrons, rapidly cleared from the circulation in remnants and converted into bile salts in the liver. However, at concentrations apparently found in vivo 7-KC is cytotoxic, inducing apoptosis and necrosis of endothelial cells and macrophages. Previous studies found that HDL could promote efflux of 7-KC from macrophages, while apoA-I had only limited ability to do so (Terasaka et al., 2007). HDL and ABCG1 have a specific role in promoting efflux of 7-KC and 7b-OH cholesterol in transfected 293 cells and macrophages, while ABCA1 and apoA-I have no ability to stimulate efflux of these oxysterols. Other oxysterols such as 25OH cholesterol can undergo efflux by both ABCA1 and ABCG1 pathways. Moreover, Abcg1
/
mice fed the Western diet showed prominent accumulation of 7-KC in macrophages. ABCG1 is also highly expressed in endothelial cells (ECs) (O’Connell et al., 2004). The role of ABCG1 in promoting efflux of 7-KC suggests that large HDL2 particles that promote sterol efflux via ABCG1 may have a particular role in protecting endothelial cells and macrophages from the deleterious effects of oxysterols consumed in the diet or formed on LDL (Terasaka et al., 2007). This could be important in maintaining normal endothelial functions and in atherosclerotic plaque stabilization (Libby et al., 2002). In addition to the severe defect in cholesterol efflux, Abca1
/
Abcg1
/
macrophages showed increased mRNA and secretion of chemokines and inflammatory cytokines (Yvan-Charvet et al., 2007a). The single deficiency of ABCG1 produced a similar though milder defect in secretion of inflammatory cytokines and chemokines, while the deficiency of ABCA1 had smaller effects (Yvan-Charvet et al., 2007a). Abca1
/
macrophages show increased tumor necrosis factor (TNF) secretion following treatment with lipopolysaccharide (LPS) (Koseki et al., 2007) and it is possible that macrophage inflammatory responses were mediated by LPS (Yvan-Charvet et al., 2007a). The increased inflammatory gene expression was abolished in macrophages deficient in Toll-like receptor 4 (TLR4) or MyD88/TRIF (Yvan-Charvet et al., 2008). TLR4 cell surface concentration was increased in the order Abca1
/
Abcg1
/
>Abcg1
/
>Abca1
/
>wild-type macrophages (Yvan-Charvet et al., 2008). Treatment of transporter-deficient cells with cyclodextrin reduced and cholesterol-cyclodextrin loading increased inflammatory gene expression. Abca1
/
Abcg1
/
bone marrow-derived macrophages showed enhanced inflammatory gene responses to TLR2, TLR3, and TLR4 ligands (YvanCharvet et al., 2008). In Abcg1
/
bone marrow-transplanted, Western dietfed Ldlr-deficient mice, there was a profound inflammatory infiltrate in the adventitia and necrotic core region of atherosclerotic lesions, consisting primarily of neutrophils after intraperitoneal injection with thioglycollate (Yvan-Charvet et al., 2008). Other studies have shown that TLR4 mutant mice have decreased atherosclerosis (Michelsen et al., 2004), and that repeated injections of LPS worsen atherosclerosis in mice and rabbits (Lehr et al., 2001; Westerterp et al., 2007). The results suggest that HDL and apoA-I exert anti-inflammatory effects by promoting cholesterol efflux via

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ABCG1 and ABCA1 with consequent attenuation of signaling via Toll-like receptors. Thus, HDL and ABC transporters may be important in protecting advanced plaques from macrophage apoptosis and inflammatory responses, suggesting a role in plaque stabilization and acute coronary syndromes.

A POTENTIAL ROLE OF HDL AND ABCG1 IN ENDOTHELIAL FUNCTION A part of the atheroprotective effect of HDL may be related to its role in preserving endothelial function (Li et al., 2000; Kuvin et al., 2003). In humans, HDL levels are correlated with flow-mediated vasodilation responses of the brachial artery (Li et al., 2000; Kuvin et al., 2003) and with decreased coronary vasoconstrictor responses (Zeiher et al., 1994). Importantly, infusion of recombinant phospholipid/apoA-I particles into Tangier disease with isolated low HDL levels reversed defective forearm blood flow measurements (Bisoendial et al., 2003). Niacin therapy has been shown to improve NO-mediated vascular relaxation in humans (Kuvin et al., 2002). The beneficial effects of HDL on ECs may include stimulation of proliferation, cell survival, migration, inhibition of the expression of VCAM-1 and ICAM-1 and NO synthesis (Collins and Cybulsky, 2001; O’Connell et al., 2004; Rohrer et al., 2004; Mineo et al., 2006). HDL may have a specific role in reversing decreased eNOS activity in human ECs treated with oxidized LDL (Uittenbogaard et al., 2000) or in reversing the decrease in eNOS dependent vascular relaxation induced by high cholesterol diets (Deckert et al., 1999). Arteries from Abcg1
/
and Abca1
/
Abcg1
/
mice fed a high cholesterol diet exhibited a marked decrease in endothelium-dependent vasorelaxation, while Abca1
/
mice had a relative milder defect (Terasaka et al., 2008). In addition, eNOS activity was reduced in aortic homogenates of Abcg1
/
mice fed either the high cholesterol diet or a Western diet, and this correlated with decreased levels of the active dimeric form of eNOS (Terasaka et al., 2008). More detailed analysis indicated that ABCG1 was expressed primarily in ECs, and that these cells accumulated 7-KC when Abcg1
/
mice were fed the Western diet (Terasaka et al., 2008). Consistent with these data, ABCG1 had a major role in promoting efflux of cholesterol and 7-KC to HDL in cultured human aortic ECs (HAECs) (Terasaka et al., 2008). Furthermore, HDL treatment of HAECs prevented 7-KC-induced reactive oxygen species production and active eNOS dimer disruption in an ABCG1-dependent manner (Terasaka et al., 2008). In addition, apoA-I transgene expression increased HDL, improved endothelial function, decreased aortic 7-KC content and increased eNOS activity in high cholesterol-fed Ldlrþ/
mice (Terasaka et al., 2008). The ability of ABCG1 to preserve endothelial function appears to be at least partly related to its role in promoting efflux of 7-oxysterols such as 7-KC to HDL.

