Plasma Lipids and Their Role in Disease
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Plasma Lipids and Their Role in Disease
Advances in Vascular Biology A series of books bringing together important advances and reviewing all areas of vascular biology. Edited by Mathew A.Vadas, The Hanson Centre for Cancer Research, Adelaide, South Australia and John Harlan, Division of Hematology, University of Washington, Seattle, USA.
Volume One Vascular Control of Hemostasis edited by Victor W.M.van Hinsbergh Volume Two Immune Functions of the Vessel Wall edited by Göran K.Hansson and Peter Libby Volume Three The Selectins: Initiators of Leukocyte-Endothelial Adhesion edited by Dietmar Vestweber Volume Four The Role of Herpesviruses in Atherogenesis edited by David P.Hajjar and Stephen M.Schwartz Volume Five Plasma Lipids and Their Role in Disease edited by Philip J.Barter and Kerry-Anne Rye Volumes in Preparation Platelets, Thrombosis and the Vessel Wall edited by Michael C.Berndt Structure and Function of Endothelial Cell to Cell Junctions edited by Elisabetta Dejana This book is part of a series. The publisher will accept continuation orders which may be cancelled at any time and which provide for automatic billing and shipping of each title in the series upon publication. Please write for details.
Plasma Lipids and Their Role in Disease edited by
Philip J.Barter University Department of Medicine Royal Adelaide Hospital Adelaide Australia and Kerry-Anne Rye Lipid Research Laboratory Hanson Centre Adelaide Australia
harwood academic publishers Australia • Canada • China • France • Germany • India • Japan Luxembourg • Malaysia • The Netherlands • Russia • Singapore Switzerland
This edition published in the Taylor & Francis e-Library, 2005. “To purchase your own copy of this or any of Taylor & Francis or Routledge's collection of thousands of eBooks please go to www.eBookstore.tandf.co.uk.” Copyright © 1999 OPA (Overseas Publishers Association) N.V. Published by license under the Harwood Academic Publishers imprint, part of The Gordon and Breach Publishing Group. All rights reserved. No part of this book may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying and recording, or by any information storage or retrieval system, without permission in writing from the publisher. Printed in Singapore. Amsteldijk 166 1st Floor 1079 LH Amsterdam The Netherlands British Library Cataloguing in Publication Data Plasma lipids and their role in disease.—(Advances in vascular biology; v. 5) 1. Blood lipids I. Barter, Philip J. II. Rye, Kerry-Anne 547.7′7 ISBN 0-203-30481-0 Master e-book ISBN
ISBN 0-203-35287-4 (Adobe eReader Format) ISBN 9057024667 (Print Edition)
CONTENTS
Series Preface Preface List of Contributors 1. Overview of Plasma Lipid Transport Kerry-Anne Rye , Moira A.Clay and Philip J.Barter 2. Abnormalities of Plasma Lipoprotein Transport Gilbert R.Thompson 3. Epidemiological Evidence Linking Plasma Lipoprotein Disorders to Atherosclerosis and Other Diseases Jacques Genest Jr. and Jeffrey Cohn 4. Atherogenicity of Low-density Lipoproteins: Mechanisms M.John Chapman 5. High Density Lipoproteins: The Anti-atherogenic Fraction Philip J.Barter , Moira A.Clay and Kerry-Anne Rye 6. Chylomicron Remnants and Atherosclerosis John C.L.Mamo and Spencer D.Proctor 7. High Density Lipoprotein Receptors Noel H.Fidge 8. Regulation of the Low Density Lipoprotein (B/E) Receptor Petri T.Kovanen and Wolfgang J.Schneider 9. The Role of the Microsomal Triglyceride Transfer Protein in the Assembly and Secretion of Plasma Lipoproteins John R.Wetterau and David A.Gordon 10. Role of Endothelial Lipases in Disease Ira J.Goldberg and Catherine H.Tuck 11. Relationship of the Cholesteryl Ester Transfer Protein (CETP) to Atherosclerosis Laurent Lagrost 12. Familial Lecithin: Cholesterol Acyltransferase Deficiency Syndromes P.Haydn Pritchard , Amir F.Ayyobi and John S.Hill 13. Apolipoprotein E and Human Disease Yadong Huang and Robert W.Mahley 14. Role of the Plasma Phospholipid Transfer Protein in Plasma Lipid Transport Matti Jauhiainen and Christian Ehnholm 15. Oxidation of Plasma Lipids and Lipoproteins J.M.Upston and Roland Stocker
viii x xii 1 19 51
83 105 138 177 211 240
261 279
301 334 373 392
16. Plasma Lipoprotein Disorders and Endothelial Function Mark R.Adams and David S.Celermajer Subject Index
429
461
SERIES PREFACE It is our privilege to live at a time when scientific discoveries are providing insights into human biology at an unprecedented rate. It is also a time when the sheer quantity of information tends to obscure underlying principles, and when hypotheses or insights that simplify and unify may be relegated to the shadow of hard data. The driving force for editing a series of books on Vascular Biology was to partially redress this balance. In inviting editors of excellence and experience, it is our aim to draw together important facts, in particular areas of vascular biology, and to allow the generation of hypotheses and principles that unite an area and define newer horizons. We also anticipate that, as is often the case in biology, the formulation and application of these principles will interrelate with other disciplines. Vascular biology is a frontier that has been recognised since at least the time of Cohnheim and Metchikoff, but has really come into prominence over the last 10–15 years, once the molecules that mediate the essential functions of the blood vessel started to be defined. The boundaries of this discipline are, however, not clear. There are intersections, for example, with hypertension and atherogenesis that bring in, respectively, neuroendocrine control of vessel tone and lipid biochemistry which exist as separate bodies of knowledge. Moreover, it would be surprising if some regional vascular biology (for example, pulmonary, renal etc.) were not to emerge as subgroups in the future. Our aims for the moment, however, are to concentrate on areas of vascular biology that have a wide impact. It is our hope to publish two books each year for the next 3–4 years. Indeed the first five books have been commissioned and address areas primarily in endothelial biology (hemostasis and thrombosis), immunology, leukocyte adhesion molecules, platelet adhesion molecules, adhesion molecules that mediate cell-cell contact. Subsequent volumes will cover the physiology and pathology of other vascular cells as well as developmental vascular biology. We thank the editors and contributors for their very hard work. Mathew VADAS John HARLAN
PREFACE Disease associated with abnormalities of plasma lipids has been recognised since the beginning of the 20th century when it was shown that diet-induced hypercholesterolaemia in rabbits resulted in the development of atherosclerosis. However, it has taken until the last two decades of the century to generate the scientific evidence that has finally persuaded the sceptics that disorders of plasma lipids are also causally related to atherosclerosis and coronary artery disease in humans. Results of human population studies have demonstrated once and for all that hyperlipidaemia is associated with an increased risk of developing atherosclerotic coronary artery disease. Subsequent large-scale intervention trials have established that this risk can be markedly reduced by reducing plasma lipid levels. The transport of lipids in plasma is complex and is subject to multiple abnormalities, most of which reflect an interaction between genes and a variety of lifestyle factors. Several of these abnormalities lead to severe clinical disease. This volume commences with an overview of plasma lipid transport, followed by a chapter describing various categories of plasma lipid disorders. The chapters that follow address the relationships of plasma lipid levels and of the different plasma lipoprotein fractions with atherosclerotic disease. There are two main cholesterol carrying lipoproteins in plasma: low density lipoproteins (LDL) and high density lipoproteins (HDL). A relationship between elevated levels of LDL and the development of atherosclerosis has been known for many years. As indicated in Chapter 4, there has been a growing understanding of the mechanism of this effect of LDL and of the factors that influence the atherogenicity of these lipoproteins. In contrast to LDL, plasma HDL do not cause atherosclerosis. Rather, there is now good evidence that they protect against development of the disease. The mechanism of the protection is not yet known with certainty but, as outlined in Chapter 5, may relate to several well documented functions of HDL. It is now known that LDL are not the only lipoproteins that cause atherosclerosis. As outlined in Chapter 6, there has been recognition over the past few years that the metabolic remnants of triglyceride-rich lipoproteins (very low density lipoproteins and chylomicrons) may also contribute substantially to the disease. Knowledge of the regulation of plasma lipoproteins has been greatly increased with the recognition and characterisation of cell surface receptors which bind plasma HDL and LDL. Receptors for HDL and LDL are described in Chapters 7 and 8. Chapters 9 to 14 are concerned with a number of key proteins that play fundamental roles in the metabolism and remodelling of plasma lipoproteins. While some of these factors have been known for many years, the past few years have provided fundamental new information about all of them. Well characterised genetic abnormalities of these proteins have been shown to impact on plasma lipid transport and, in some cases, to predispose to clinical disease states.
There has been much recent interest in the oxidation of plasma lipids and lipoproteins. It has been suggested by some that LDL become atherogenic only after being oxidatively modified. The growing body of information about plasma lipid and lipoprotein oxidation is presented in Chapter 15. The possible relationship between lipid oxidation and the development of atherosclerosis has led a number of researchers to postulate that antioxidants may protect against the development of coronary heart disease. While there is still no conclusive evidence from large-scale end-point studies, it has been shown that antioxidants do improve endothelial function. Thus, the volume concludes with a topical chapter on the effects of disorders of plasma lipids and lipoproteins on endothelial function. We believe that this volume provides up to date information for physicians and scientists who are interested in plasma lipid transport and how abnormalities of plasma lipids and lipoproteins relate to disease states such as atherosclerosis. We owe a major debt to our co-authors who have not only given freely of their time and knowledge to the generation of the volume but who have also managed somehow to meet the necessary deadlines. Philip BARTER Kerry-Anne RYE
LIST OF CONTRIBUTORS Adams, Mark R. Department of Cardiology Royal Prince Alfred Hospital Missenden Road Camperdown New South Wales 2050 Australia Ayyobi, Amir F. Healthy Heart Program St. Paul’s Hospital 1101 Burrara Street Vancouver, BC Canada V6Z 1Y6 Barter, Philip J. University Department of Medicine Royal Adelaide Hospital North Terrace Adelaide, South Australia 5000 Australia Celermajer, David S. Department of Cardiology Royal Prince Alfred Hospital Missenden Road Camperdown New South Wales 2050 Australia Chapman, M.John INSERM Unit 321, Hopital de la Pitie Pavillon Benjamin Delessert 83, Boulevard de L’Hopital 75651 Paris, Cedex 13 France Clay, Moira A. Lipid Research Laboratory
Hanson Centre Frome Road Adelaide, South Australia 5000 Australia Cohn, Jeffrey Cardiovascular Genetics Laboratory Clinical Research Institute of Montreal 110 Avenue des Pins Quest Montreal, Quebec Canada H2W 1R7 Ehnholm, Christian Department of Biochemistry National Public Health Institute Mannerheimintie 166 FIN-00300 Helsinki Finland Fidge, Noel H. Baker Medical Research Institute PO Box 348 Prahrah, Victoria 3181 Australia Genest, Jr., Jacques Cardiovascular Genetics Laboratory Clinical Research Institute of Montreal 110 Avenue des Pins Quest Montreal, Quebec Canada H2W 1R7 Goldberg, Ira J. Department of Medicine Columbia University College of Physicians and Surgeons 630 West 168th Street New York, NY 10032 USA Gordon, David A. Department of Metabolic Diseases Bristol-Myers Squibb P.O. Box 4000 Princeton, NJ 08543 USA
Hill, John S. Healthy Heart Program St. Paul’s Hospital 1101 Burrara Street Vancouver, BC Canada V6Z 1Y6 Huang, Yadong Gladstone Institute of Cardiovascular Disease University of California P.O. Box 419100 San Francisco, CA 94141–9100 USA Jauhiainen, Matti Department of Biochemistry National Public Health Institute Mannerheimintie 166 FIN-00300 Helsinki Finland Kovanen, Petri T. Wihuri Research Institute Kalliolinnantie 4 FIN-00140 Helsinki Finland Lagrost, Laurent Laboratoire de Biochimie des Lipoprotéines INSERM U498 Hôpital du Bocage BP 1542 21034 Dijon Cedex France Mahley, Robert W. Departments of Pathology and Medicine University of California PO Box 419100 San Francisco, CA 94141–9100 USA Mamo, John C.L. University Department of Medicine
Medical Research Foundation Building Rear of 50 Murray Street Perth, Western Australia 6000 Australia Pritchard, P.Haydn Healthy Heart Program St. Paul’s Hospital 1101 Burrara Street Vancouver, BC Canada, V6Z 1Y6 Proctor, Spencer D. University Department of Medicine Medical Research Foundation Building Rear of 50 Murray Street Perth, Western Australia 6000 Australia Rye, Kerry-Anne Lipid Research Laboratory Hanson Centre Frome Road Adelaide, South Australia 5000 Australia Schneider, Wolfgang J. Department of Molecular Genetics Vienna Biocenter University of Vienna Dr. Bohrgasse 9 1030 Vienna Austria Stocker, Roland Biochemistry Group The Heart Research Institute 145 Missenden Road Camperdown, New South Wales 2050 Australia Thompson, Gilbert R. MRC Lipoprotein Team Clinical Sciences Centre Imperial College School of Medicine
Hammersmith Hospital DuCane Road London W12 ONN UK Tuck, Catherine H. Department of Medicine Columbia University College of Physicians and Surgeons 630 West 168th Street New York, NY 10032 USA Upston, J.M. Biochemistry Group The Heart Research Institute 145 Missenden Road Camperdown, New South Wales 2050 Australia Wetterau, John R. Department of Metabolic Diseases Bristol-Myers Squibb P.O. Box 4000 Princeton, NJ 08543 USA
1 Overview of Plasma Lipid Transport Kerry-Anne Rye 1 , Moira A.Clay 1 and Philip J.Barter 2 1
Lipid Research Laboratory, Hanson Centre, Frome Road, Adelaide, South Australia 5000, Australia 2 University Department of Medicine, Royal Adelaide Hospital, North Terrace, Adelaide, South Australia 5000, Australia
Cholesterol and triglyceride are normal components of plasma. The fact that abnormalities of their transport in plasma underlie a substantial proportion of premature coronary heart disease has stimulated enormous interest in their metabolism and regulation. This overview chapter describes the transport of cholesterol and triglyceride in plasma lipoproteins and summarises the roles played by the different lipoprotein fractions in the process of atherosclerosis. KEYWORDS: Cholesterol, triglyceride, chylomicrons, VLDL, LDL, HDL.
INTRODUCTION Cholesterol, triglyceride and the plasma lipoproteins that transport them are normal, physiological components of plasma. The lipoproteins in plasma are responsible for redistributing cholesterol and triglyceride between tissues in processes that are fundamental to energy metabolism and cell membrane homeostasis. There are three functional pathways of cholesterol transport in plasma: a pathway that delivers dietary cholesterol from the intestine to the liver, a pathway that delivers cholesterol from the liver to extrahepatic tissues and a pathway in which extrahepatic cholesterol is transported back to the liver. The third pathway is often referred to as reverse cholesterol transport. There are two main pathways of triglyceride transport in plasma, each of which is concerned with the transport of energy between tissues. One involves triglyceride of dietary origin. The other involves triglyceride which has been newly synthesised in the liver. In each case the triglyceride is transported as a component of lipoproteins in the plasma to tissues throughout the body, where it is used either as an immediate source of energy or, in the case of adipose tissue, is stored for later use.
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PLASMA LIPOPROTEINS Plasma lipoproteins are the vehicles by which cholesterol and triglyceride are transported in plasma. About 70% of plasma cholesterol exists as cholesteryl esters.