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CONCLUSION AND PERSPECTIVE ABCA1 and ABCG1 sterol efflux pathways show mutual compensation, and deficiency of both transporters results in a dramatic increase in macrophage inflammatory and apoptotic responses and in atherosclerosis (Yvan-Charvet et al., 2007a). Treatments that increase cholesterol efflux via ABCA1 and/or ABCG1 are likely to be beneficial for atherosclerosis, though perhaps with a different spectrum of clinical effects: the ABCG1 pathway might have a particular role in protecting vulnerable plaques as a result of its anti-apoptotic, anti-inflammatory functions and improvement of endothelial function. It is possible that in vivo CETP inhibitors and niacin increase cholesterol efflux via ABCG1, but also, by decreasing catabolism of HDL in the kidney and liver, lead to an increase in apoA-I levels which may be made available to foam cell ABCA1 in the arterial wall. A variety of different approaches to raising HDL are likely to be further developed. Drugs that increase expression of apoA-I in the liver, or infusion of apoA-I or peptides derived from apoA-I that have been optimized for interaction with ABCA1 or LCAT are also likely to be beneficial (Belalcazar et al., 2003; Nissen et al., 2003; Linsel-Nitschke and Tall, 2005; Navab et al., 2006; Rader, 2007). These approaches may also lead to increased formation and levels of HDL particles that can in turn promote efflux of sterols via ABCG1. Therapeutic strategies that upregulate both ABCA1 and ABCG1 transporters themselves may also be beneficial as low levels of the transporters themselves may be rate-limiting for cellular cholesterol efflux in some subjects with coronary heat disease (Trogan et al., 2006). Thus, assuming problems of increased triglycerides can be solved (Repa et al., 2000; Schultz et al., 2000), LXR agonists might be ideal agents for increasing a variety of molecules involved in sterol efflux (Bradley et al., 2007; Scott, 2007).

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atherosclerosis progression and remodels atherosclerotic plaques in a mouse model of familial hypercholesterolemia. Circulation, 107, 2726e2732. Bisoendial, R. J., Hovingh, G. K., Levels, J. H., et al. (2003). Restoration of endothelial function by increasing high-density lipoprotein in subjects with isolated low high-density lipoprotein. Circulation, 107, 2944e2948. Bodzioch, M., Orso, E., Klucken, J., et al. (1999). The gene encoding ATP-binding cassette transporter 1 is mutated in Tangier disease. Nat Genet, 22, 347e351. Bradley, M. N., Hong, C., Chen, M., et al. (2007). Ligand activation of LXR beta reverses atherosclerosis and cellular cholesterol overload in mice lacking LXR alpha and apoE. J Clin Invest, 117, 2337e2346. Brooks-Wilson, A., Marcil, M., Clee, S. M., et al. (1999). Mutations in ABC1 in Tangier disease and familial high-density lipoprotein deficiency. Nat Genet, 22, 336e345. Brown, A. J., & Jessup, W. (1999). Oxysterols and atherosclerosis. Atherosclerosis, 142, 1e28. Brown, A. J., Dean, R. T., & Jessup, W. (1996). Free and esterified oxysterol: formation during copper-oxidation of low density lipoprotein and uptake by macrophages. J Lipid Res, 37, 320e335. Buechler, C., Boettcher, A., Bared, S. M., Probst, M. C., & Schmitz, G. (2002). The carboxyterminus of the ATP-binding cassette transporter A1 interacts with a beta2-syntrophin/ utrophin complex. Biochem Biophys Res Commun, 293, 759e765. Castelli, W. P., Garrison, R. J., Wilson, P. W., Abbott, R. D., Kalousdian, S., & Kannel, W. B. (1986). Incidence of coronary heart disease and lipoprotein cholesterol levels. The Framingham Study. J Am Med Assoc, 256, 2835e2838. Cohen, J. C., Kiss, R. S., Pertsemlidis, A., Marcel, Y. L., McPherson, R., & Hobbs, H. H. (2004). Multiple rare alleles contribute to low plasma levels of HDL cholesterol. Science, 305, 869e872. Collins, T., & Cybulsky, M. I. (2001). NF-kappaB: pivotal mediator or innocent bystander in atherogenesis? J Clin Invest, 107, 255e264. Costet, P., Luo, Y., Wang, N., & Tall, A. R. (2000). Sterol-dependent transactivation of the ABC1 promoter by the liver X receptor/retinoid X receptor. J Biol Chem, 275, 28240e28245. Cui, D., Thorp, E., Li, Y., et al. (2007). Pivotal advance: macrophages become resistant to cholesterol-induced death after phagocytosis of apoptotic cells. J Leukoc Biol, 82, 1040e1050. Deckert, V., Lizard, G., Duverger, N., et al. (1999). Impairment of endothelium-dependent arterial relaxation by high-fat feeding in ApoE-deficient mice. Circulation, 100, 1230e1235. Fitzgerald, M. L., Mendez, A. J., Moore, A. J., Andersson, L. P., Panjeton, H. A., & Freeman, M. W. (2001). ATP-binding cassette transporter A1 contains an NH2-terminal signal anchor sequence that translocates the protein’s first hydrophilic domain to the exoplasmic space. J Biol Chem, 276, 15137e15145. Francis, G. A., Knopp, R. H., & Oram, J. F. (1995). Defective removal of cellular cholesterol and phospholipids by apolipoprotein A-I in Tangier disease. J Clin Invest, 96, 78e87. Frikke-Schmidt, R., Nordestgaard, B. G., Jensen, G. B., & Tybjaerg-Hansen, A. (2004). Genetic variation in ABC transporter A1 contributes to HDL cholesterol in the general population. J Clin Invest, 114, 1343e1353. Fu, X., Menke, J. G., Chen, Y., et al. (2001). 27-Hydroxycholesterol is an endogenous ligand for liver X receptor in cholesterol-loaded cells. J Biol Chem, 276, 38378e38387. Gelissen, I. C., Harris, M., Rye, K. A., et al. (2006). ABCA1 and ABCG1 synergize to mediate cholesterol export to apoA-I. Arterioscler Thromb Vasc Biol, 26, 534e540.