Table 1.1 Plasma lipoprotein classes
Lipoprotein Class Main Lipid Constituents
Main apolipoproteins
Hydrated Density (g/ml)
Diameter (nm)
Chylomicrons
Triglyceride
apoB-48
<0.95
100–1000
Chylomicron remnants
Triglyceride, Cholesterol
apoB-48, apoE
<1.006
50–150
VLDL
Triglyceride
apoB-100, apoE
<1.006
35–50
IDL
Triglyceride, Cholesterol
apoB-100, apoE
1.006–1.019
28–35
LDL
Cholesterol
apoB-100
1.019–1.063
20–26
HDL
Phospholipids, Cholesterol
apoA-I, apoA-II
1.063–1.21
7–12
Since both cholesteryl esters and triglyceride are insoluble in water they must be “solubilised” before they can be transported in plasma (which is predominantly water). This is achieved by their inclusion into lipid-protein complexes known as lipoproteins. There are several classes of plasma lipoproteins which differ markedly in size, density and composition (Table 1.1). All the classes have the same general structure (Figure 1.1). The hydrophobic cholesteryl esters and triglyceride occupy a core that is surrounded by a hydrophilic monomolecular surface layer of apolipoproteins, phospholipids and unesterified cholesterol. The hydrophilic surface enables the water-insoluble triglyceride and cholesteryl esters in the lipoprotein core to be transported in the aqueous blood plasma. Plasma lipoproteins are classified primarily on the basis of hydrated density into chylomicrons and very low density lipoproteins (VLDL) (particles of d<1.006 g/ml which originate, respectively, in the intestine and liver), intermediate density lipoproteins (IDL) (1.006
Overview of plasma lipid transport
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Chylomicrons and Chylomicron Remnants Chylomicrons include a range of particles that are synthesised in the intestine. They are the largest and least dense of the plasma lipoprotein classes. The largest chylomicrons are visible when viewed with the light microscope. Their main function is to transport dietary triglyceride from the intestine to tissues throughout the body. As such, their predominant core lipid is triglyceride. Chylomicrons also provide a vehicle for the delivery of dietary cholesterol from the intestine to the liver. The main apolipoprotein in chylomicrons is apoB-48 (Kane, 1983). They also contain substantial amounts of the C-apolipoproteins (McConathy and Alaupovic, 1986). Chylomicrons are catabolised to chylomicron remnants, which also contain apoE. The apoE in chylomicron remnants is transferred from HDL during chylomicron catabolism (Havel et al., 1973).
Figure 1.1 Schematic diagram of a plasma lipoprotein.
Very Low Density Lipoproteins VLDL are synthesised in the liver from which they transport triglyceride to tissues throughout the body. Thus, like chylomicrons, they have a core which is rich in triglyceride. They also have an additional function as transport vehicles for cholesterol in a process which delivers hepatic cholesterol into the plasma. However, VLDL are smaller and denser than chylomicrons. They also contain apoB-100, rather than apoB-48, as their main apolipoprotein (Kane, 1983). Most VLDL contain apoE and the C-apolipoproteins (McConathy and Alaupovic, 1986).
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Table 1.2 Plasma apolipoproteins
Apolipoprotein Tissue of Origin
Number of Amino Acids
Constituent of Lipoproteins
Plasma Concentration (mg/L)
ApoA-I
Liver, Intestine
243
HDL, Chylomicrons
900–1600
ApoA-II
Liver
77 (mostly present as HDL a 154 aa dimer)
250–450
ApoA-IV
Liver, Intestine
376
HDL, VLDL, Chylomicrons
100–200
ApoB-100
Liver
4536
VLDL, LDL
500–1500
ApoB-48
Intestine
2152
Chylomicrons
0–100
ApoC-I
Liver, Intestine
57
Chylomicrons, VLDL, HDL
50–60
ApoC-II
Liver, Intestine
79
Chylomicrons, VLDL, HDL
30–50
ApoC-III
Liver, Intestine
79
Chylomicrons, VLDL, HDL
100–140
ApoD
Many Tissues
169
HDL
40–70
ApoE
Most Tissues
299
Chylomicrons, VLDL, HDL
20–80
ApoJ
Most Tissues
427
HDL
100
Intermediate Density Lipoproteins IDL are catabolic products of VLDL and are intermediates in the pathway in which VLDL are converted to LDL (Havel, 1984; Nilsson-Ehle et al., 1980). IDL are smaller and denser than VLDL and their core contains approximately equal amounts of triglyceride and cholesteryl esters. The main apolipoproteins in IDL are apoB-100 and apoE (Kane, 1983; McConathy and Alaupovic, 1986). IDL are normally present in plasma at low concentration and are of uncertain function. Low Density Lipoproteins LDL are catabolic products of IDL. They are what remain after IDL have lost most of their triglyceride and all of their C and E apolipoproteins. LDL are smaller and denser than IDL and their core contains predominantly cholesteryl esters. Each LDL particle
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5
contains a single molecule of apoB-100 as its sole apolipoprotein constituent (Kane, 1983). The LDL fraction comprises a number of particles of varying size and density (Austin, 1991; Chapman et al., 1988; Krauss and Burke, 1982; Opplt and Holzberg, 1994; Shen et al., 1981). One of the major functions of LDL is the delivery of cholesterol to cells. The LDL fraction also includes a group of lipoproteins known as Lp(a). In Lp(a) a protein designated apo(a) is covalently linked to apoB-100 (Albers et al., 1990). A possible relationship between Lp(a) and coronary heart disease (CHD) (Scanu et al., 1991; Utermann, 1989) has stimulated enormous interest in this lipoprotein. Its function, however, remains unknown. High Density Lipoproteins HDL are the smallest, most dense plasma lipoproteins. They originate in the liver (Hamilton et al., 1976; Marsh, 1976) and intestine (Green et al., 1978) as lipid-poor particles which rapidly become cholesterol-enriched as they acquire cholesterol from tissues throughout the body. The predominant HDL core lipid is cholesteryl ester. The main HDL apolipoproteins, apoA-I and apoA-II, account for more than 90% of the apolipoproteins in HDL. HDL also contain smaller amounts of apoA-IV, the Capolipoproteins and apoE. HDL may be subdivided on the basis of size and density into smaller and more dense HDL3 and larger and less dense HDL2 (Blanche et al., 1991; DeLalla and Gofman, 1954; Havel et al., 1955). They may be separated on the basis of apolipoprotein composition into two main subpopulations: one containing apoA-I without apoA-II (A-I HDL) and the other containing both apoA-I and apoA-II (A-I/A-II HDL) (Cheung and Albers, 1984). There is also a minor population of HDL which contains apoA-II without apoA-I (Bekaert et al., 1992). A major function of HDL is to act as a recipient of the cholesterol which is transferred from extrahepatic tissues into the plasma via the pathway termed reverse cholesterol transport (Barter, 1993; Fielding and Fielding, 1995; Pieters et al., 1994). HDL also have important anti-inflammatory properties (Calabresi et al., 1997; Cockerill et al., 1995; Maier et al., 1994; Navab et al., 1991).
CONCENTRATIONS OF LIPIDS IN PLASMA LIPOPROTEINS Plasma triglyceride is found mainly in chylomicrons and VLDL. Chylomicrons are present in plasma for up to 10 hours after eating a meal containing fat and, under normal conditions, may transiently increase the plasma triglyceride level by 0.5 to 2 mmol/L. VLDL triglyceride concentrations are normally less than 1 mmol/L and account for about 70% of the triglyceride in fasting plasma (Table 1.3). Smaller proportions of plasma triglyceride are transported in IDL, LDL and HDL. Increased concentrations of plasma triglyceride are generally the result of increased levels of VLDL.
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Table 1.3 Concentrations of lipids in plasma lipoproteins
Lipoprotein class
Triglyceride (mmol/L fasted plasma) a
Cholesterol (mmol/L fasted plasma) a
VLDL
0.2–1.2
0.1–0.4
IDL
0.1–0.3
0.1–0.3
LDL
0.2–0.4
1.5–3.5
HDL
0.1–0.2
1.1–1.6
a These values
are typical for normal human plasma.
Most of the cholesterol in human plasma is carried as a component of LDL. The concentration of LDL cholesterol in healthy adults generally ranges between 2 and 3 mmol/L. HDL cholesterol concentrations are typically 1.1 to 1.6 mmol/L, while the VLDL cholesterol concentration varies with the level of VLDL triglyceride. In general, for every 1.0 mmol/L of VLDL triglyceride, there is about 0.4 mmol/L VLDL cholesterol. Chylomicrons, on the other hand, contribute very little to the overall concentration of plasma cholesterol, even in the postprandial state. Increased plasma cholesterol concentrations are generally the result of increased levels of LDL.
PHYSIOLOGY OF PLASMA LIPIDS AND LIPOPROTEINS Cholesterol Metabolism Cholesterol exists in the cell membranes of all tissues. Its concentration in membranes is tightly regulated, with membrane structure and function being compromised if there is either too little or too much cholesterol. Delivery of cholesterol both from lipoproteins in the plasma to tissues and from tissues back into the plasma can influence the amount of cholesterol in membranes. Thus, the transport of cholesterol in plasma plays an important role in the maintenance of cell membrane structure and function in tissues throughout the body. There are two sources of cholesterol in extrahepatic cells: that which is transferred to cells from the plasma and that which is synthesised within the cell. This ensures that cells always have sufficient amounts of this essential component, even when there is an increased demand for the sterol during periods of tissue growth. Both sources of cell cholesterol are subject to feed-back inhibition when the cell cholesterol content is increased (Brown and Goldstein, 1986; Goldstein and Brown, 1990). The removal of cholesterol from extrahepatic cells, the first step in the reverse cholesterol transport pathway, involves delivery of cholesterol from cell membranes to lipoproteins in the extracellular space (Barter, 1993; Fielding and Fielding, 1995; Pieters et al., 1994). The acceptor lipoproteins then transport the cholesterol through the
Overview of plasma lipid transport
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lymphatics to the plasma compartment. Once in plasma the cholesterol is either recycled to extrahepatic tissues or transported to the liver where it may be eliminated from the body in bile (Grundy and Metzger, 1972). This pathway of reverse cholesterol transport is essential in order to prevent the accumulation of cholesterol in cells. The pathway is thus crucial for the maintenance of cell membrane homeostasis. Triglyceride Metabolism Most of the energy that is stored in the body is in the form of triglyceride in adipose tissue. This triglyceride has its origin either as a component of the diet or is synthesised de novo in the liver. It is necessary, therefore, to transport triglyceride from both the intestine and liver, via the plasma, to adipose tissue for storage as well as to other tissues for use as a source of energy. As outlined below, plasma transport of triglyceride is linked closely to that of cholesterol. Each may be considered in terms of whether the lipid originates as a component of the diet (exogenous lipid transport) or is synthesised in the body (endogenous lipid transport). Exogenous Lipid Transport Most of the fat in the diet is triglyceride, which provides 30–35% of the daily energy requirements for most human subjects in developed countries. While in the intestine, triglyceride is acted upon by pancreatic lipase, which breaks it down to non-esterified fatty acids (NEFA) and glycerol. The NEFA are absorbed into intestinal cells where they are reformed into triglyceride. The triglyceride subsequently interacts with apoB-48 in intestinal cells to form chylomicrons (Figure 1.2). Chylomicrons also accommodate any dietary cholesterol (generally less than 0.5 g per day) that is absorbed into intestinal cells. It should be emphasised that cholesterol is only a minor component of chylomicrons. Chylomicrons are released from intestinal cells into the intestinal lymphatics from which, several hours later, they enter the plasma. Within minutes of reaching the plasma they are acted upon by the enzyme, lipoprotein lipase (LPL). LPL resides on the surface of endothelial cells which line the blood vessels of most tissues. When LPL is activated by the apoC-II in chylomicrons (Wang et al., 1992), most of the chylomicron triglyceride is hydrolysed and NEFA are released for uptake by tissues. As a consequence of the loss of triglyceride, the chylomicrons are converted into smaller particles called chylomicron remnants (Mahley and Hussain, 1991). Chylomicron remnants are subsequently taken up by the liver, which is therefore the initial recipient of dietary cholesterol (Mahley and Hussain, 1991; Windler et al., 1988). Hepatic uptake of chylomicron remnants is dependent on the presence of apoE on the remnant surface (Mahley and Hussain, 1991), and is probably mediated by the binding of the apoE to a cell surface receptor. The most likely candidate for this receptor is the LDL-receptor-related-protein (Strickland et al., 1995). The rate of catabolism of chylomicrons by LPL and the rate of hepatic uptake of chylomicron remnants are both very rapid. Thus, under normal circumstances, neither fraction accounts for more than a small proportion of the plasma cholesterol, even in the non-fasting state.
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Figure 1.2 Exogenous lipid transport. The pathway by which triglyceride and cholesterol of dietary origin are delivered to tissues is shown. TG, triglyceride; CE, cholesteryl esters; LPL, lipoprotein lipase; NEFA, non-esterified fatty acids.
Endogenous Lipid Transport A proportion of the carbohydrate eaten each day is converted into triglyceride in the liver. The liver also produces triglyceride by re-esterification of NEFA that it extracts from the plasma. However, the liver has a very limited capacity for storing triglyceride. Rather, the triglyceride is incorporated into VLDL and secreted into
Overview of plasma lipid transport
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Figure 1.3 Endogenous lipid transport. The pathway by which triglyceride and cholesterol of hepatic origin are delivered to tissues is shown. TG, triglyceride; CE, cholesteryl esters; LPL, lipoprotein lipase; HL, hepatic lipase; LCAT, lecithin cholesterol acyltransferase; CETP, cholesteryl ester transfer protein; NEFA, non-esterified fatty acids; B-100, apolipoprotein B-100; E, apolipoprotein E.
plasma (Figure 1.3). A proportion of the hepatic cholesterol, whether of dietary origin or synthesised de novo in liver cells, is also incorporated into VLDL. Once in plasma, VLDL acquire C apolipoproteins by transfer from HDL (Mjøs et al.,
Plasma lipids and their role in disease
10
1975). As with chylomicrons, apoC-II on the VLDL surface activates LPL (Wang et al., 1992) which hydrolyses VLDL triglyceride and releases free fatty acids to tissues for energy or storage. Coincident with the loss of triglyceride, VLDL acquire cholesteryl esters and apoE from HDL (Barter et al., 1982; Mjøs et al., 1975) and are converted into IDL (also known as VLDL remnants). IDL are enriched in cholesteryl esters and apoE and depleted of triglyceride relative to their precursor, VLDL. As IDL are catabolised rapidly, their concentration in plasma is usually low. They are either taken up directly by the liver or are catabolised further and converted into LDL. The precise mechanism of the conversion of IDL to LDL is uncertain, but is known to involve the loss of apoE and the C apolipoproteins and further hydrolysis of triglyceride by another endothelial enzyme known as hepatic lipase (Nilsson-Ehle et al., 1980). The end products of this catabolism are cholesterol-rich LDL which are removed only slowly from the circulation. As a result, relatively high concentrations of LDL can accumulate in plasma. LDL are removed from plasma following their binding to cell surface receptors. The most important of these receptors is the LDL, or B/E, receptor (Brown and Goldstein, 1986). This receptor binds the apoB-100 in LDL and causes the LDL particle to be internalised and degraded, thus allowing the LDL to deliver its cholesterol to the cell. Most of the LDL receptor activity is found in the liver (Koelz et al., 1982; Pittman et al., 1982). However, a proportion of the uptake of plasma LDL may be via LDL receptors in extrahepatic tissues. Activity of the LDL receptor is determined in part by the intracellular cholesterol concentration (Brown and Goldstein, 1986). If the level of cholesterol in a cell is high and the cell has no need for additional cholesterol, the receptor is down-regulated. If, on the other hand, the cell cholesterol level is low, the receptor up-regulates so that the cell can acquire additional cholesterol. It should be noted, however, that uptake of LDL by extra-hepatic tissues is probably not essential, as cells in every tissue of the body have the ability to synthesise cholesterol de novo. Most extrahepatic tissues cannot catabolise the cholesterol molecule. Nor can tissues other than the liver and intestine incorporate cholesterol into lipoproteins prior to secretion into the plasma. Thus, there must exist an alternate mechanism for extrahepatic cells to eliminate cholesterol which would otherwise accumulate and compromise cell function. This alternate mechanism involves the transfer of cholesterol from cell membranes to an extracellular acceptor in the first step of the reverse cholesterol transport pathway. Reverse Cholesterol Transport The precise details of reverse cholesterol transport remain to be elucidated. In broad terms the pathway may be divided into four steps (Barter, 1993; Fielding and Fielding, 1995; Pieters et al., 1994). (i) Cholesterol is transferred from cell membranes down a concentration gradient to extracellular HDL (Barter, 1993; Fielding and Fielding, 1995; Pieters et al., 1994). (ii) Esterification of HDL cholesterol is catalysed by lecithin: cholesterol acyltransferase (LCAT), which converts unesterified cholesterol into cholesteryl esters (Glomset, 1968). As cholesteryl esters are not miscible with water they cannot remain on the lipoprotein surface. Instead, they move to the interior, or core, of
Overview of plasma lipid transport
11
the lipoprotein, leaving the surface depleted of cholesterol. This generates the concentration gradient down which more cholesterol flows from cell membranes, (iii) A substantial proportion of the cholesteryl esters in the core of HDL is subsequently transferred by a plasma protein known as cholesteryl ester transfer protein (CETP) to VLDL, IDL and LDL (Barter et al., 1982). (iv) The cholesteryl esters are finally delivered to the liver, predominantly by receptor-mediated uptake of LDL (Brown and Goldstein, 1986). A smaller proportion of the cholesteryl esters are also delivered to the liver directly by HDL (Glass et al., 1983; Glass et al., 1985). Once in the liver, the cholesterol may be excreted in bile, either as unmetabolised sterol or after conversion to bile acids. Cholesterol in the liver can also be recycled back into the plasma by incorporation into VLDL.