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Gillotte-Taylor, K., Nickel, M., Johnson, W. J., et al. (2002). Effects of enrichment of fibroblasts with unesterified cholesterol on the efflux of cellular lipids to apolipoprotein A-I. J Biol Chem, 277, 11811e11820. Haghpassand, M., Bourassa, P. A., Fancone, O. L., & Aiello, R. J. (2001). Monocyte/macrophage expression of ABCA1 has minimal contribution to plasma HDL levels. J Clin Invest, 108, 1315e1320. Haidar, B., Denis, M., Krimbou, L., Marcil, M., & Genest, J., Jr. (2002). cAMP induces ABCA1 phosphorylation activity and promotes cholesterol efflux from fibroblasts. J Lipid Res, 43, 2087e2094. Haidar, B., Denis, M., Marcil, M., Krimbou, L., & Genest, J., Jr. (2004). ApolipoproteinA-I activates cellular cAMP signaling through the ABCA1 transporter. J Biol Chem, 279, 9963e9969. Joseph, S. B., McKilligin, E., Pei, L., et al. (2002). Synthetic LXR ligand inhibits the development of atherosclerosis in mice. Proc Natl Acad Sci USA, 99, 7604e7609. Kennedy, M. A., Venkateswaran, A., Tarr, P. T., et al. (2001). Characterization of the human ABCG1 gene: liver X receptor activates an internal promoter that produces a novel transcript encoding an alternative form of the protein. J Biol Chem, 276, 39438e39447. Kennedy, M. A., Barrera, G. C., Nakamura, K., et al. (2005). ABCG1 has a critical role in mediating cholesterol efflux to HDL and preventing cellular lipid accumulation. Cell Metab, 1, 121e131. Kobayashi, A., Takanezawa, Y., Hirata, T., et al. (2006). Efflux of sphingomyelin, cholesterol, and phosphatidylcholine by ABCG1. J Lipid Res, 47, 1791e1802. Koseki, M., Hirano, K., Masuda, D., et al. (2007). Increased lipid rafts and accelerated lipopolysaccharide-induced tumor necrosis factor-alpha secretion in Abca1-deficient macrophages. J Lipid Res, 48, 299e306. Kuvin, J. T., Ramet, M. E., Patel, A. R., Pandian, N. G., Mendelsohn, M. E., & Karas, R. H. (2002). A novel mechanism for the beneficial vascular effects of highdensity lipoprotein cholesterol: enhanced vasorelaxation and increased endothelial nitric oxide synthase expression. Am Heart J, 144, 165e172. Kuvin, J. T., Patel, A. R., Sidhu, M., et al. (2003). Relation between high-density lipoprotein cholesterol and peripheral vasomotor function. Am J Cardiol, 92, 275e279. Lange, Y., Dolde, J., & Steck, T. L. (1981). The rate of transmembrane movement of cholesterol in the human erythrocyte. J Biol Chem, 256, 5321e5323. Lehr, H. A., Sagban, T. A., Ihling, C., et al. (2001). Immunopathogenesis of atherosclerosis: endotoxin accelerates atherosclerosis in rabbits on hypercholesterolemic diet. Circulation, 104, 914e920. Li, X. P., Zhao, S. P., Zhang, X. Y., Liu, L., Gao, M., & Zhou, Q. C. (2000). Protective effect of high density lipoprotein on endothelium-dependent vasodilatation. Int J Cardiol, 73, 231e236. Libby, P., Ridker, P. M., & Maseri, A. (2002). Inflammation and atherosclerosis. Circulation, 105, 1135e1143. Linsel-Nitschke, P., & Tall, A. R. (2005). HDL as a target in the treatment of atherosclerotic cardiovascular disease. Nat Rev Drug Discov, 4, 193e205. Martinez, L. O., Agerholm-Larsen, B., Wang, N., Chen, W., & Tall, A. R. (2003). Phosphorylation of a pest sequence in ABCA1 promotes calpain degradation and is reversed by ApoA-I. J Biol Chem, 278, 37368e37374.

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Matsuura, F., Wang, N., Chen, W., Jiang, X. C., & Tall, A. R. (2006). HDL from CETP-deficient subjects shows enhanced ability to promote cholesterol efflux from macrophages in an apoEand ABCG1-dependent pathway. J Clin Invest, 116, 1435e1442. Michelsen, K. S., Wong, M. H., Shah, P. K., et al. (2004). Lack of Toll-like receptor 4 or myeloid differentiation factor 88 reduces atherosclerosis and alters plaque phenotype in mice deficient in apolipoprotein E. Proc Natl Acad Sci USA, 101, 10679e10684. Mineo, C., Deguchi, H., Griffin, J. H., & Shaul, P. W. (2006). Endothelial and antithrombotic actions of HDL. Circ, Res, 98, 1352e1364. Munehira, Y., Ohnishi, T., Kawamoto, S., et al. (2004). Alpha1-syntrophin modulates turnover of ABCA1. J Biol Chem, 279, 15091e15095. Nakamura, K., Kennedy, M. A., Baldan, A., Bojanic, D. D., Lyons, K., & Edwards, P. A. (2004). Expression and regulation of multiple murine ATP-binding cassette transporter G1 mRNAs/ isoforms that stimulate cellular cholesterol efflux to high density lipoprotein. J Biol Chem, 279, 45980e45989. Navab, M., Anantharamaiah, G. M., Reddy, S. T., & Fogelman, A. M. (2006). Apolipoprotein A-I mimetic peptides and their role in atherosclerosis prevention. Nat Clin Pract Cardiovasc Med, 3, 540e547. Nissen, S. E., Tsunoda, T., Tuzcu, E. M., et al. (2003). Effect of recombinant ApoA-I Milano on coronary atherosclerosis in patients with acute coronary syndromes: a randomized controlled trial. J Am Med Assoc, 290, 2292e2300. O’Connell, B. J., Denis, M., & Genest, J. (2004). Cellular physiology of cholesterol efflux in vascular endothelial cells. Circulation, 110, 2881e2888. Okuhira, K., Fitzgerald, M. L., Sarracino, D. A., et al. (2005). Purification of ATP-binding cassette transporter A1 and associated binding proteins reveals the importance of beta1-syntrophin in cholesterol efflux. J Biol Chem, 280, 39653e39664. Oram, J. F., Lawn, R. M., Garvin, M. R., & Wade, D. P. (2000). ABCA1 is the cAMP inducible apolipoprotein receptor that mediates cholesterol secretion from macrophages. J Biol Chem, 275, 34508e34511. Ou, J., Tu, H., Shan, B., et al. (2001). Unsaturated fatty acids inhibit transcription of the sterol regulatory element-binding protein-1c (SREBP-1c) gene by antagonizing ligand-dependent activation of the LXR. Proc Natl Acad Sci USA, 98, 6027e6032. Out, R., Hoekstra, M., Habets, K., et al. (2007). Combined deletion of macrophage ABCA1 and ABCG1 leads to massive lipid accumulation in tissue macrophages and distinct atherosclerosis at relatively low plasma cholesterol levels. Arterioscler Thromb Vasc Biol, 28, 258e264. Out, R., Jessup, W., Le Goff, W., et al. (2007a). Coexistence of foam cells and hypocholesterolemia in mice lacking the ABC transporters A1 and G1. Circ Res, 102, 113e120. Rader, D. J. (2006). Molecular regulation of HDL metabolism and function: implications for novel therapies. J Clin Invest, 116, 3090e3100. Rader, D. J. (2007). Mechanisms of disease: HDL metabolism as a target for novel therapies. Nat Clin Pract, 4, 102e109. Ranalletta, M., Wang, N., Han, S., Yvan-Charvet, L., Welch, C., & Tall, A. R. (2006). Decreased atherosclerosis in low-density lipoprotein receptor knockout mice transplanted with Abcg1
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Rhoads, G. G., Gulbrandsen, C. L., & Kagan, A. (1976). Serum lipoproteins and coronary heart disease in a population study of Hawaii Japanese men. N Engl J Med, 294, 293e298. Rohrer, L., Hersberger, M., & von Eckardstein, A. (2004). High density lipoproteins in the intersection of diabetes mellitus, inflammation and cardiovascular disease. Curr Opin Lipidol, 15, 269e278. Roosbeek, S., Peelman, F., Verhee, A., et al. (2004). Phosphorylation by protein kinase CK2 modulates the activity of the ATP binding cassette A1 transporter. J Biol Chem, 279, 37779e37788. Rust, S., Rosier, M., Funke, H., et al. (1999). Tangier disease is caused by mutations in the gene encoding ATP-binding cassette transporter. Nat Genet, 22, 352e355. Santamarina-Fojo, S., Peterson, K., Knapper, C., et al. (2000). Complete genomic sequence of the human ABCA1 gene: analysis of the human and mouse ATP-binding cassette A promoter. Proc Natl Acad Sci USA, 97, 7987e7992. Schultz, J. R., Tu, H., Luk, A., et al. (2000). Role of LXRs in control of lipogenesis. Genes Dev, 14, 2831e2838. Scott, J. (2007). The liver X receptor and atherosclerosis. N Engl J Med, 357, 2195e2197. Singaraja, R. R., Fievet, C., Castro, G., et al. (2002). Increased ABCA1 activity protects against atherosclerosis. J Clin Invest, 110, 35e42. Small, D. M. (2003). Role of ABC transporters in secretion of cholesterol from liver into bile. Proc Natl Acad Sci USA, 100, 4e6. Smith, J. D., Le Goff, W., Settle, M., et al. (2004). ABCA1 mediates concurrent cholesterol and phospholipid efflux to apolipoprotein A-I. J Lipid Res, 45, 635e644. Tabas, I. (2005). Consequences and therapeutic implications of macrophage apoptosis in atherosclerosis: the importance of lesion stage and phagocytic efficiency. Arterioscler Thromb Vasc Biol, 25, 2255e2264. Tall, A. R. (2003). Role of ABCA1 in cellular cholesterol efflux and reverse cholesterol transport. Arterioscler Thromb Vasc Biol, 23, 710e711. Tall, A. R., Costet, P., & Wang, N. (2002). Regulation and mechanisms of macrophage cholesterol efflux. J Clin Invest, 110, 899e904. Tang, C., Vaughan, A. M., & Oram, J. F. (2004). Janus kinase 2 modulates the apolipoprotein interactions with ABCA1 required for removing cellular cholesterol. J Biol Chem, 279, 7622e7628. Terasaka, N., Wang, N., Yvan-Charvet, L., & Tall, A. R. (2007). High-density lipoprotein protects macrophages from oxidized low-density lipoprotein-induced apoptosis by promoting efflux of 7-ketocholesterol via ABCG1. Proc Natl Acad Sci USA, 104, 15093e15098. Terasaka, N., Hiroshima, A., Koieyama, T., et al. (2003). T-0901317, a synthetic liver X receptor ligand, inhibits development of atherosclerosis in LDL receptor-deficient mice. FEBS Lett, 536, 6e11. Terasaka, N., Yu, S., Yvan-Charvet, L., et al. (2008). ABCG1 and HDL protect against endothelial dysfunction in mice fed a high-cholesterol diet. J Clin Invest, 118, 3701e3713. Timmins, J. M., Lee, J. Y., Boudyguina, E., et al. (2005). Targeted inactivation of hepatic Abca1 causes profound hypoalphalipoproteinemia and kidney hypercatabolism of apoA-I. J Clin Invest, 115, 1333e1342. Tontonoz, P., & Mangelsdorf, D. J. (2003). Liver X receptor signaling pathways in cardiovascular disease. Mol Endocrinol, 17, 985e993. Trogan, E., Feig, J. E., Dogan, S., et al. (2006). Gene expression changes in foam cells and the role of chemokine receptor CCR7 during atherosclerosis regression in ApoE-deficient mice. Proc Natl Acad Sci USA, 103, 3781e3786.