ATHEROGENICITY OF PLASMA LIPOPROTEINS Chylomicrons and Chylomicron Remnants There is little to suggest that chylomicrons are atherogenic (Table 1.4), as evidenced by the lack of accelerated atherosclerosis in subjects in whom a genetic deficiency of LPL is accompanied by massive elevations of plasma chylomicrons (Hayed et al., 1991). Chylomicron remnants, by contrast, may be highly atherogenic. This may explain why the magnitude of postprandial lipaemia following a standard fat meal is predictive of CHD (Groot et al., 1991; Miesenböck and Patsch, 1992). It should be noted, however, that there is currently no easy way of measuring the concentration of Chylomicron remnants in routine clinical practice.
Table 1.4 Atherogenicity of plasma lipoproteins
Lipoprotein class
Atherogenicity
Chylomicrons
−
Chylomicron remnants
+
VLDL
+/−
IDL
+
LDL
+
HDL
Anti-atherogenic
Lp(a)
+
Plasma lipids and their role in disease
12
Very Low Density Lipoproteins It is still uncertain whether VLDL are atherogenic (Table 1.4). While subjects with elevated levels of VLDL, as reflected by hypertriglyceridaemia, tend also to have an increased risk of developing CHD (Carlson and Bottiger, 1985), it has been argued that the relationship may be indirect. For example, elevated VLDL levels are often associated with reduced concentrations of HDL cholesterol (Brunzell et al., 1983) and with the presence of small, dense LDL (Austin, 1991), each of which may be responsible for the CHD. Furthermore, subjects with elevated VLDL levels may also have increased concentrations of potentially atherogenic IDL particles (Krauss et al., 1987). It is also possible that there is variations in the atherogenicity of different VLDL subpopulations, with large, triglyceride-rich, cholesterol-poor VLDL being much less atherogenic than smaller, cholesterol-enriched particles. Intermediate Density Lipoproteins There are several lines of evidence linking IDL to CHD (Table 1.4): (i) they promote accumulation of cholesterol in macrophages in vitro (Mahley, 1982); (ii) they appear to contribute to the development of atherosclerosis in a number of animal species which have been fed cholesterol-rich diets (Mahley, 1982); (iii) they are present in increased concentrations in human subjects with dysbetalipoproteinaemia and familial combined hyperlipidaemia, conditions in which there is a very high frequency of CHD (Mahley, 1983); (iv) it has been reported that the presence (Tatami et al., 1981), the severity (Reardon et al., 1985) and the progression (Krauss, 1987) of CHD in humans are all predicted by the concentration of IDL; (v) mice which have been genetically engineered to lack apoE accumulate high concentrations of IDL and develop spontaneous atherosclerosis (Plump et al., 1992; Piedrahita et al., 1992; Zhang et al., 1992). Low Density Lipoproteins Observations in patients with familial hypercholesterolaemia have established beyond reasonable doubt that elevated levels of LDL predispose to atherosclerosis (Table 1.4). These subjects have well defined genetic defects of the LDL receptor which result in marked increases in the plasma concentration of LDL (Brown and Goldstein, 1986). They also have an extremely high incidence of premature CHD. A genetically determined defect in the LDL receptor, with accompanying increased concentrations of LDL cholesterol, has also been described in Watanabe Heritable Hyperlipidaemic Rabbits (Tanzawa et al., 1980). Like their human counterparts, such animals develop aggressive atherosclerosis spontaneously. The mechanism by which LDL cause atherosclerosis is uncertain. For example, when studied in vitro, unmodified LDL do not promote the accumulation of cholesterol in macrophages (Carew et al., 1980) and do not lead to the formation of the foam cells which are believed to initiate the development of atherosclerosis. If, however, LDL are first modified by oxidation (Austin et al., 1988) or self-aggregation (Khoo et al., 1988) they are able to promote foam cell formation. Evidence is accumulating that such
Overview of plasma lipid transport
13
modifications of LDL may take place within the artery wall, thus providing the link between LDL and atherosclerosis (Steinberg et al., 1989). It has been suggested that LDL subpopulations may vary in their atherogenic potential. For example, subjects with CHD frequently have an increased concentration of small, dense LDL (Austin, 1991; Howard, 1987; Reaven, 1991). However, the presence of such particles is often associated with hypertriglyceridaemia and low concentrations of HDL (Austin, 1991; Brunzell et al., 1983), especially in subjects with non-insulin dependent diabetes mellitus (NIDDM) (Howard, 1987) or the insulin resistance that accompanies truncal obesity (Reaven, 1991). It is uncertain whether it is the small, dense LDL or other factors which are the true cause of the CHD. On the other hand, small, dense LDL have been shown to be more readily oxidised in vitro than larger and less dense LDL (de Graaf et al., 1991). This observation led to the proposition that the presence of small, dense LDL in plasma and their consequent accumulation and oxidative modification in the artery wall may be related directly to the development of atherosclerosis. Lipoprotein(a) The concentration of Lp(a) is a powerful predictor of the development of CHD (Scanu et al., 1991; Utermann, 1989). This raises the possibility that Lp(a) may be atherogenic. Although the mechanism of the atherogenesis is unknown, speculation centres on the apo (a) moiety. In support of this proposition, transgenic mice that express human apo(a) develop atherosclerosis when fed a high fat diet (Lawn et al., 1992). High Density Lipoproteins Human population studies have shown consistently that the concentration of HDL cholesterol correlates inversely with the development of CHD. These observations suggest that HDL protect against CHD (Enger et al., 1979; Goldbourt and Medalie, 1979; Gordon et al., 1977; Jacobs et al., 1990; Miller et al., 1977; Miller et al., 1992; Pekkanen et al., 1990). This possibility has gained strong support from recent studies of transgenic mice in which over-expression of human apoA-I increases the concentration of HDL and protects against both diet-induced (Rubin et al., 1991) and spontaneous atherosclerosis (Paszty et al., 1994; Plump et al., 1994). However, it is also possible that the relationship between HDL levels and CHD in human subjects may have an indirect component which reflects the well documented association of low HDL levels with other factors (eg. smoking, obesity, NIDDM and increased concentrations of atherogenic VLDL subpopulations) which may be the true cause of the CHD (Brunzell et al., 1983; Howard, 1987; Reaven, 1991). The mechanism by which HDL protect against atherosclerosis is uncertain, but may relate to some of their known functions. These include the ability of HDL to promote the efflux of cholesterol from cells (Barter, 1993; Fielding and Fielding, 1995; Pieters et al., 1994) and the capacity of HDL to function as an anti-inflammatory agent (Calabresi et al., 1997; Cockerill et al., 1995; Maier et al., 1994; Navab et al., 1991). There is evidence from transgenic mouse studies (Schultz et al., 1993) that specific HDL subpopulations may differ in their ability to inhibit the development of
Plasma lipids and their role in disease
14
atherosclerosis, but the evidence from human studies is not yet convincing (Amouyel et al., 1993).
CONCLUSION Plasma lipid and lipoprotein transport is a normal physiological process that is fundamental to the delivery of energy to tissues and the maintenance of cell membrane homeostasis. Deficiencies of plasma lipoproteins may compromise these functions and result in serious disease states. However, the main consequence of disordered plasma lipoprotein transport is the development of premature vascular disease which relates to states in which there is an excess of pro-atherogenic lipoproteins such as chylomicron remnants, IDL, LDL and Lp(a), or a deficiency of the anti-atherogenic HDL fraction. References Albers, J.J., Marcovina, S.M. and Lodge, M.S. (1990). The unique lipoprotein(a): properties and immunochemical measurement. Clin. Chem. , 36 , 2019–2026. Amouyel, P., Isorez, D., Bard, J.-M., Goldman, M., Level, P., Zylberg, G. et al. (1993). Parenteral history of early myocardial infarction is associated with decreased levels of lipoparticle AI in adolescents. Arterioscler. Thromb. , 13 , 1640–1644. Austin, M.A. (1991). Plasma triglyceride and coronary heart disease. Arterioscler. Thromb. , 11 , 2–14. Austin, M.A., Breslow, J.L., Hennekens, C.H., During, J.E., Willett, W.C. and Krauss, R.M. (1988). Low-density lipoprotein subclass patterns and risk of myocardial infarction. J.Amer. Med. Assoc. , 260 , 1917–1921. Barter, P.J. (1993). High-density lipoproteins and reverse cholesterol transport. Curr. Opin. Lipidol. , 4 , 210–217. Barter, P.J., Hopkins, G.J. and Calvert, G.D. (1982). Transfers and exchanges of esterified cholesterol between plasma lipoproteins. Biochem. J., 208 , 1–7. Bekaert, E.D., Alaupovic, P., Knight-Gibson, C., Norum, R.A., Laux, M.J. and AyraultJarrier, M. (1992). Isolation and partial characterization of lipoprotein A-II (LP-A-II) particles of human plasma. Biochim. Biophys. Acta. , 1126 , 105–113. Blanche, P.J., Gong, E.L., Forte, T.M. and Nichols, A.V. (1981). Characterization of human high-density lipoproteins by gradient gel electrophoresis. Biochim. Biophys. Acta. , 665 , 408–419. Brown, M.S. and Goldstein, J.L. (1986). A receptor-mediated pathway for cholesterol homeostasis. Science , 232 , 34–47. Brunzell, J.D., Albers, J.J., Chait, A., Grundy, S.M., Groszek, E. and McDonald, G.B. (1983). Plasma lipoproteins in familial combined hyperlipidaemia and monogenic familial hypertriglyceridemia. J.Lipid Res. , 24 , 147–155. Calabresi, L., Franceschini, G., Sirtori, C.R., DePalma, A., Sarasella, M., Ferrante, P. et al. (1997). Inhibition of VCAM-1 expression in endothelial cells by reconstituted high density lipoproteins. Biochem. Biophys. Res. Commun. , 238 , 61–65. Carew, T.E., Chapman, M.J., Goldstein, S. and Steinberg, D. (1980). Enhanced degradation of trypsin-treated low density lipoproteins by fibroblasts from a patient with homozygous familial hypercholesterolemia. Biochim. Biophys. Acta. , 529 , 171– 175.
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Carlson, L.A. and Bottiger, L.E. (1985). Risk factors for ischemic heart disease in men and women. Acta Med. Scand. , 218 , 207–211. Chapman, M.J., Laplaud, P.M., Luc, G., Forgez, P., Bruckert, E., Goulinet, S. et al. (1988). Further resolution of the low density lipoprotein spectrum in normal human plasma: physicochemical characteristics of discrete subspecies separated by density gradient ultracentrifugation. J.Lipid Res. , 29 , 442–458. Cheung, M.C. and Albers, J.J. (1984). Characterization of lipoprotein particles isolated by immunoaffinity chromatography. Particles containing A-I and A-II and particles containing A-I but no A-II. J.Biol. Chem. , 259 , 12201–12209. Cockerill, G.W., Rye, K.-A., Gamble, J.R., Vadas, M.A. and Barter, P.J. (1995). High density lipoproteins inhibit cytokine-induced expression of endothelial cell adhesion molecules. Arterioscler. Thromb. Vasc. Biol. , 15 , 1987–1994. de Graaf, J., Hak-Lemmers, H.L.M., Hectors, M.P.C., Demacker, P.N., Hendriks, J.C. and Stalenhoef, A.F. (1991). Enhanced susceptibility to in vitro oxidation of the dense low density lipoprotein subfraction in healthy subjects. Arterioscler. Thromb. , 11 , 298–306. DeLalla, O.F. and Gofman, J.W. (1954). Ultracentrifugal analysis of serum lipoproteins. Methods Biochem. Anal. , 1 , 459–478. Enger, S.C., Hjermann, I., Foss, O.P., Helgeland, A., Holme, I., Leren, P. et al. (1979). High density lipoprotein cholesterol and m yocardial infarction or sudden death: a prospective case-control study in middle-aged men of the Oslo Study . Artery , 5 , 170– 181. Fielding, C.J. and Fielding, P.E. (1995). Molecular physiology of reverse cholesterol transport. J.Lipid Res. , 36 , 211–228. Glass, C., Pittman, R.C., Civen, M. and Steinberg, D. (1985). Uptake of high-density lipoprotein-associated apoprotein A-I and cholesteryl esters by 16 tissues of the rat in vivo and by adrenal glands and hepatocytes in vitro. J.Biol. Chem. , 260 , 744–750. Glass, C., Pittman, R.C., Weinstein, D.B. and Steinberg, D. (1983). Dissociation of tissue uptake of cholesterol from that of apoprotein A-I of rat high density lipoproteins: selective delivery of cholesterol to liver, adrenal and gonad. Proc. Natl. Acad. Sci. USA , 80 , 5435–5439. Glomset, J.A. (1968). The plasma lecithin: cholesterol acyltransferase reaction. J.Lipid Res. , 9 , 155–167. Goldbourt, U. and Medalie, J.H. (1979). High density lipoprotein cholesterol and incidence of coronary heart disease: the Israeli Ischemic Heart Disease Study. Am. J.Epidemiol , 109 , 296–308. Goldstein, J.L. and Brown, M.S. (1990). Regulation of the mevalonate pathway. Nature , 343 , 425–430. Gordon, T., Castelli, W.P., Hjortland, M.C., Kannel, W.B and Dawber, T.R. (1977). High density lipoprotein as a protective factor against coronary heart disease: the Framingham Study. Am. J.Med. , 62 , 707–714. Green, P.H.R., Tall, A.R. and Glickman, R.M. (1978). Rat intestine secretes discoid high density lipoproteins. J.Clin. Invest. , 61 , 528–534. Groot, P.H.E., van Stiphout, W.A.H.J., Krauss, X.H., Jansen, H., vanTol, A., vanRamshorst, E. et al. (1991). Postprandial lipoprotein metabolism in normolipidemic men with and without coronary artery disease. Arterioscler. Thromb. , 11 , 656–662. Grundy, S.M. and Metzger, A.L. (1972). A physiological method for estimation of hepatic secretion of biliary lipids in man. Gastroenterology , 7 , 1200–1217. Hamilton, R.L., Williams, M.C., Fielding, C.J. and Havel, R.J. (1976). Discoidal bilayer
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structures of nascent high density lipoproteins from perfused rat liver. J.Clin. Invest. , 58 , 667–680. Hatch, F.T. and Lees, R.S. (1968). Practical methods for lipoprotein analysis. Adv. Lipid Res. , 6 , 1–68. Havel, R.J. (1984). The formation of LDL: mechanisms and regulation. J.Lipid Res. , 25 , 1570–1576. Havel, R.J., Eder, H.A. and Bragdon, J.H. (1955). The distribution and chemical composition of ultra-centrifugally separated lipoproteins in human serum. J.Clin. Invest. , 34 , 1345–1353. Havel, R.J., Kane, J.P. and Kashyap, M.L. (1973). Interchange of apolipoproteins between chylomicrons and high density lipoproteins during alimentary lipemia in man. J.Clin. Invest. , 52 , 32–38. Hayden, M.R., Ma, Y., Brunzell, J. and Henderson, H.E. (1991). Genetic variants affecting human lipoprotein and hepatic lipases. Curr. Opin. Lipidol. , 2 , 104–109. Howard, B.V. (1987). Lipoprotein metabolism in diabetes mellitus. J.Lipid Res. , 28 , 613–628. Jacobs, D.R., Jr., Mebane, I.L., Bangdiwala, S.I., Criqui, M.H. and Tyroler, H.A. (1990). High density lipoprotein cholesterol as a predictor of cardiovascular disease mortality in men and women: the follow-up study of the Lipid Research Clinics Prevalence Study. Am. J.Epidemiol. , 131 , 32–47. Kane, J.P. (1983). Apolipoprotein B: structural and metabolic heterogeneity. Ann. Rev. Physiol. , 45 , 637–650. Khoo, J.C., Miller, E., McLoughlin, P. and Steinberg, D. (1988). Enhanced macrophage uptake of low-density lipoprotein after self-aggregation. Arteriosclerosis , 8 , 348–358. Koelz, H.R., Sherrill, B.C., Turley, S.D. and Dietschy, J.M. (1982). Correlation of low and high density lipoprotein binding in vivo with rates of lipoprotein degradation in the rat. J.Biol. Chem. , 257 , 8061–8072. Krauss, R.M. (1987). Relationship of intermediate and low-density lipoproteins to risk of coronary artery disease. Am. Heart J. , 113 , 578–583. Krauss, R.M., Lindgren, F.T., Williams, P.T., Brensike, J., Detre, K.M. et al. (1987). Intermediate-denstiy lipoproteins and progression of coronary artery disease in hypercholesterolaemic men. Lancet , 2 , 62–66. Krauss, R.M. and Burke, D.J. (1982). Identification of multiple subclasses of plasma low density lipoproteins in normal humans. J.Lipid Res. , 23 , 97–104. Lawn, R.M., Wade, D.P., Hammer, R.E., Chiesa, G., Verstuyft, J.G. and Rubin, E.M. (1992). Atherogenesis in transgenic mice expressing human apolipoprotein [a]. Nature , 360 , 670–672. Mahley, R.W. (1982). Atherogenic hyperlipoproteinemia. Med. Clin. North Am. , 66 , 375–402. Mahley, R.W. (1983). Development of accelerated atherosclerosis: concepts derived from cell biology and animal model studies. Arch. Pathol. Lab. Med. , 107 , 393–399. Mahley, R.W. and Hussain, M.M. (1991). Chylomicron and chylomicron remnant catabolism. Curr. Opin. Lipidol. , 2 , 170–176. Maier, J.A.M., Barenghi, L., Pagani, F. and Bradamante, S. (1994). The protective role of high-densty lipoprotein on oxidized-low-density-lipoprotein-induced U937/endothelial cell interactions. Eur. J. Biochem. , 221 , 35–41. Marsh, J.B. (1976). Apoproteins of the lipoprotein in a nonrecirculating perfusate of rat liver. J.Lipid Res. , 17 , 85–90. McConathy, W.J. and Alaupovic, P. (1986). Isolation and characterization of other
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apolipoproteins. Meth. Enzymol. , 128 , 297–310. Miesenböck, G. and Patsch, J.R. (1992). Postprandial hyperlipidemia: the search for the atherogenic lipoprotein. Curr. Opin. Lipidol. , 3 , 196–201. Miller, M., Seidler, A., Kwiterovich, P.O. and Pearson, T.A. (1992). Long-term predictors of subsequent cardiovascular events with coronary artery disease and “desirable” levels of plasma total cholesterol. Circulation , 86 , 1165–1170. Miller, N.B., Forde, O.H., Thelle, D.S. and Mjøs, O.D. (1977). The Tromso Heart Study: high-density lipoprotein and coronary heart disease: a prospective case-control study. Lancet , 1 , 965–968. Mjøs, O.D., Faergeman, O., Hamilton, R.L. and Havel, R.J. (1975). Characterization of remnants produced during the metabolism of triglyceride-rich lipoproteins of blood plasma and intestinal lymph in the rat. J.Clin. Invest. , 56 , 603–615. Navab, M., Imes, S.S., Hama, S.Y., Hough, G.P., Ross, L.A., Bork, R.W. et al. (1991). Monocyte transmigration induced by modification of low density lipoprotein in cocultures of human aortic wall cells is due to induction of monocyte chemotactic protein I synthesis and is abolished by high density lipoprotein. J.Clin. Invest. , 88 , 2039–2046. Nilsson-Ehle, P., Garfinkel, A.S. and Schotz, M.C. (1980). Lipolytic enzymes and plasma lipoprotein metabolism. Ann. Rev. Biochem. , 49 , 667–693. Opplt, J.J. and Holzberg, E.S. (1994). Ultracentrifugal subclasses of low and intermediate density lipoproteins. J.Lipid Res. , 35 , 510–523. Paszty, C., Maeda, N., Verstuyft, J. and Rubin, E.M. (1994). Apolipoprotein AI transgene corrects apolipoprotein E deficiency-induced atherosclerosis in mice. J.Clin. Invest. , 94 , 899–903. Pekkanen, J., Linn, S., Heiss, G., Suchindran, C.M., Leon, A., Rifkind, B.M. et al. (1990). Ten-year mortality from cardiovascular disease in relation to cholesterol level among men with and without preexisting cardiovascular disease. N.Engl. J.Med. , 322 , 1700–1707. Piedrahita, J.A., Zhang, S.H., Hagaman, J.R., Oliver, P.M. and Maeda, N. (1992). Generation of mice carrying a mutant apolipoprotein E gene inactivated by gene targeting in embryonic stem cells. Proc. Natl. Acad. Set. USA , 89 , 4471–4475. Pieters, M.N., Schouten, D. and Van Berkel, T.J.C. (1994). In vitro and in vivo evidence for the role of HDL in reverse cholesterol transport. Biochim. Biophys. Acta. , 1225 , 125–134. Pittman, R.C., Carew, T.E., Attie, A.D., Witztum, J.L., Watanabe, Y. and Steinberg, D. (1982). Receptor-dependent and receptor-independent degradation of low density lipoprotein in normal rabbits and in receptor-deficient mutant rabbits. J.Biol. Chem. , 257 , 7994–8000. Plump, A.S., Scott, C.J. and 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 , 9607–9611. Plump, A.S., Smith, J.D., Hayek, T., Aalto-Setala, K., Walsh, A., Verstuyft, J.G. et al. (1992). Severe hypercholesterolemia and atherosclerosis in apolipoprotein E-deficient mice created by homologous recombination in ES cells. Cell , 71 , 343–353. Reardon, M.F., Nestel, P., Craig, I.H. and Harper, R.W. (1985). Lipoprotein predictors of the severity of coronary artery disease in men and women. Circulation , 71 , 881–888. Reaven, G.M. (1991). Insulin resistance and compensatory hyperinsulinemia: role in hypertension, dyslipidemia, and coronary heart disease. Am. Heart J. , 121 , 1283– 1288.
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Rubin, E.M., Krauss, R.M., Spangler, E.A., Verstuyft, J.G. and Clift, S.M. (1991). Inhibition of early atherogenesis in transgenic mice by human apolipoprotein AI. Nature , 353 , 265–267. Scanu, A.M., Lawn, R.M. and Berg, K. (1991). Lipoprotein[a] and atherosclerosis. Ann. Intern. Med. , 115 , 209–218. Schultz, J.R., Verstuyft, J.G., Gong, E.L., Nichols, A.V. and Rubin, E.M. (1993). Protein composition determines the anti-atherogenic properties of HDL in transgenic mice. Nature , 365 , 762–764. Shen, M.M.S., Krauss, R.M., Lindgren, F.T. and Forte, T.M. (1981). Heterogeneity of serum low density lipoproteins in normal human subjects. J.Lipid Res. , 22 , 236–244. Steinberg, D., Parthasarathy, S., Carew, T.E., Khoo, J.C. and Witzum, J.L. (1989). Beyond cholesterol: modifications of low density lipoprotein that increase its atherogenicity. N.Engl. J.Med. , 320 , 915–924. Strickland, D.K., Kounnas, M.Z. and Argraves, W.S. (1995). LDL receptor-related protein: a multiligand receptor for lipoprotein and proteinase catabolism. FASEB J. , 9 , 890–898. Tanzawa, K., Shimada, Y., Kuroda, M., Tsujita, V., Arai, M. and Watanabe, H. (1980). WHHL-rabbit: a low density lipoprotein receptor-deficient animal model for familial hypercholesterolemia. FEBS Lett . 118 , 81–84. Tatami, R., Mabuchi, H., Ueda, K., Haba, T., Kametani, T., Ito, S. et al. (1981). Intermediate-density lipoprotein and cholesterol-rich very low density lipoprotein in angiographically determined coronary artery disease. Circulation , 64 , 1174–1184. Utermann, G. (1989). The mysteries of lipoprotein[a]. Science , 246 , 904–910. Wang, C.-S., Hartsuck, J. and McConathy, W.J. (1992). Structure and functional properties of lipoprotein lipase. Biochim. Biophys. Acta. , 1123 , 1–7. Windler, E.E., Greeve, J., Daerr, W.H. and Greten, H. (1988). Binding of rat chylomicrons and their remnants to the hepatic low-density-lipoprotein receptor and its role in remnant removal. Biochem. J. , 252 , 553–561. Zhang, S.H., Reddick, R.L., Piedrahita, J.A. and Maeda, N. (1992). Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E. Science , 258 , 468–471.
2 Abnormalities of Plasma Lipoprotein Transport Gilbert R.Thompson MRC Lipoprotein Team, Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital, DuCane Road, London W12 ONN, U.K.
Abnormalities of plasma lipoprotein transport are expressed as either hyperlipidaemia or hypolipidaemia. Nowadays the hyperlipidaemias are usually classified phenotypically as hypercholesterolaemia, hypertriglyceridaemia and mixed hyperlipidaemia whereas the hypolipidaemias are sub-divided into disorders characterised by either hypo lipoproteinaemia or by hypo lipoproteinaemia. Recent advances in molecular biology have enabled an increasing number of primary lipoprotein disorders to be defined genotypically in terms of the mutation of the receptor, ligand or enzyme responsible. Genetic predisposition also plays a role in the expression of some secondary forms of dyslipidaemia. The causes of several forms of primary hyperlipidaemia and hypolipidaemia have yet to be defined, exemplified by Familial Combined Hyperlipidaemia and Tangier disease respectively. The increasing use of transgenic and gene knockout animal models may provide clues to the mechanisms underlying these and other lipoprotein disorders of unknown aetiology. KEYWORDS: Cholesterol, Dyslipidaemia, Hyperlipidaemia, Hypolipidaemia, Triglyceride.
DEFINITION AND CLASSIFICATION The term dyslipidaemia is useful in that it encompasses abnormalities of lipoprotein transport which result in a lack of lipids in plasma, hypolipidaemia, as well as those causing an excess, hyperlipidaemia. The relevance of both to atherosclerosis is illustrated by increasing evidence that deficiency of HDL may be as important a risk factor for coronary heart disease (CHD) as is an excess of LDL. The definition of dyslipidaemia has gradually evolved with advances in the understanding of underlying mechanisms. Many monogenically-inherited disorders can now be defined in terms of the specific mutation(s) responsible for encoding the dysfunctional receptor, ligand or enzyme causing the dyslipidaemia. In contrast, most
Plasma lipids and their role in disease
20
polygenic and secondary forms of dyslipidaemia are still defined by arbitrary cut-offs such as the 5th and 95th percentile of the distribution of the lipid or lipoprotein variable in question or, alternatively, by “ideal” values shown to be associated with a minimal risk of CHD. In a similar manner the classification of dyslipidaemia has evolved from the Fredrickson and WHO classifications of lipoprotein phenotypes devised 30 years ago to the much simpler system now in use. This includes both hypolipidaemia and hyperlipidaemia and differentiates the latter into hypercholesterolaemia, hypertriglyceridaemia and mixed hyperlipidaemia. Although more user-friendly the current system is less informative in some respects than its predecessors which described 5 and 6 distinct lipoprotein phenotypes respectively. Some of these are still used; for example type III hyperlipoproteinaemia is often preferred to the term familial dysbetalipoproteinaemia when describing the accumulation of remnant particles which results from inheritance of certain apoE isoforms.
PREVALENCE As implied in the previous section the prevalence of dyslipidaemia will be influenced to a large extent by the criteria used to define increases and decreases of the various lipoprotein classes. Reference values vary according to ethnicity, locality and lifestyle. This is exemplified by the apparent infrequency of heterozygous familial hypercholesterolaemia (FH) in Tunisia if Western reference values of serum cholesterol are used as an index of hypercholesterolaemia (Slimane et al., 1993). The prevalence of obligatory heterozygotes, based on the frequency of homozygotes in the population, is much higher however, which suggests that estimates based on raised serum cholesterol values are spuriously low, presumably reflecting the masking effect of dietary influences.
Table 2.1 Inherited lipoprotein disorders predisposing to premature atherosclerosis
Disorder
Phenotype
Familial hypercholesterolaemia
VLDL remnants and LDL increased
2:1000
Familial defective apoB-100
LDL increased
1:1000
Familial combined hyperlipidaemia VLDL or LDL or both increased
Prevalence
5:1000
Type III hyperlipoproteinaemia
Chylomicron and VLDL remnants increased
0.1:1000
Polygenic hypercholesterolaemia
LDL moderately increased
42:1000
Familial hypo lipoproteinaemia
HDL decreased
50:1000
Table 2.1 shows the prevalence of the major genetically-determined forms of dyslipidaemia predisposing to premature CHD. The frequency of polygenic hypercholesterolaemia is based on the premise that it is responsible for all serum cholesterol levels above the 95th percentile not accounted for by FH, familial defective
Abnormalities of plasma lipoprotein transport
21
apoB100 (FDB) or familial combined hyperlipidaemia (FCH). The prevalence of FH is increased in parts of the world where an imported founder gene effect has been operative, such as South Africa and French Canada, whereas the rarity of FDB in Japan probably reflects that country’s isolation from the West in previous times.
Table 2.2 Transgenic (tg) and gene knockout (ko) mouse models of human dyslipidaemia
Manipulation
Phenotype
Source
LDL receptor ko and high cholesterol diet
Hyper lipoproteinaemia and atherosclerosis
Ishibashi et al., a
ApoB ko
Hypo lipoproteinaemia
Homanics et al., b
ApoB tg (human) and high fat diet
Hyper lipoproteinaemia and atherosclerosis
Purcell-Huynh et al., c
LDL receptor and LRP ko
Impaired chylomicron remnant clearance Willnow et al., d
Apo(a) tg (human) and high fat diet
Apo(a)aemia and atherosclerosis
ApoB and apo(a) tg (human) and Lp(a)aemia and atherosclerosis high fat diet
Lawn et al., e Callow et al., f
ApoA-I tg (human) and high fat diet
Hyper lipoproteinaemia and inhibition of atherogenesis
Rubin et al., g
ApoA-I ko
Hypo lipoproteinaemia
Williamson et al., h
ApoE ko
Hypercholesterolaemia and atherosclerosis
Zhang et al., i
ApoEArg112,Cys142 tg (human)
Type III hyperlipoproteinaemia
Fuzio et al., j
ApoC-III tg (human)
Hypertriglyceridaemia
Ito et al., k
ApoC-III ko
Improved fat tolerance
Maeda et al., l
ApoC-III tg (human) and LDL receptor ko
Mixed (combined) hyperlipidaemia
Masucci-Magoulas et al., m
ApoC-I tg (human)
Mixed (combined) hyperlipidaemia
Schacter et al., n
Lipoprotein lipase ko
Hypertriglyceridaemia (and neonatal death)
Weinstock et al., o
Lipoprotein lipase tg (human)
Resistance to dietary hyperlipidaemia
Shimada et al., p
Hepatic lipase ko
Mild dyslipidaemia
Homanics et al., q
Hepatic lipase tg (human)
Hypo lipoproteinaemia
Busch et al., r
a Journal of Clinical Investigation, 1994, 93, 1885–1893. b Proceedings of the National Academy of Sciences USA, 1993, c
Journal of Clinical Investigation, 1995, 95, 2246–2257.