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Tsukamoto, T., Hirano, K., Tsujii, K., et al. (2001). ATPbinding cassette transporter-1 induces rearrangement of actin cytoskeletons possibly through Cdc42/N-WASP. Biochem Biophys Res Commun, 287, 757e765. Uittenbogaard, A., Shaul, P. W., Yuhanna, I. S., Blair, A., & Smart, E. J. (2000). High density lipoprotein prevents oxidized low density lipoprotein-induced inhibition of endothelial nitricoxide synthase localization and activation in caveolae. J Biol Chem, 275, 11278e11283. Vaisman, B. L., Lambert, G., Amar, M., et al. (2001). ABCA1 overexpression leads to hyperalphalipoproteinemia and increased biliary cholesterol excretion in transgenic mice. J Clin Invest, 108, 303e309. van Eck, M., Bos, I. S., Kaminski, W. E., et al. (2002). Leukocyte ABCA1 controls susceptibility to atherosclerosis and macrophage recruitment into tissues. Proc Natl Acad Sci USA, 99, 6298e6303. Vaughan, A. M., & Oram, J. F. (2005). ABCG1 redistributes cell cholesterol to domains removable by HDL but not by lipid-depleted apolipoproteins. J Biol Chem, 280, 30150e30157. Vedhachalam, C., Duong, P. T., Nickel, M., et al. (2007). Mechanism of ATP-binding cassette transporter A1-mediated cellular lipid efflux to apolipoprotein A-I and formation of high density lipoprotein particles. J Biol Chem, 282, 25123e25130. Vine, D. F., Mamo, C. L., Beilin, L. J., Mori, T. A., & Croft, K. D. (1998). Dietary oxysterols are incorporated in plasma triglyceride-rich lipoproteins, increase their susceptibility to oxidation and increase aortic cholesterol concentration of rabbits. J Lipid Res, 39, 1995e2004. Wang, Y., & Oram, J. F. (2002). Unsaturated fatty acids inhibit cholesterol efflux from macrophages by increasing degradation of ATP-binding cassette transporter A1. J Biol Chem, 277, 5692e5697. Wang, Y., & Oram, J. F. (2005). Unsaturated fatty acids phosphorylate and destabilize ABCA1 through a phospholipase D2 pathway. J Biol Chem, 280, 35896e35903. Wang, N., Silver, D. L., Costet, P., & Tall, A. R. (2000). Specific binding of ApoA-I, enhanced cholesterol efflux, and altered plasma membrane morphology in cells expressing ABC1. J Biol Chem, 275, 33053e33058. Wang, N., Lan, D., Chen, W., Matsuura, F., & Tall, A. R. (2004). ATP-binding cassette transporters G1 and G4 mediate cellular cholesterol efflux to high-density lipoproteins. Proc Natl Acad Sci USA, 101, 9774e9779. Wang, N., Ranalletta, M., Matsuura, F., Peng, F., & Tall, A. R. (2006). LXR induced redistribution of ABCG1 to plasma membrane in macrophages enhances cholesterol mass efflux to HDL. Arterioscler Thromb Vasc Biol, 26, 1310e1316. Westerterp, M., Berbee, J. F., Pires, N. M., et al. (2007). Apolipoprotein C-I is crucially involved in lipopolysaccharide-induced atherosclerosis development in apolipoprotein E-knockout mice. Circulation, 116, 2173e2181. Yancey, P. G., Bortnick, A. E., Kellner-Weibel, G., de la Llera-Moya, M., Phillips, M. C., & Rothblat, G. H. (2003). Importance of different pathways of cellular cholesterol efflux. Arterioscler Thromb Vasc Biol, 23, 712e719. Yvan-Charvet, L., Ranalletta, M., Wang, N., et al. (2007a). Combined deficiency of ABCA1 and ABCG1 promotes foam cell accumulation and accelerates atherosclerosis in mice. J Clin Invest, 117, 3900e3908. Yvan-Charvet, L., Matsuura, F., Wang, N., et al. (2007b). Inhibition of cholesteryl ester transfer protein by torcetrapib modestly increases macrophage cholesterol efflux to HDL. Arterioscler Thromb Vasc Biol, 27, 1132e1138. Yvan-Charvet, L., Pagler, T. A., Wang, N., et al. (2008). SR-BI inhibits ABCG1-stimulated net cholesterol efflux from cells to plasma HDL. J Lipid Res, 49, 107e114.