90, 2389–2393.
Plasma lipids and their role in disease
d
22
Science, 1994, 264, 1471–1474.
e Nature, 1992, 360, 670–672. f Journal of Clinical Investigation, 1995, 96,
1639–1646. Nature, 1991, 353, 265–267. Proceedings of the National Academy of Sciences USA, 1992, 89, 7134–7138. i Science, 1992, 258, 468–471. j Journal of Clinical Investigation, 1993, 92, 1497–1503. k Science, 1990, 249, 790–793. l Journal of Biological Chemistry, 1994, 269, 23610–23616. m Science, 1997, 275, 391–394. n Journal of Clinical Investigation, 1996, 98, 846–855. o Journal of Clinical Investigation, 1995, 96, 2555–2568. p Journal of Biological Chemistry, 1993, 268, 17924–17929. q Journal of Biological Chemistry, 1995, 270, 2974–2980. r Journal of Biological Chemistry, 1994, 169, 16376–16382. g h
MECHANISMS The advent of transgenic and gene knock-out technology has enabled the creation of mouse models of many genetically-determined forms of dyslipidaemia (Table 2.2). In most instances these have simply confirmed what was already known from studies in humans. However, some of the mouse models have provided possible clues to disorders where the mechanism of dyslipidaemia is unknown or poorly understood. One example is the apoC-III transgenic mouse, which suggests that increased levels of apoC-III can play a causal role in hypertriglyceridaemia and are not just the consequence of a raised level of VLDL. Another example is the apoC-III transgenic and LDL receptor gene knockout mouse model of FCH, although turnover studies suggest that overproduction rather than defective catabolism is responsible for the raised level of apoB-containing lipoproteins which characterise that disorder in humans. Hence this is a model of mixed dyslipidaemia rather than of FCH.
THE PRIMARY HYPERCHOLESTEROLAEMIAS These comprise several disorders due to increases in LDL or HDL known to be genetic in origin or presumed to be so in the absence of any cause of secondary hypercholesterolaemia. This section also includes genetic disorders characterised by the accumulation in plasma and tissues of abnormal amounts of other sterols, notably plant sterols (phytosterolaemia) and cholestanol (cerebro-tendinous xanthomatosis), and also cholesterol ester storage disease. Familial Hypercholesterolaemia (FH) This disorder affects approximately 0.2% of the population (see Table 2.1) and is due to
Abnormalities of plasma lipoprotein transport
23
inheritance of one mutant gene encoding the LDL receptor (heterozygous familial hypercholesterolaemia), or rarely to inheritance of two mutant alleles (homozygous familial hypercholesterolaemia). To date more than 250 distinct mutations of the LDL receptor gene have been described (Brown et al., 1997). The wide variety of mutations which have been identified in FH patients in Britain are shown in Figure 2.1. Deficient expression or defective function of LDL receptors in FH results in accumulation of LDL, causing hypercholesterolaemia from birth. Serum total cholesterol ranges between 8 and 15 mmol/l in adult heterozygotes and between 15 and 25 mmol/1 in homozygotes. Triglyceride levels are usually normal in affected children, most of whom exhibit a type IIa phenotype, but a type IIb phenotype is not uncommon in adults. High density lipoprotein cholesterol is normal or reduced. A definitive diagnosis of familial hypercholesterolaemia depends upon identifying the mutant gene or demonstrating a deficiency of LDL receptors in fresh or cultured cells. However, the presence of tendon xanthomata in an individual with a type IIa or IIb phenotype or a raised LDL cholesterol in someone with a hypercholesterolaemic firstdegree relative with tendon xanthomata is presumptive proof of heterozygous familial hypercholesterolaemia. Homozygotes result from unions between two heterozygotes, which are often consanguineous. Homozygous FH This rare condition is characterised by extreme hypercholesterolaemia and the early onset of cutaneous planar or tuberose xanthomata, tendon xanthomata and corneal arcus. Atheromatous involvement of the aortic root is evident by puberty, manifested by an aortic systolic murmur, a gradient across the aortic valve and narrowing of the aortic root (Allen et al., 1980). Coronary ostial stenosis is common leading to sudden death from acute coronary insufficiency during early adulthood. At post-mortem, the aortic valve, sinuses of Valsalva and ascending arch of the aorta are grossly infiltrated with atheroma. Less severe changes are found in the abdominal aorta, pulmonary artery, carotid arteries and circle of Willis. Histologically, the lesions are typical of advanced atherosclerosis (Buja et al., 1979). The age of onset and severity of cardiovascular involvement depends upon the nature of the gene defect (Goldstein and Brown, 1983), being earlier and worse when the abnormality is an inability to produce mature receptors (receptor-negative) than when mature but abnormal receptors are formed (receptor-defective). Inheritance of two different mutations results in a genetically compound heterozygote but presents clinically as homozygous FH. Heterozygous FH may be detected early when screening an affected family but often remains undiagnosed until the onset of cardiovascular symptoms in adult life. There is a marked increase in the risk of premature coronary heart disease in both males and females (Scientific Steering Committee on behalf of the Simon Broome Register Group, 1991). In addition to hypercholesterolaemia, heterozygotes often show signs of cholesterol deposition, such as corneal arcus, xanthelasma and tendon xanthomata. Characteristic sites for the latter are the extensor tendons on the back of the hands and elbows, the Achilles tendon and the patellar tendon insertion into the pretibial tuberosity.
Plasma lipids and their role in disease
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Some patients exhibit a systolic ejection murmur, indicative of atherosclerotic involvement of the aortic root.
Figure 2.1 Schematic diagram of LDL receptor illustrating the nature and site of mutations identified in patients referred to Hammersmith Hospital (compiled from Sun, X-M. et al. (1992). Arteriosclerosis and Thrombosis, 12, 762–770; Webb, J.C. et al. (1996). Journal of Lipid Research, 37, 368–381; Sun, X-M. et al. (1997). Arteriosclerosis, Thrombosis, and Vascular Biology, 17, 3092–3101 and Bourbon, M., Sun, X-M. and Soutar, A.K. (personal communication)).
Heterozygous Familial Hypercholesterolaemia LDL levels in FH are determined by both genetic and environmental influences. For example in children with heterozygous FH in Quebec the LDL cholesterol was significantly lower in children with a receptor-defective missense mutation than in those
Abnormalities of plasma lipoprotein transport
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with the receptor-negative French Canadian deletion (Torres et al., 1996). Similarly mutations in exon 4 of the gene for the LDL receptor, which encodes its apoB/E binding domain, are associated with a higher LDL cholesterol than mutations in other exons (Gudnason et al., 1994). In Norwegian children with FH the degree of obesity was an important determinant of variations in LDL level among those with any given mutation of the LDL receptor (Tonstad et al., 1995). It has been estimated that the onset of coronary heart disease occurs about 20 years earlier in FH, if left untreated, than in the remainder of the population. One factor influencing the presence of vascular disease is the HDL cholesterol, high levels being protective (Streja et al., 1978); another factor is smoking, the age of onset of coronary heart disease in women with familial hypercholesterolaemia who smoke being similar to that in men. On angiography, over 70% of male heterozygotes have triple vessel disease, including 32% with disease of the left main stem (Sugrue et al., 1981). Lesions occur in both the proximal and distal portions of affected vessels compared with normocholesterolaemic smokers, in whom distal lesions affecting a single vessel are quite common. Completely occluded vessels and coronary ectasia were also found to be much commoner in familial hypercholesterolaemic patients. Familial Defective ApoB-100 This inherited disorder, often abbreviated to FDB, was originally described as due to a single amino acid substitution (glutamine for arginine) at residue 3500 in apoB (Innerarity et al., 1990). This results in an almost complete loss of ability of LDL to bind to its receptor. Recently, however, a milder form of FDB has been described due to substitution of cysteine for arginine at residue 3531 (Pullinger et al., 1995). Estimates of frequency vary but FDB3500 is probably about half as common as FH. Affected individuals usually have moderate hypercholesterolaemia but some heterozygotes present with clinical features which are indistinguishable from FH (Myant, 1993). However, homozygotes are not so severely affected as are FH homozygotes (Marz et al., 1992) and a recent study suggests that in heterozygotes too the phenotypic expression of FDB is milder than FH, at least in children (Pimstone et al., 1997). Polygenic Hypercholesterolaemia Plasma cholesterol levels are under the control of many different genes and environmental factors the summated effects of which give a near-Gaussian distribution of cholesterol levels in the population. The clustering in an individual and within families of several genes which together induce moderate elevations of plasma cholesterol is termed polygenic or sporadic hypercholesterolaemia. Most of these genes have yet to be identified but in patients with primary hypercholesterolaemia not due to FH the frequency of the apoE4 allele is significantly increased. This allele is known to be associated with higher LDL-cholesterol levels in the general population than are the apoE2 and apoE3 alleles and is one of the many genetic factors contributing to polygenic hypercholesterolaemia (Utermann, 1988).
Plasma lipids and their role in disease
26
The classical clinical features of familial hypercholesterolaemia do not occur in polygenic hypercholesterolaemia but the disorder does appear to be associated with premature atherosclerosis. Estimates of the prevalence of polygenic hypercholesterolaemia will vary according to the arbitrary definition of the upper limit of normal for serum cholesterol. Goldstein et al. (1973) identified it in 14% of their hyperlipidaemic survivors of myocardial infarction, using a value of 7.4mmol/l as their cut-off, as compared with the 10% in that study who had FH. Obviously the lower the cut-off value that is used the higher the estimated frequency of polygenic hypercholesterolaemia will be in the population. Familial Hyper
lipoproteinaemia
Hyper lipoproteinaemia, defined as an HDL cholesterol > 2mmol/l, sometimes occurs on a familial basis. Familial hyper lipoproteinaemia is a heterogeneous entity in that some families show a clear-cut autosomal dominant pattern of inheritance while in others the features suggest interaction between polygenic influences and common environmental factors within the household, such as alcohol. The increase in HDL cholesterol reflects rises in both HDL2 and HDL3 (Iselius and Lalouel, 1982). The syndrome often tends to be associated with a decreased frequency of CHD and with longevity (Glueck et al., 1975). In most instances the mechanism of hyper lipoproteinaemia is unknown but an association has been described with a polymorphism of the promoter region of the apoA-I gene (Pagani et al., 1990). Recently a sub-group of patients has been described with familial hyper lipoproteinaemia due to deficiency of cholesterol ester transfer activity in plasma (Yamashita et al., 1988). This results in smaller than normal LDL particles and enlarged HDL particles, rich in apoE. Such individuals may exhibit corneal opacities and can develop CHD despite their high HDL (see Chapter 12). Cholesterol Ester Storage Disease A rare cause of primary hypercholesterolaemia is inherited deficiency of cholesterol ester hydrolase, which gives rise to cholesterol ester storage disease. Mutations of this lysosomal enzyme (Ameis et al., 1995) impair hydrolysis of cholesterol ester, resulting in failure of down regulation of HMG CoA reductase. The consequent increase in cholesterol synthesis leads to increased secretion of VLDL (Cummings and Watts, 1995). In plasma LDL cholesterol is increased whereas HDL cholesterol is reduced. Clinically the disorder is characterised by hepatic and splenic enlargement but xanthomata are absent. Diagnosis depends upon demonstrating excessive amounts of cholesterol ester in liver biopsies and deficiency of cholesterol ester hydrolase activity in cultured fibroblasts or finding a mutation in the gene. Treatment with an HMG CoA reductase inhibitor lowers LDL cholesterol and may prevent the accelerated atherosclerosis which has been described in this disorder (Ginsberg et al., 1987). Phytosterolaemia (Sitosterolaemia) This recessively-inherited disorder is characterised by excessive absorption of plant
Abnormalities of plasma lipoprotein transport
27
sterols, with a consequent increase in plasma levels of sitosterol and campesterol. This is accompanied by a moderate increase in LDL cholesterol and the early onset of tendon xanthomas and atherosclerosis (Berger et al., 1994). Cholesterol and bile acid synthetic rates are both decreased but these changes are probably secondary to the accumulation of plant sterols. The site of the primary defect is uncertain but the most likely candidate is an inherited abnormality of the putative pathway which in normal individuals discriminates between the absorption of cholesterol and other sterols from the small intestine (Gregg et al., 1986). Affected individuals respond well to treatment with bile acid sequestrants, with a reduction in plasma levels of both cholesterol and plant sterols. The disorder is not expressed in heterozygotes. Cerebro-tendinous Xanthomatosis (CTX) This rare, recessively inherited disorder is characterised by tendon xanthomas, cataracts and neurological dysfunction. The disease is due to mutations affecting sterol 27 hydroxylase (Kim et al., 1994; Garuti et al., 1997), an enzyme involved in catalysing the conversion of cholesterol to bile acids. Deficiency of this enzyme results in accumulation of cholestanol in plasma and tissues, notably the central nervous system and tendons. Affected individuals are usually normolipidaemic but concomitant mixed hyperlipidaemia has been described (Watts et al., 1996). Untreated these patients develop dementia and are at increased risk of premature atherosclerosis. Hyperlipoprotein(a)aemia Lipoprotein(a), or Lp(a), consists of an LDL particle covalently linked to a molecule of apolipoprotein(a). The latter has close homology with plasminogen (McLean et al., 1987) and is polymorphic. At least 34 isoforms have been described (Marcovina et al., 1993), which vary markedly in size. The distribution of Lp(a) in Caucasian populations is skewed, high plasma levels being associated with low molecular weight isoforms of apo (a). Variation in molecular weight and size of apo(a) are determined by the number of Kringle IV repeats in the molecule, which range from 11 to more than 50 (Kraft et al., 1996). The inverse correlation between Lp(a) concentration in plasma and particle size is explained by higher rates of secretion of small versus large isoforms (Rader et al., 1994). Based on its structural features Lp(a) would be expected to have both atherogenic and thrombogenic potential. However, its importance as a risk factor for cardiovascular disease remains controversial in that the results of prospective studies have been equivocal, in contrast to those of case control studies. The latter have suggested that risk increases with Lp(a) levels above 30 mg/dl whereas in the prospective GRIP study risk of future myocardial infarction increased steeply only with levels of > 60 mg/dl (Cremer et al., 1994). It is possible that the conflict of evidence as to the role of Lp(a) as a risk factor is explained by qualitative differences between isoforms of similar size, notably their lysine-binding affinity, a property which is also exhibited by plasminogen and which mediates its interaction with fibrin (Hoover Plow et al., 1993). Thus isoforms of apo(a) with a high affinity for lysine could be expected to competitively inhibit binding of
Plasma lipids and their role in disease
28
plasminogen to fibrin and thereby impair fibrinolysis. Perhaps the most convincing evidence for the pathogenicity of Lp(a) relates to its ability to promote smooth muscle cell proliferation in vitro, by reducing the concentration of active TGF- (Grainger et al., 1993). Lp(a) has been shown to accumulate in arteries at the site of experimental balloon injury (Nielsen et al., 1996) and these two lines of evidence could well explain the association between raised levels of Lp(a) and re-stenosis after angioplasty (Desmarais et al., 1995; Miyata et al., 1996).
THE PRIMARY HYPERTRIGLYCERIDAEMIAS This section considers disorders characterised by predominant hypertriglyceridaemia due to increases in fasting plasma of chylomicrons and/or VLDL but without any obvious secondary cause. Evidence of the hereditary basis of some of these disorders is often presumptive in the absence of an identifiable genetic abnormality but investigation of first degree relatives may reveal partial metabolic defects, suggestive of heterozygous inheritance. Familial Lipoprotein Lipase Deficiency This rare disorder, also known as familial type I hyperlipoproteinaemia, is characterised by marked hypertriglyceridaemia and chylomicronaemia and usually presents in childhood (Nikkila, 1983). It is due to homozygous or compound heterozygous inheritance of mutations of the gene for lipoprotein lipase, located on chromosome 8 (Hayden et al., 1991). A large number of mutations have now been identified (Santamarina-Fojo, 1992), any of which cause complete or subtotal deficiency of the extrahepatic enzyme. This results in a failure of hydrolysis and accumulation of chylomicrons in plasma. The main clinical features are recurrent episodes of abdominal pain, often resembling acute pancreatitis, eruptive xanthomata, hepatosplenomegaly and lipaemia retinalis, associated with serum triglycerides in the region of 50–100mmol/l. There appears to be no increased susceptibility to atheroslcerosis in this condition. The gross chylomicronaemia results in an increase in serum cholesterol as well as triglyceride, but the plasma triglyceride:cholesterol mass ratio often exceeds 9:1. VLDL levels are usually normal or decreased, whereas LDL and HDL levels are both markedly reduced. The diagnosis depends upon demonstrating that plasma lipoprotein lipase levels, following an intravenous dose of heparin 5000 iu, are less than 10% of normal. Measurement of post-heparin lipolytic activity (PHLA) can be misleading since plasma levels of hepatic lipase, phospholipase and monoglyceride lipase often rise normally after heparin administration. Lipoprotein lipase can be distinguished from these other enzymes by various techniques including inhibition by sodium chloride, protamine sulphate or specific antibodies, or by chromatographic separation on heparin-sepharose affinity columns. Heterozygous carriers occur with a frequency of 1:500 except in Quebec, where the rate is 1:40, and tend to have higher triglyceride and apoB levels and lower levels of HDL cholesterol and PHLA than their unaffected relatives (Hayden et al., 1991). These
Abnormalities of plasma lipoprotein transport
29
features suggest that, unlike homozygotes, heterozygotes may be at increased risk of CHD (Bijvoet et al., 1996). Familial ApoC-II Deficiency This disorder is due to recessively inherited mutations of the gene for apoC-II which, in the homozygous state, result in the absence from plasma of normal apoC-II, with a consequent defect of lipolysis and hypertriglyceridaemia (Breckenridge et al., 1978). A number of distinct functionally inactive apoC-II variants have been described (Santamarina-Fojo, 1992). Lipoprotein lipase is present in normal amounts but cannot hydrolyse chylomicrons or VLDL in the absence of normal apoC-II, which activates the enzyme in plasma. Addition of apoC-II in vitro restores to normal the PHLA in patients’ plasma, which is inactive as a substrate for lipoprotein lipase unless apoC-II is present. The diagnosis can sometimes be made by demonstrating an absent or anomalous band of apoC-II on isoelectric focusing of delipidated VLDL. Homozygotes have triglycerides in the range of 15–107 mmol/l, with either a type I or type V phenotype, and often develop acute pancreatitis (Cox et al., 1978). Premature vascular disease is unusual but has been described. Heterozygotes exhibit a 30–50% decrease in apoC-II levels and a tendency to raised triglycerides (Santamarina-Fojo, 1992). Familial Hepatic Lipase Deficiency Two pairs of brothers have been described with this syndrome in Canada and Sweden respectively (Breckenridge et al., 1982; Carlson et al., 1987). The Canadian index patient had raised levels of both serum cholesterol and triglyceride, the latter ranging from 4.5– 93mmol/l depending upon his diet. Corneal arcus, eruptive xanthomata and palmar striae were present together with clinical and electrocardiographic evidence of myocardial ischaemia. Despite a type III pattern on lipoprotein electrophoresis isoelectric focussing showed the presence of apoE3. However, both sets of brothers had levels of post-heparin hepatic lipase which were only 5% of normal. This deficiency was presumed responsible for the accumulation of -VLDL and IDL as well as for the abnormally large, triglyceride-enriched HDL2 and absence of HDL3 particles which were observed. Hepatic lipase deficiency has also been recorded in a third family. A point mutation in exon 8 of the hepatic lipase gene has been identified in six affected individuals who are presumed to be compound heterozygotes for a second, unidentified mutation (Hayden et al., 1991). Familial Hypertriglyceridaemia This disorder is sub-divided according to whether the predominant abnormality in affected individuals is an excess of VLDL alone (type IV) or together with an excess of chylomicrons (type V). However, there is sometimes overlap within families and it is probable that similar genetic abnormalities are responsible for both varieties of the disorder but with a more severe expression in those with a type V phenotype. For
Plasma lipids and their role in disease
30
example the presence of chylomicrons in patients with a type V phenotype could simply reflect impaired chylomicron clearance due to saturation of lipolytic pathways by high concentrations of VLDL. Familial type IV hyperlipoproteinaemia is characterised by moderate hypertriglyceridaemia due to increased levels of VLDL, with an autosomal dominant pattern of inheritance. The frequency of the disorder in the adult population has been estimated at 0.2–0.3% but it is expressed less frequently in childhood. Fasting values of serum cholesterol and triglyceride averaged 6.2 and 3.0mmol/l respectively in one series of patients. The majority exhibited a type IV phenotype but some families also had members with a type V phenotype. In contrast with familial combined hyperlipidaemia none of the families contained individuals with type IIa or IIb phenotypes (Goldstein et al., 1973). Affected subjects have larger than normal VLDL particles with an increased triglyceride:apoB ratio, accompanied by a decrease in HDL cholesterol (Brunzell et al., 1983). Turnover studies show that VLDL triglyceride synthesis is increased to a greater extent than VLDL-apoB synthesis and that the fractional catabolic rate of both VLDL components is reduced (Chait et al., 1980). FFA flux into triglyceride is increased in type IV subjects, especially when on a high carbohydrate intake (Quarfordt et al., 1970). The increase in VLDL synthesis is accompanied by a decrease in the proportion of VLDL converted to LDL (Packard et al., 1980) which maintains plasma LDL cholesterol and apoB levels within the normal range. The underlying mechanism for the overproduction of VLDL triglyceride remains to be determined but affected members of type IV families have higher insulin levels than unaffected members, suggesting that insulin resistance may be involved. Administration of corticosteroids or oestrogens accentuates the hypertriglyceridaemia and can lead to acute pancreatitis. There are conflicting data as to whether the risk of myocardial infarction is increased (Brunzell et al., 1976). Familial type V hyperlipoproteinaemia is an uncommon disorder characterised by an increase in both VLDL and chylomicrons. Unlike type I hyperlipoproteinaemia it seldom presents in childhood and post-heparin lipoprotein lipase and hepatic lipase activities are usually normal. However, there is a similar liability to develop acute pancreatitis (Nikkila, 1983). Other features are eruptive xanthomata, glucose intolerance, hyperuricaemia and peripheral neuropathy. Hypertriglyceridaemia in type V subjects is accentuated by obesity and alcohol consumption. The mode of inheritance is uncertain but as mentioned previously there seems to be an overlap with familial type IV hyperlipoproteinaemia. In obese patients a type V phenotype will often change to a type IV pattern following successful introduction of a weight-reducing diet. Turnover studies show similar increases in VLDL apoB synthesis in type IV and V patients but a more marked decrease in fractional catabolic rate among the latter (Packard et al., 1980). There have been isolated reports of coronary disease in type V patients but there was no evidence of undue predisposition to CHD in a large study of 32 families (Greenberg et al., 1977).
Abnormalities of plasma lipoprotein transport
31
PRIMARY MIXED HYPERLIPIDAEMIAS Under this heading come two disorders with little in common other than the presence of a mixed form of hyperlipidaemia. Hypertriglyceridaemia and hypercholesterolaemia are both equally prominent in type III hyperlipoproteinaemia, whereas familial combined hyperlipidaemia (FCH) is characterised by increases in cholesterol or triglyceride or both these lipids in related individuals. However, type III hyperlipoproteinaemia and other genetic disorders associated with apolipoprotein E polymorphism are dealt with in Chapter 14, and only FCH will be considered here. Familial Combined Hyperlipidaemia This entity was first described in the families of hyperlipidaemic patients who survived a myocardial infarction (Goldstein et al., 1973). The authors showed that 30% of these patients had elevations of both cholesterol and triglyceride, and that roughly 50% of their relatives were hyperlipidaemic, of whom a third had hypercholesterolaemia (type IIa), a third had hypertriglyceridaemia (type IV or V) and a third had both abnormalities (type IIb). They concluded that the disorder was inherited in a monogenic manner but this has been disputed on the grounds that the pattern of transmission is more consistent with polygenic inheritance (Nikkila and Aro, 1973). Whatever its mode of inheritance FCH is a relatively common disorder, occurring in up to 0.5% of the general population. FCH differs from FH in that affected children are never hypercholesterolaemic, hypertriglyceridaemia being the earliest manifestation of the disorder, and differs from familial hypertriglyceridaemia in that the latter is never associated with an elevated LDL cholesterol. Although the nature of the genetic defect is unknown the disorder is characterised by increased secretion of apoB100, both as VLDL and LDL (Grundy et al., 1987), resulting in raised plasma apoB levels. The LDL cholesterol:apoB ratio and HDL cholesterol concentration both tend to be low, especially in hypertriglyceridaemic subjects. Metabolic abnormalities which have been described in FCH include an association with specific haplotypes of the apoA-I/C-III/A-IV gene complex (Dallinga-Thie et al., 1997); insulin resistance, raised free fatty acid levels and delayed chylomicron remnant clearance (Cabezas et al., 1993); and heterozygosity for lipoprotein lipase deficiency in a third of affected individuals (Kwiterovich, 1993). It has been speculated that lipoprotein lipase regulates the re-uptake of nascent hepatic lipoproteins, thereby down regulating secretion of apoB-containing lipoproteins, and that deficiency of that enzyme could explain the overproduction of apoB which characterises FCH (Williams et al., 1991). There seems to be no evidence of a defect of the apoB gene in this disorder and a primary abnormality of lipid metabolism offers a more plausible explanation for the overproduction of apoB. Overproduction of apoB is also a feature of hyperapo lipoproteinaemia (hyperapoB), a term first coined by Sniderman et al. (1980) to describe a syndrome characterised by an increased concentration of LDL-apoB in plasma despite a normal concentration of LDL cholesterol. Affected individuals are often hypertriglyceridaemic, and manifest
Plasma lipids and their role in disease
32
atherosclerosis of the coronary, cerebral and peripheral arteries. The increase in apoB occurs without a corresponding increase in LDL-cholesterol because the major subfraction of LDL, “heavy” LDL, has a subnormal cholesterol:protein ratio and is smaller and denser than normal in hyperapoB subjects, especially those with hypertriglyceridaemia (Teng et al., 1983). These features are also found in FCH and there is considerable overlap between these two entities. There are no distinctive clinical features in FCH and the diagnosis depends upon family studies. Cabezas et al. (1993) have proposed the following criteria for diagnosing FCH: the presence of primary hyperlipidaemia, with a serum cholesterol of > 6.5mmol/l and/or triglyceride > 2mmol/l and an apoB > 90 mg/dl in the patient; at least one first degree relative with a different lipoprotein phenotype; and a history of coronary or cerebrovascular disease in a first or second degree relative before the age of 60. The condition is undoubtedly associated with an increased risk of atherosclerosis and it is estimated that it occurs in at least 15% of patients with CHD below the age of 60 (Grundy et al., 1987).
SECONDARY HYPERLIPIDAEMIA Hyperlipidaemia can be secondary to a number of diseases, hormonal disturbances and iatrogenic agents and can present with any of the phenotypes associated with primary hyperlipoproteinaemia. Although many of the disorders listed can cause hyperlipidaemia in their own right the most marked abnormalities tend to occur in those individuals who have an underlying genetic susceptibility to dyslipidaemia. Hormonal Influences Pregnancy Pregnancy is normally accompanied by moderate rises in both cholesterol and triglyceride which reflect increases in VLDL, LDL and HDL, due mainly to the increase in oestrogens. Marked rises in cholesterol are usual in FH during pregnancy which can also markedly exacerbate hypertriglyceridaemia, especially where this is due to lipoprotein lipase deficiency. Exogenous sex-hormones The LRC Prevalence Study showed that women below the age of 45 on oral contraceptives had higher serum cholesterol and triglycerides than women not taking them. These differences reflected increases in VLDL and LDL but were not apparent in women over the age of 45 on oestrogen replacement therapy, who instead had higher HDL cholesterol levels than those of similar age not on oestrogens (Wallace et al., 1979). In some instances oral oestrogens, whether given as a contraceptive or replacement therapy, or for the treatment of prostatic cancer, cause marked hypertriglyceridaemia
Abnormalities of plasma lipoprotein transport
33
(Molitch et al., 1974), severe enough to precipitate acute pancreatitis. Hypothyroidism Hypothyroidism has long been recognised as an important and relatively common cause of hyperlipidaemia. Usually this presents as hypercholesterolaemia but it can also manifest itself as a mixed hyperlipidaemia. HDL cholesterol levels are sometimes elevated as well as LDL cholesterol. The increase in LDL is due to a decrease in receptor-mediated catabolism, reversible by replacement therapy with L-thyroxine. Hypothyroidism can also precipitate type III hyperlipoproteinaemia in those with an ε2 allele and accentuate the hypercholesterolaemia of FH. Growth hormone Replacement therapy with recombinant human growth hormone in growth hormonedeficient adults results in a decrease in LDL cholesterol but increases in HDL cholesterol and Lp(a) (Eden et al., 1993). The decrease in LDL cholesterol has been attributed to restoration of LDL receptor activity, which appears to be a growth hormone-dependent function. Metabolic Disorders Diabetes mellitus Untreated juvenile onset, type I or insulin-dependent diabetes mellitus (IDDM) is accompanied by marked hypertriglyceridaemia, often with a type V phenotype. The latter is partly due to deficiency of lipoprotein lipase consequent on insulin lack and partly due to an increased flux of FFA from adipose tissue, which promotes hepatic triglyceride synthesis. Maturity onset, type II or non-insulin-dependent diabetes mellitus (NIDDM) usually comes on after the age of 40 and is often associated with obesity. It seems to be particularly common in Asians and is characterised by insulin resistance. The commonest lipid abnormality is hypertriglyceridaemia, usually type IV, due mainly to increased synthesis of VLDL. This is the result of an increased input of substrates for triglyceride synthesis into the liver and leads to the production of large VLDL particles. Clearance of triglyceride is also impaired, due to decreased lipoprotein lipase activity but the proportion of VLDL converted to LDL is decreased and LDL levels are often normal. However, its content of triglyceride tends to be higher than normal and up to 5% of the lysine residues in apoB may be glycosylated. Gout Hypertriglyceridaemia is a common accompaniment of gout but there appears to be no direct metabolic link between hyperuricaemia and hypertriglyceridaemia. The association may simply reflect the fact that obesity, alcohol and thiazides are common causes of both
Plasma lipids and their role in disease
34
abnormalities. Obesity Hypertriglyceridaemia, glucose intolerance, hyperinsulinism and vascular disease all commonly accompany obesity, which in these respects resembles maturity onset diabetes. HDL cholesterol is also low, being inversely correlated with body weight, and rises with weight reduction. Total cholesterol and LDL levels are often normal but turnover studies show increased rates of synthesis of both cholesterol and apoB (Kesaniemi and Grundy, 1983). These metabolic abnormalities are especially common in association with the central or abdominal pattern of obesity, which has been shown to be an independent risk factor for cardiovascular mortality in middle-aged men (Kannel et al., 1991). Progressive partial lipodystrophy This rare disorder, which is sometimes familial, usually affects females and is characterised by the progressive loss of subcutaneous fat from the upper half of the body. Sometimes this is associated with apparent redistribution of fat, resulting in gross obesity of the lower limbs. Other features are glucose intolerance, which may progress to frank diabetes, hepatic dysfunction, severe hypertriglyceridaemia and glomerulo-nephritis (Bennett et al., 1977). The cause of the disorder is unknown. Storage disorders Hypertriglyceridaemia is a feature of both Gaucher’s disease and glycogen storage disease type I (Levy et al., 1988). Renal Dysfunction Nephrotic syndrome Hyperlipidaemia, often severe, is common in the nephrotic syndrome. Hypoalbuminaemia appears to play a central role probably by diverting increased amounts of FFA to the liver and thus stimulating apoB secretion (Joven et al., 1990). LDL cholesterol is inversely correlated with serum albumin and falls temporarily after albumin infusions. Lp(a) levels are increased also in the nephrotic syndrome (Wanner et al., 1993). Accelerated vascular disease can be a major consequence of persistent hyperlipidaemia in such patients. Chronic renal failure, on dialysis or post-transplant Hyperlipidaemia is common in patients with chronic renal failure, but in contrast to the nephrotic syndrome hypertriglyceridaemia is much commoner than hypercholesterolaemia. This appears to be secondary to impaired lipolysis, possibly
Abnormalities of plasma lipoprotein transport
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because of inhibition of lipoprotein lipase by a non-dialysable factor present in uraemic plasma. Increased concentrations of remnant particles and decreases in HDL occur in patients with chronic renal failure including those on haemodialysis. Lp(a) levels are increased two to fourfold in patients on haemodialysis (Dieplinger et al., 1993). Hyperlipidaemia also appears to be common in patients on chronic ambulatory peritoneal dialysis (CAPD), but the pattern differs from that seen with haemodialysis, possibly reflecting the absence of heparin administration in CAPD. Hyperlipidaemia often persists after successful renal transplantation although elevations in LDL as well as VLDL (type IIb) are commoner than in haemodialysed patients. Immunosuppressive drugs probably play an important role in post-transplant hyperlipidaemia, especially steroids. Dyslipidaemia not only predicts the likelihood of CHD in patients with renal disease (Tschope et al., 1993) but may also play a role in aggravating the underlying glomerular lesions (Diamond and Karnovsky, 1992). Obstructive Liver Disease Primary biliary cirrhosis or prolonged cholestasis from other causes is accompanied by marked hyperlipidaemia, due to the presence of high concentrations of Lp-X (Seidel et al., 1970). In biliary obstruction there is reflux of biliary lecithin into plasma, which interacts with free cholesterol, albumin and apoC in plasma. If these events occur at a rate which exceeds the cholesterol-esterifying capacity of LCAT, Lp-X is formed. Xanthelasma can be a prominent accompaniment of the hypercholesterolaemia of primary biliary cirrhosis, as was first documented almost one and a half centuries ago (Figure 2.2). Excessive consumption of ethanol is a common cause of secondary hypertriglyceridaemia, especially in males, and often results in a type V phenotype. Even moderate consumption of alcohol on a regular basis results in significantly higher serum triglycerides than are found in total abstainers. One postulated mechanism is that ethanol is preferentially oxidised in the liver, which results in sparing of FFA and an increased availability of the latter for triglyceride synthesis. Withdrawal of alcohol results in a rapid decrease in triglyceride levels. An increased level of HDL cholesterol is a commoner consequence of heavy consumption of alcohol than is hypertriglyceridaemia and reflects increases in both HDL2 and HDL3, which are due to the increase in lipoprotein lipase activity which accompanies regular drinking. Coffee Frequent consumption of boiled or percolated coffee, but not the instant or filtered versions, can result in increases in serum cholesterol and triglyceride. These effects are mediated by diterpenes contained in oils leached out from coffee beans but the mechanism is unclear (Mensink et al., 1995).