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Yvan-Charvet, L., Welch, C., Pagler, T. A., et al. (2008). Increased inflammatory gene expression in ABC transporter-deficient macrophages: free cholesterol accumulation, increased signaling via toll-like receptors, and neutrophil infiltration of atherosclerotic lesions. Circulation, 118, 1837e1847. Zeiher, A. M., Schachlinger, V., Hohnloser, S. H., Saurbier, B., & Just, H. (1994). Coronary atherosclerotic wall thickening and vascular reactivity in humans. Elevated high-density lipoprotein levels ameliorate abnormal vasoconstriction in early atherosclerosis. Circulation, 89, 2525e2532.

Chapter 11

Functional Change in the HDL Particle by Oxidative Modification and its Contribution to Atherogenesis Toshiyuki Matsunaga 1, Akira Hara 1 and Tsugikazu Komoda 2 1 Laboratory of Biochemistry, Gifu Pharmaceutical University, 2 Department of Biochemistry, Saitama Medical University, Japan

INTRODUCTION In epidemiological studies, low concentrations of high density lipoprotein (HDL) have been shown to inversely correlate with the risk of atherosclerosis and the associated cardiovascular diseases (Reichl and Miller, 1989; Rader, 2003; Assmann and Gotto, 2004). HDL is involved in the transportation of cholesterol from extrahepatic tissues to the liver. In the system of so-called reverse cholesterol transport, HDL can also enhance the cholesterol efflux from macrophage-derived foam cells (Brown et al., 1980). Therefore, the system is considered to be a major anti-atherogenic function of HDL (Steinberg, 1978; Tall, 1990; Johnson et al., 1991; Rader, 2003; Assmann and Gotto, 2004). Recent studies have, in addition, found that HDL has other abilities to protect from atherogenic events. Those beneficial abilities of HDL are to inhibit low density lipoprotein (LDL) oxidation, vascular cell apoptosis and endothelial dysfunction (O’Connell and Genest, 2001; Rohrer et al., 2004). In order to protect from the endothelial dysfunction observed at the onset and development of atherosclerosis, HDL appears to exert a variety of actions including enhanced production of nitric oxide (NO) and downregulation of adhesion molecules (Collins and Cybulsky, 2001; O’Connell and Genest, 2001; Rohrer et al., 2004). Based on multiple investigations, HDL has newly been reported to possess antiviral activity (Singh and Baron, 2000; Van Lenten et al., 2001), anti-thrombotic effects (Saku et al., 1985) and prostacyclin stabilizing activity (Fleisher et al., 1982; Yui et al., 1988). The HDL Handbook. ISBN: 978-0-12-382171-3 Copyright Ó 2010 Elsevier Inc. All rights of reproduction in any form reserved.