Plasma lipids and their role in disease
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Figure 2.2 Postmenopausal woman with obstructive jaundice and extensive xanthelasma (from Addison, T. and Gull, W. (1851). Guy’s Hospital Review, 7, 265–276).
Beverages and Toxins Alcohol Chlorinated hydrocarbons Chlorinated hydrocarbons such as the insecticide DDT can markedly elevate HDL cholesterol. In contrast, o,p’DDD, which is structurally related to DDT but is used to treat refractory cases of Cushing’s syndrome, markedly increases LDL cholesterol (Maher et al., 1992).
Abnormalities of plasma lipoprotein transport
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Iatrogenic Thiazides and
-blockers
Administration of thiazide diuretics such as chlorthalidone and hydrochlorothiazide has long been recognised to increase total cholesterol and triglyceride. HDL cholesterol changes little but VLDL and LDL cholesterol both increase. These changes probably reflect the adverse effects of these drugs on glucose tolerance and tend to be accompanied by hyperuricaemia. Long-term administration of -blockers without intrinsic sympathomimetic activity (ISA) is associated with increases in serum triglyceride and decreases in HDL cholesterol, -blockers with ISA have a much less marked influence on serum triglyceride and, like -blockers, cause an increase in HDL cholesterol. The mechanism of the hypertriglyceridaemic and HDL lowering effects of -blockers may involve a decrease in lipoprotein lipase due to inhibition of adenyl cyclase in adipocytes. Removal of triglycerides from plasma is impaired during -blockade and can occasionally lead to marked increases in serum triglyceride in genetically-predisposed individuals. Immunosuppressants Corticosteroids cause insulin resistance and impaired glucose tolerance which leads to hypertriglyceridaemia and a reduction in HDL cholesterol. Experimental studies suggest that a steroid-induced increase in VLDL synthesis is one of the mechanisms involved, as can occur spontaneously in patients with Cushing’s syndrome. Studies in renal transplant patients on cyclosporin show that this drug causes an increase in serum cholesterol, reflecting an increase in LDL cholesterol. It has been suggested that the latter stems from a hepatotoxic effect of the drug, which impairs receptor-mediated LDL catabolism. Other drugs An increase in HDL cholesterol has been well documented in epileptics receiving phenytoin. A similar effect as been reported with barbiturates and with cimetidine, but not ranitidine. Retinoids induce a marked increase in serum triglycerides, especially in patients with pre-existing hypertriglyceridaemia. Amiodarone can cause hypercholesterolaemia independently of its effects on thyroid function (Wiersinga et al., 1991).
Plasma lipids and their role in disease
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PRIMARY HYPOLIPOPROTEINAEMIA Abetalipoproteinaemia This rare, recessively inherited disease is characterised by the onset during infancy of malabsorption and anaemia accompanied by the development in later childhood of progressively severe ataxia and retinitis pigmentosa. Examination of the blood shows the presence of acanthocytes and the absence of chylomicrons, VLDL and LDL. Serum cholesterol and triglyceride levels are both very low, usually in the range 0.5–2mmol/l, and apoB is undetectable. Nearly all the cholesterol in plasma is present as HDL, mainly as HDL2. The majority of patients described seem to be males and at least half of them were the result of consanguineous unions. Obligate heterozygotes, however, show no signs of disease and have normal serum lipids. Homozygotes usually present with steatorrhoea in early childhood and jejunal biopsy shows the characteristic lipid-filled but otherwise normal looking villi (Isselbacher et al., 1964). The liver too contains excess fat. It had been assumed that the underlying defect was an inherited inability to synthesise both apoB48 and apoB100, and thus a failure to form either intestinal chylomicrons or hepatic VLDL. However, it has now been shown that the disorder is due to mutations of the gene encoding microsomal triglyceride transfer protein (MTP), which is essential for the incorporation of non-polar lipids into apoB-containing lipoproteins and for secretion of the latter by the liver and small intestine (Sharp et al., 1993) (see Chapter 10). Malabsorption of triglyceride, although less marked than that of fat-soluble vitamins, leads to decreases in the linoleate and arachidonate content of plasma lipids. Osteomalacia has been reported but deficiency of vitamin D is less common than of vitamins A, E and K. Abetalipoproteinaemia represents the most severe vitamin E deficiency state known in humans but vitamin E supplementation prevents the development of neurological symptoms if given in childhood, and prevents further deterioration if disability is present prior to starting treatment (Muller et al., 1977). If left untreated patients develop dysarthria, cerebellar ataxia, areflexia, and loss of vibration and joint position sense. The disorder is progressive and relatively few patients are still ambulant after the age of 30. Retinitis pigmentosa is usually evident during the second decade of life. The peripheral part of the retina contains hyper-pigmented and depigmented areas which later spread to affect the macula, with a resulting loss of visual acuity and progressive restriction of the visual fields. Familial Hypobetalipoproteinaemia The homozygous form of this disorder presents in a manner either identical to or as a milder version of abetalipoproteinaemia. It differs in that heterozygotes have hypocholesterolaemia and LDL levels which are about 25% of normal. Thus the disorder appears to be inherited in an autosomal dominant manner. It has been suggested that heterozygotes are protected from coronary heart disease by their low LDL levels and that this leads to enhanced longevity. Reduced synthesis of apoB has been demonstrated in
Abnormalities of plasma lipoprotein transport
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such individuals, in whom fatty liver has also been described (Ogata et al., 1997; Targui et al., 1996). This is illustrated in Figure 2.3.
Figure 2.3 Fatty liver in 16 year old boy with heterozygous familial hypo lipoproteinaemia, stained with haematoxylin and eosin, magnification X 400 (by courtesy of Dr. J.O’Grady, Dr. B.Portmann and Dr. M.Pignatelli). Jejunal biopsy was normal.
Table 2.3 ApoA-I mutations associated with hypo lipoproteinaemia (adapted from Tilly-Kiesi et al., 1995)
Amino acid defect
Clinical findings
Cys173
None
Gly26 Pro165 Gln84
Arg Arg Iowa Arg stop Tsukuba
Systemic amyloidosis None CHD; xanthomas
Gua202
0
Corneal opacities
Glu146
Arg160 Seattle
Arcus senilis
Absence of apoA-I Q[-2]X
Xanthelasma; premature CHD
Lys107
0 Helsinki
Premature CHD in kindred
Leu141
Arg Pisa
Corneal opacities; CHDa
a
Miccoli, R. et al. (1996) Circulation, 94, 1622–1628.
Plasma lipids and their role in disease
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Recently, it has been shown that hypobetalipoproteinaemia can result from mutations of the apoB gene, the severity of clinical manifestations correlating inversely with the length and amount of apoB synthesised (Innerarity, 1990). At least 10 different mutations have been described so far, resulting in truncated forms of apoB which represent between 2– 89% of normal apoB-100 (Schonfeld, 1995). It has been shown that the shorter the length of truncated apoB, the lower is the rate at which it is synthesised (Parhofer et al., 1996). In some affected families truncated forms of apoB have not been found but linkage studies confirm involvement of the apoB gene. In others the apoB gene appears not to be involved and the hypo lipoproteinaemia presumably results from inherited abnormalities of other metabolic pathways. Hypo
lipoproteinaemia
ApoA-I mutations Familial absence or deficiency of HDL is a relatively rare disorder which is usually due to either Tangier disease, familial deficiency of lecithin cholesterol acyl transferase (LCAT) or mutations of the apoA-I gene (Funke, 1997). Most apoA-I mutations are silent but a minority are associated with HDL deficiency, as shown in Table 2.3. Clinical features associated with such mutations include corneal opacities, xanthomas and premature coronary heart disease. ApoA-I Iowa differs from the other mutations listed in causing systemic amyloidosis (Nicholls et al., 1990). Not listed in Table 2.3 are mutations of the apoA-I/C-III/A-IV gene complex, which predispose to atherosclerosis (Norum et al., 1982) nor apoA-I Milano, which appears to be protective (Franceschini et al., 1980). A similar mutation which was described subsequently, apoAIArg151 cysteine, seems also to have protective properties against atherosclerosis in that affected individuals tend to be long lived despite their low HDL levels (Bruckert et al., 1997). In a recent survey 10% of individuals with hypo lipoproteinaemia were found to have a dysfunctional mutation of lipoprotein lipase (Reymer et al., 1995). However, familial hypo lipoproteinaemia has also been reported in the absence of any detectable abnormality of apoA-I, lipoprotein lipase or LCAT nor any evidence of Tangier disease (Marcil et al., 1995). Tangier disease This disorder was first described by Fredrickson et al. (1961) in two young siblings who presented with enlargement of liver, spleen, lymph
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nodes and tonsils, which were orange-coloured, together with hypocholesterolaemia. Further investigation revealed the presence in these organs of numerous cholesterol ester containing macrophages, or foam cells, and an almost complete absence of HDL in plasma. A survey of over 50 cases of Tangier disease shows that peripheral neuropathy is a common complication and also suggests that there is an increased frequency of cardiovascular disease in middle-age (Serfaty Lacrosniere et al., 1994). Despite intensive investigation over the intervening 35 years the molecular basis of Tangier disease remains unknown. Turnover studies show very rapid removal of apoA-I from the circulation (Schaefer et al., 1982), possibly due to enhanced catabolism of HDL by reticuloendothelial cells. Secondary Hypolipoproteinaemia Hypocholesterolaemia secondary to malabsorption was first described in adults by Adlersberg et al. (1957). Similar findings were reported by Thompson and Miller (1973) with decreases in both LDL and HDL cholesterol in the face of normal or increased levels of VLDL. Patients with steatorrhoea were subsequently shown to have reduced amounts of linoleic acid in their plasma and in three instances clinical essential fatty acid deficiency was documented following massive intestinal resection (Press et al., 1974). Hypocholesterolaemia can be induced surgically by ileal resection or by creating a partial ileal bypass (Buchwald, 1964). The latter procedure lowers LDL levels by preventing reabsorption of bile acids, which stimulates receptor-mediated LDL catabolism (Spengel et al., 1981). Hypocholesterolaemia due to an increased rate of catabolism of LDL has also been reported in patients with metastatic carcinoma of the prostate (Henriksson et al., 1989). An unexpected fall in serum cholesterol in an hyperlipidaemic patient is sometimes an indication of the development of malignant disease. References Adlersberg, D., Wang, C.I. and Bossak, E.T. (1957). Disturbances in protein and lipid metabolism in malabsorption syndrome. Journal of the Mount Sinai Hospital , 24 , 206. Allen, J.M., Thompson, G.R., Myant, N.B., Steiner, R. and Oakley, C.M. (1980). Cardiovascular complications of homozygous familial hypercholesterolaemia. British Heart Journal , 44 , 361–368. Ameis, D., Brockmann, G., Knoblich, R., Merkel, M., Ostlund, R.E., Jr., Yang, J.W. et al. (1995). A 5' splice-region mutation and a dinucleotide deletion in the lysosomal acid lipase gene in two patients with cholesteryl ester storage disease. Journal of Lipid
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Research , 36 , 241–250. Bennett, W.M., Bardana, E.J., Wuepper, K., Houghton, D., Border, W.A., Gotze, O. et al. (1977). Partial lipodystrophy, C3 nephritic factor and clinically inapparent mesangiocapillary glomerulonephritis. American Journal of Medicine , 62 , 757–760. Berger, G.M., Deppe, W.M., Marais, A.D. and Biggs, M. (1994). Phytosterolaemia in three unrelated South African families. Postgraduate Medical Journal , 70 , 631–637. Bijvoet, S., Gagne, S.E., Moorjani, S., Gagne, C., Henderson, H.E., Fruchart, J. et al. (1996). Alterations in plasma lipoproteins and apolipoproteins before the age of 40 in heterozygotes for lipoprotein lipase deficiency . Journal of Lipid Research , 37 , 640–650. Breckenridge, W.C., Little, J.A., Steiner, G., Chow, A. and Poapst, M. (1978). Hypertriglyceridemia associated with deficiency of apolipoprotein C-II. New England Journal of Medicine , 298 , 1265– 1273. Breckenridge, W.C., Little, J.A., Alaupovic, P., Wang, C.S., Kuksis, A., Kakis, G. et al. (1982). Lipoprotein abnormalities associated with a familial deficiency of hepatic lipase. Atherosclerosis , 45 , 161– 179. Brown, M.S., Herz, J. and Goldstein, J.L. (1997). Calcium cages, acid baths and recycling receptors. Nature , 388 , 629–630. 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 , 121–128. Brunzell, J.D., Schrott, H.G., Motulsky, A.G. and Bierman, E.L. (1976). Myocardial infarction in the familial forms of hypertriglyceridemia. Metabolism , 25 , 313–320. Brunzell, J.D., Albers, J.J., Chait, A., Grundy, S.M., Groszek, E. and McDonald, G.B. (1983). Plasma lipoproteins in familial combined hyperlipidemia and monogenic familial hypertriglyceridemia. Journal of Lipid Research , 24 , 147–155. Buchwald, H. (1964). Lowering of cholesterol absorption and blood levels by ileal exclusion. Circulation , 29 , 713–720. Buja, L.M., Kovanen, P.T. and Bilheimer, D.W. (1979). Cellular pathology of homozygous familial hypercholesterolemia. American Journal of Pathology , 97 , 327–357. Cabezas, M.C., de Bruin, T.W.A., de Valk, H.W., Shoulders, C.C., Jansen, H. and Erkelens, D.W. (1993). Impaired fatty acid metabolism in familial combined hyperlipidemia. A mechanism assocating hepatic apolipoprotein B overproduction and insulin resistance. Journal of Clinical Investigation , 92 , 160–168. Carlson, L.A., Holmquist, L. and Nilsson-Ehle, P. (1987). Deficiency of hepatic lipase activity in postheparin plasma in familial hypertriglyceridaemia. Acta Medica Scandinavica , 219 , 435–447. Chait, A., Albers, J.J. and Brunzell, J.D. (1980). Very low density lipoprotein overproduction in genetic forms of hypertriglyceridaemia. European Journal of Clinical Investigation ,