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HDL contains antioxidant molecules and enzymes in the particle; nevertheless, it is oxidized more rapidly than LDL during in vitro oxidation (Nakajima et al., 1995; Hurtado et al., 1996). Ohmura et al. (1999) demonstrated that HDL from patients with coronary artery spasm has a higher susceptibility to lipid peroxidative modification than LDL. This suggests a possibility that oxidized lipoproteins, including both HDL and LDL, are involved in the genesis of coronary artery diseases and reflect an oxidative stress state of the diseases. An immunohistochemical study by Vollmer et al. (1991) also revealed that apoprotein A-I (apoA-I) is present in lumen-adjacent layers of the intima in the earlier stage and in deeper layers of vascular wall of patients with the advanced stage of the diseases. Within the intimal layer, apoproteins in HDL are detected either in an intracellular (mainly in foam cells) or extracellular location, depending on the stage of atherosclerosis. The altered localization of apoproteins in the lesion suggests the contribution of oxidized HDL (ox-HDL) to each step in the atherogenic processes. Greilberger and Ju¨rgens (1998) showed that mild oxidation enhances the ability of HDL to strongly compete with binding of ox-LDL to type I and III collagens, whereas native and highly oxidized HDL are weak competitors. This may imply that longer retention in the atheromatous lesion enables ox-HDL to participate in atherogenic events, because an increased binding of ox-LDL to type I, III and IV collagens is thought to lead to the progression of atherosclerosis (Jimi et al., 1994). Our recent analyses using ox-HDL-specific antibody have shown that ox-HDL is present in the intima of atheromatous plaques in the human abdominal aorta (Nakajima et al., 2000), and its level is increased in patients with coronary artery diseases, type-II diabetes mellitus and chronic renal failure (Tsumura et al., 2001; Nakajima et al., 2004). Thus, the oxidative modification of HDL could occur in patients with a variety of vascular diseases. In addition to verification of the in vivo presence of ox-HDL, recent studies using different oxidants have proposed that oxidative modification of HDL raises multiple alterations of its components and conformation (Nagano et al., 1991; Salmon et al., 1992a; Morel, 1994; Bonnefont-Rousselot et al., 1995). In this chapter, we review the current literature on the structural and functional alterations in HDL by oxidation and discuss the clinical relevance of ox-HDL for atherogenic events.

STRUCTURAL ALTERATIONS IN HDL COMPONENTS BY OXIDATION Oxidation of HDL HDL and its lipidic components are oxidized rapidly and preferably, compared with LDL during in vitro oxidation (Bowry et al., 1992; Nakajima et al., 1995; Hurtado et al., 1996). In HDL subclasses, HDL2 is more susceptible to Cu2þ-mediated oxidation than HDL3 (Stojanovic et al., 2006). Accordingly,

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determination of oxidized lipids in HDL2 particles might be a useful marker for the earlier stages of atherogenetic events, while in vivo oxidation of HDL2 lipids has been little investigated. HDL can readily diffuse into the subendothelial fraction because its particle size is smaller than that of LDL (Sloop et al., 1987; Bjornheden et al., 1996). The long retention in the arterial intima might result in an oxidation of HDL, dependent on enhanced expression of oxidant enzymes, and lack of free radical scavengers and reducing agents. Immunohistochemical study using a monoclonal antibody against ox-HDL has shown that ox-HDL is localized in the surface of the endothelial layer and arterial intima of atheromatous plaques of the human abdominal aorta, in contrast to the localization of ox-LDL in the whole area of the plaques (Nakajima et al., 2000). This different localization between ox-HDL and oxLDL may be of crucial importance in order to clarify the role of oxidized lipoprotein in atherogenic events. In previous investigations, numerous potential mechanisms for in vivo oxidation of lipoproteins were proposed (Berliner and Heinecke, 1996). Possible in vivo mechanisms of LDL oxidation are well-documented, and include the myeloperoxidase pathway (Carr et al., 2000; Gaut et al., 2002; Zhang et al., 2002; Brennan and Hazen, 2003; Brennan et al., 2003), the 12/15-lipoxygenase pathway (Harats et al., 2000; Cyrus et al., 2001; George et al., 2001; Zhao et al., 2002), the 5-lipoxygenase pathway (Mehrabian et al., 2002; Mehrabian and Allayee, 2003; Dwyer et al., 2005), the cyclooxygenase pathway (Linton and Fazio, 2004; Natarajan and Nadler, 2004; Vila, 2004), and the NADPH oxidase pathway (Sorescu et al., 2001; Cathcart, 2004). Although there is little evidence for how ox-HDL is formed, Nagano et al. (1991) suggested that HDL is oxidized in the same manner as LDL. Myeloperoxidase, an enzyme in macrophages and neutrophils, may be one of the most potential enzymes concerning in vivo oxidant generation. This enzyme system generates reactive species including hypochlorous acid/ hypochlorite (HOCl/OCl), tyrosyl radicals, chloramines, and nitrogen dioxides (NO2) (Weiss et al., 1982; Kettle et al., 1997). Recent investigations have provided direct evidence for activation of the enzyme in human atherosclerotic lesions (Daugherty et al., 1994), and increased levels of hypochlorite-modified proteins and dityrosines in early and advanced lesions of atherosclerosis (Heinecke et al., 1993; Hazell et al., 1996; Hazen and Heinecke, 1997; Leeuwenburgh et al., 1997a). Lipoxygenase, which is produced by endothelial cells and macrophages, may also participate in the in vivo oxidation of lipoproteins, because treatment with the enzyme inhibitors prevents cell-mediated oxidation of LDL (Parthasarathy et al., 1989; Rankin et al., 1991). The oxidative mechanism of lipoproteins by lipoxygenase is mediated by conversion of polyunsaturated fatty acids into lipid peroxides including 13S-hydroxy-9,11-octadecadienoic acid, and the metabolites are detected in the early stage of atherosclerosis, accordingly suggesting the positive relationship of the enzyme with atherogenesis

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(Kuhn et al., 1997). Cyrus et al. (1999) have also reported that the inhibition of 12/15-lipoxygenase diminishes atherosclerotic lesions in apoE-deficient mice. Peroxynitrite, a potent oxidant biologically produced by the interaction of nitric oxide with superoxide anion, is thought to be relevant to cardiovascular diseases. Peroxynitrite can directly oxidize polyunsaturated fatty acids, tocopherols, carbohydrates, DNA and proteins (Christen et al., 1997; Pannala et al., 1998; Ducrocq et al., 1999). Peroxynitrite-induced nitration generates high amounts of 3-nitrotyrosine in proteins (Ischiropoulos et al., 1992; Matsunaga et al., 2001). A high level of 3-nitrotyrosine in the LDL isolated from human atherosclerotic intima suggests that 3-nitrotyrosine-containing LDL promotes atherogenesis by counteracting the anti-atherogenic effect of NO (Leeuwenburgh et al., 1997b). Interestingly, the content of 3-nitrotyrosine in HDL isolated from human aortic atherosclerotic intima is six-fold higher than that in circulating HDL (Pennathur et al., 2004). Thus, the formation of peroxynitrite during atherogenic events may also promote oxidative modification of HDL. Tocopheroxy radical (Neuzil et al., 1997), metal ions (Parthasarathy et al., 1990; Reyftmann et al., 1991; Nagano et al., 1991) and ultraviolet irradiation (Alomar et al., 1992; Salmon et al., 1992b) are also proposed as oxidants for HDL, although in vivo oxidation mechanisms have not been established. It is recognized that LDL from patients with hyperglycemia is more susceptible to oxidation than that from healthy volunteers, and that, in the LDL particles, the oxidative rate is positively related with the rate of glycation (Bowie et al., 1993). This oxidative modification of LDL is presumably due to oxygen radicals derived from amadori products, intermediates of non-enzymatic glycosylation (i.e., Maillard reaction) (Yim et al., 2000). Previous clinical investigation detected high amounts of ox-HDL in sera from subjects with type II diabetes as described above (Nakajima et al., 2004). In addition, other studies demonstrated that exposure to glucose makes HDL more susceptible to oxidation, and the oxidative inhibition of paraoxonase (PON) is responsible for the resultant HDL dysfunction such as a loss of antioxidative capacity (Hedrick et al., 2000; Matsunaga et al., 2003a; Zhou et al., 2008). These findings indicate a possibility that ox-HDL is involved in accelerating diabetic complications, besides ox-LDL, although mechanisms underlying the HDL oxidation and PON inactivation during the incubation with glucose are not fully understood. Molecular mechanisms of oxidation of the HDL particle appear to be different among oxidants. In the system including metal ions and lipoxygenase, oxidants primarily attack lipidic components, especially phospholipids and cholesterols, in the HDL particle and subsequent oxidation of apoproteins is mediated by the oxidized lipids (Marcel et al., 1989; Morel, 1994; Garner et al., 1998a). In contrast, a low dose of tyrosyl radical, which is generated by the myeloperoxidase system, oxidizes lipid-free apoproteins in HDL (Wang et al., 1998), and the apoproteins in HDL are more highly oxidized than the lipidic components (Panzenboeck et al., 1997; Wang et al., 1998). Possible

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HDL Oxidation myeloperoxidase, lipoxygenase, cyclooxygenase, peroxynitrite, tocopheroxy radical, metals and ultraviolet irradiation

ox-HDL

Structural alterations in ox-HDL Oxidation of amino acids (Lys, Trp and Met) in apoproteins Cross-linking of apoproteins Conversion of phosphatidylcholine (PC) into the lyso form Reduction in polyunsaturated fatty acids in PC Increase in hydroperoxide forms of PC and cholesterol Alteration of fluidity at surface of particle Inactivation of antioxidant enzymes (paraoxonase, lecithin: cholesterol acyltransferase and so on) Depletion of antioxidant molecules

SCHEME 11.1 Possible mechanisms for in vivo oxidation of HDL and structural alterations in ox-HDL.

mechanisms for in vivo oxidation of HDL and structural alterations previously found in ox-HDL are shown in Scheme 11.1.

Alteration in apoproteins by oxidation Cu2þ-mediated oxidation increases the net negative charge in HDL particles. This is speculated to be due to modification of positively charged residues such as Lys in apoproteins (Nagano et al., 1991). The modification in residues of Lys and Trp during oxidation appears to be involved in the reduction of the binding affinity of HDL for the receptor and in the reduced activity of the particle to remove cholesterol from the cells (Nagano et al., 1991; Reyftmann et al., 1991). Indeed, Duell et al. (1991) demonstrated the importance of the Lys residues in the HDL receptor-dependent efflux of intracellular cholesterol. In addition, mild oxidation of HDL by 2,2’-azo-bis(2-amidinopropane) dihydrochloride (AAPH), a generator of aqueous peroxyl radical, is capable of converting Met residues of HDL apoproteins into Met sulfoxides (MetO) (Garner et al.,

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1998a,b; Pankhurst et al., 2003). Lipid hydroperoxides formed during the AAPH-induced oxidation of HDL convert two Met residues (Met86 and Met112) in apoA-I molecules to MetO in a step-wise manner, while Met148 is not oxidized. In the case of homodimeric apoA-II, two Met26 residues of the subunits are oxidized to MetO. These studies also suggest the clinical importance of apoA-I containing MetO as a new marker for coronary artery diseases because there is a relationship between its circulating concentration and the risk for the vascular diseases. It should be noted that the high molar ratio of hypochlorite to HDL induces oxidative modification of amino acids other than Met (Panzenboeck et al., 1997), although at low ratios of the oxidant/HDL (