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10 , 17–22. Cox, D.W., Breckenridge, W.C. and Little, J.A. (1978). Inheritance of apolipoprotein C-II deficiency with hypertriglyceridemia and pancreatitis. New England Journal of Medicine , 299 , 1421–1424. Cremer, P., Nagel, D., Labrot, B., Mann, H., Muche, R., Elster, H. et al. (1994). Lipoprotein Lp(a) as predictor of myocardial infarction in comparison to fibrinogen, LDL cholesterol and other risk factors: results from the prospective Gottingen Risk Incidence and Prevalence Study (GRIPS). European Journal of Clinical Investigation , 24 , 444–453. Cummings, M.H. and Watts, G.F. (1995). Increased hepatic secretion of very-low-density lipoprotein apolipoprotein B-100 in cholesteryl ester storage disease. Clinical Chemistry , 41 , 111–114. Dallinga-Thie, G.M., Van Linde-Sibenius Trip, M., Rotter, J.I., Cantor, R.M., Bu, X., Lusis, A.J. et al. (1997). Complex genetic contribution of the apo AI-CIII-AIV gene cluster to familial combined hyperlipidemia. Journal of Clinical Investigation , 99 , 953–961. Desmarais, R.L., Sarembock, I.J., Ayers, C.R., Vernon, S.M., Powers, E.R. and Gimple, L.W. (1995). Elevated serum lipoprotein(a) is a risk factor for clinical recurrence after coronary balloon angioplasty. Circulation , 91 , 1403–1409. Diamond, J.R. and Karnovsky, M.J. (1992). A putative role of hypercholesterolemia in progressive glomerular injury. Annual Review of Medicine , 43 , 83–92. Dieplinger, H., Lackner, C., Kronenberg, F., Sandholzer, C., Lhotta, K., Hoppichler, F. et al. (1993). Elevated plasma concentrations of lipoprotein(a) in patients with end-stage renal disease are not related to the size polymorphism of apolipoprotein(a). Journal of Clinical Investigation , 91 , 397–401. Eden, S., Wiklund, O., Oscarsson, J., Rosen, T. and Bengtsson, B.A. (1993). Growth hormone treatment of growth hormone-deficient adults results in a marked increase in Lp(a) and HDL cholesterol concentrations. Arteriosclerosis and Thrombosis , 13 , 296–301. Franceschini, G., Sirtori, C.R., Capurso, A., 2d, Weisgraber, K.H. and Mahley, R.W. (1980). A-IMilano apoprotein. Decreased high density lipoprotein cholesterol levels with significant lipoprotein modifications and without clinical atherosclerosis in an Italian family . Journal of Clinical Investigation , 66 , 892–900. Fredrickson, D.S., Altrocchi, P.H., Avioli, L.V., Goodman, D.S. and Goodman, H.C. (1961). Tangier disease. Combined clinical staff conference at the National Institutes of Health. Annals of Internal Medicine , 55 , 1016–1031. Funke, H. (1997). Genetic determinants of high density lipoprotein levels. Current Opinion in Lipidology , 8 , 189–196. Garuti, R., Croce, M.A., Tiozzo, R., Dotti, M.T., Federico, A., Bertolini, S. et al. (1997). Four novel mutations of sterol 27hydroxylase gene in Italian patients with cerebrotendinous xanthomatosis. Journal of Lipid Research , 38 , 2322–2334. Ginsberg, H.N., Le, N.A., Short, M.P., Ramakrishnan, R. and Desnick,
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R.J. (1987). Suppression of apolipoprotein B production during treatment of cholesteryl ester storage disease with lovastatin. Implications for regulation of apolipoprotein B synthesis. Journal of Clinical Investigation , 80 , 1692–1697. Glueck, C.J., Fallat, R.W., Millett, F., Gartside, P., Elston, R.C. and Go, R.C. (1975). Familial hyperalpha-lipoproteinemia: studies in eighteen kindreds. Metabolism , 24 , 1243–1265. Goldstein, J.L., Schrott, H.G., Hazzard, W.R., Bierman, E.L. and Motulsky, A.G. (1973). Hyperlipidemia in coronary heart disease. II. Genetic analysis of lipid levels in 176 families and delineation of a new inherited disorder, combined hyperlipidemia. Journal of Clinical Investigation , 52 , 1544–1568. Goldstein, J.L. and Brown, M.S. (1983). Familial hypercholesterolemia. In The Metabolic Basis of Inherited Disease , 5th edn, edited by Stanbury, J.B., Wyngaarden, J.B., Fredrickson, D.S., Goldstein, J.L. and Brown, M.S., New York: McGraw Hill, pp. 672–712. Grainger, D.J., Kirschenlohr, H.L., Metcalfe, J.C., Weissberg, P.L., Wade, D.P. and Lawn, R.M. (1993). Proliferation of human smooth muscle cells promoted by lipoprotein(a). Science , 260 , 1655–1658. Greenberg, B.H., Blackwelder, W.C. and Levy, R.I. (1977). Primary type V hyperlipoproteinemia. A descriptive study in 32 families. Annals of Internal Medicine , 87 , 526–534. Gregg, R.E., Connor, W.E., Lin, D.S. and Brewer, H.B.J. (1986). Abnormal metabolism of shellfish sterols in a patient with sitosterolemia and xanthomatosis. Journal of Clinical Investigation , 77 , 1864–1872. Grundy, S.M., Chait, A. and Brunzell, J.D. (1987). Familial combined hyperlipidaemia Workshop. Arteriosclerosis , 7 , 203–207. Gudnason, V., Day, I.N. and Humphries, S.E. (1994). Effect on plasma lipid levels of different classes of mutations in the low-density lipoprotein receptor gene in patients with familial hypercholesterolemia. Arteriosclerosis and Thrombosis , 14 , 1717– 1722. Hayden, M., Ma, Y., Brunzell, J. and Henderson, N.E. (1991). Genetic variants affecting human lipoprotein and hepatic lipases. Current Opinion in Lipidology , 2 , 104–109. Henriksson, P., Ericsson, S., Stege, R., Eriksson, M., Rudling, M., Berglund, L. et al. (1989). Hypocholesterolaemia and increased elimination of low-density lipoproteins in metastatic cancer of the prostate. Lancet , 1178–1180. Hoover Plow, J.L., Miles, L.A., Fless, G.M., Scanu, A.M. and Plow, E.F. (1993). Comparison of the lysine binding functions of lipoprotein(a) and plasminogen. Biochemistry , 32 , 13681–13687. Innerarity, T.L. (1990). Familial apolipoprotein B100: genetic disorders associated with apolipoprotein B. Current Opinion in Lipidology , 1 , 104–109. Innerarity, T.L., Mahley, R.W., Weisgraber, K.H., Bersot, T.P., Krauss, R.M., Vega, G.L. et al. (1990). Familial defective apolipoprotein B-
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100: a mutation of apolipoprotein B that causes hypercholesterolemia. Journal of Lipid Research , 31 , 1337–1349. Iselius, L. and Lalouel, J.M. (1982). Complex segregation analysis of hyperalphalipoproteinemia. Metabolism , 31 , 521–523. Isselbacher, K.J., Scheig, R., Plotkin, G.R. and Caulfield, J.B. (1964). Congenital beta-lipoprotein deficiency: An hereditary disorder involving a defect in the absorption and transport of lipids. Medicine (Baltimore) , 43 , 347. Joven, J., Villabona, C., Vilella, E., Masana, L., Alberti, R. and Valles, M. (1990). Abnormalities of lipoprotein metabolism in patients with the nephrotic syndrome. New England Journal of Medicine , 323 , 579–584. Kannel, W.B., Cupples, L.A., Ramaswami, R., Stokes, J., III, Kreger, B.E. and Higgins, M. (1991). Regional obesity and risk of cardiovascular disease; the Framingham Study. Journal of Clinical Epidemiology , 44 , 183–190. Kesaniemi, Y.A. and Grundy, S.M. (1983). Increased low density lipoprotein production associated with obesity. Arteriosclerosis , 3 , 170–177. Kim, K.S., Kubota, S., Kuriyama, M., Fujiyama, J., Bjorkhem, I., Eggertsen, G. et al. (1994). Identification of new mutations in sterol 27-hydroxylase gene in Japanese patients with cerebrotendinous xanthomatosis (CTX). Journal of Lipid Research , 35 , 1031–1039. Kraft, H.G., Lingenhel, A., Kochl, S., Hoppichler, F., Kronenberg, F., Abe, A. et al. (1996). Apolipoprotein(a) kringle IV repeat number predicts risk for coronary heart disease. Arteriosclerosis, Thrombosis, and Vascular Bioliogy , 16 , 713–719. Kwiterovich, P.O. (1993). Genetics and molecular biology of familial combined hyperlipidemia. Current Opinion in Lipidology , 4 , 133– 143. Levy, E., Thibault, L.A., Roy, C.C., Bendayan, M., Lepage, G. and Letarte, J. (1988). Circulating lipids and lipoproteins in glycogen storage disease type I with nocturnal intragastric feeding. Journal of Lipid Research , 29 , 215–226. Maher, V.M., Trainer, P.J., Scoppola, A., Anderson, J.V., Thompson, G.R. and Besser, G.M. (1992). Possible mechanism and treatment of o,p’DDD-induced hypercholesterolaemia. Quarterly Journal of Medicine , 84 , 671–679. Marcil, M., Boucher, B., Krimbou, L., Solymoss, B.C., Davignon, J., Frohlich, J. et al. (1995). Severe familial HDL deficiency in FrenchCanadian kindreds. Clinical, biochemical, and molecular characterization. Arteriosclerosis, Thrombosis, and Vascular Bioliogy , 15 , 1015–1024. Marcovina, S.M., Zhang, Z.H., Gaur, V.P. and Albers, J.J. (1993). Identification of 34 apolipoprotein(a) isoforms: differential expression of apolipoprotein(a) alleles between American blacks and whites. Biochemical and Biophysical Research Commununications , 191 , 1192–1196. Marz, W., Ruzicka, C., Pohl, T., Usadel, K.H. and Gross, W. (1992).
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Familial defective apolipoprotein B-100: mild hypercholesterolaemia without atherosclerosis in a homozygous patient. Lancet , 340 , 1362. McLean, J.W., Tomlinson, I.E., Kuang, W.J., Eaton, D.L., Chen, E.Y., Fless, G.M. et al. (1987). cDNA sequence of human apolipoprotein (a) is homologous to plasminogen. Nature , 330 , 132–137. Mensink, R.P., Lebbink, W.J., Lobbezoo, I.E., Weusten Van der Wouw, M.P., Zock, P.L. and Katan, M.B. (1995). Diterpene composition of oils from Arabica and Robusta coffee beans and their effects on serum lipids in man. Journal of Internal Medicine , 237 , 543–550. Miyata, M., Biro, S., Arima, S., Hamasaki, S., Kaieda, H., Nakao, S. et al. (1996). High serum concentration of lipoprotein(a) is a risk factor for restenosis after percutaneous transluminal coronary angioplasty in Japanese patients with single-vessel disease. American Heart Journal , 132 , 269–273. Molitch, M.E., Oill, P. and Odell, W.D. (1974). Massive hyperlipemia during estrogen therapy. Journal of the American Medical Association , 227 , 522–525. Muller, D.P., Lloyd, J.K. and Bird, A.C. (1977). Long-term management of abetalipoproteinaemia. Possible role for vitamin E. Archives of Disease in Childhood , 52 , 209–214. Myant, N.B. (1993). Familial defective apolipoprotein B-100: a review, including some comparisons with familial hypercholesterolaemia (published erratum appears in Atherosclerosis (1994), 105, 253). Atherosclerosis , 104 , 1–18. Nicholls, W.C., Gregg, R.E., Brewer, H.B., Jr. and Benson, M.D. (1990). A mutation in apolipoprotein A-I in the Iowa type of familial amyloidotic polyneuropathy. Genomics , 8 , 318–323. Nielsen, L.B., Slender, S., Kjeldsen, K. and Nordestgaard, B.G. (1996). Specific accumulation of lipoprotein(a) in balloon-injured rabbit aorta in vivo. Circulation Research , 78 , 615–626. Nikkila, E.A. (1983). Familial lipoprotein lipase deficiency and related disorders of chylomicron metabolism. In Metabolic Basis of Inherited Disease , 5th edn, edited by Stanbury, J.B., Wyngaarden, J.B., Fredrickson, D.S., Goldstein, J.L. and Brown, M.S., New York: McGraw Hill, pp. 622–642. Nikkila, E.A. and Aro, A. (1973). Family study of serum lipids and lipoproteins in coronary heart-disease. Lancet , 1 , 954–959. Norum, R.A., Lakier, J.B., Goldstein, S., Angel, A., Goldberg, R.B., Block, W.D. et al. (1982). Familial deficiency of apolipoproteins A-I and C-III and precocious coronary-artery disease. New England Journal of Medicine , 306 , 1513–1519. Ogata, H., Akagi, K., Baba, M., Nagamatsu, A., Suzuki, N., Nomiyama, K. et al. (1997). Fatty liver in a case with heterozygous familial hypobetalipoproteinemia. American Journal of Gastroenterology , 92 , 339–342. Packard, C.J., Shepherd, J., Joerns, S., Gotto, A.M. and Taunton, O.D. (1980). Apolipoprotein B metabolism in normal, type IV and type V hyperlipoproteinemic subjects. Metabolism , 29 , 213–222.
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Pagani, F., Sidoli, A., Giudici, G.A., Barenghi, L., Vergani, C. and Baralle, F.E. (1990). Human apolipoprotein A-I gene promoter polymorphism: association with hyperalphalipoproteinemia. Journal of Lipid Research , 31 , 1371–1377. Parhofer, K.G., Barren, P.H.R., Aguilar-Salinas, C.A. and Schonfeld, G. (1996). Positive linear correlation between the length of truncated apolipoprotein B and its secretion rate: in vivo studies in human apoB-89, apoB-75, apoB-54.8, and apoB-31 heterozygotes. Journal of Lipid Research , 37 , 844–852. Pimstone, S.N., Defesche, J.C., Clee, S.M., Bakker, H.D., Hayden, M.R. and Kastelein, J.J. (1997). Differences in the phenotype between children with familial defective apolipoprotein B-100 and familial hypercholesterolemia. Arteriosclerosis, Thrombosis, and Vascular Bioliogy , 17 , 826–833. Press, M., Kikuchi, H., Shimoyama, T. and Thompson, G.R. (1974). Diagnosis and treatment of essential fatty acid deficiency in man. British Medical Journal , 2 , 247–250. Pullinger, C.R., Hennessy, L.K., Chatterton, J.E., Liu, W., Love, J.A., Mendel, C.M. et al. (1995). Familial ligand-defective apolipoprotein B. Identification of a new mutation that decreases LDL receptor binding affinity. Journal of Clinical Investigation , 95 , 1225–1234. Quarfordt, S.H., Frank, A., Shames, D.M., Berman, M. and Steinberg, D. (1970). Very low density lipoprotein triglyceride transport in type IV hyperlipoproteinemia and the effects of carbohydrate-rich diets. Journal of Clinical Investigation , 49 , 2281–2297. Rader, D.J., Cain, W., Ikewaki, K., Talley, G., Zech, L.A., Usher, D. et al. (1994). The inverse association of plasma lipoprotein(a) concentrations with apolipoprotein(a) isoform size is not due to differences in Lp(a) catabolism but to differences in production rate. Journal of Clinical Investigation , 93 , 2758–2763. Reymer, P.W., Gagne, E., Groenemeyer, B.E., Zhang, H., Forsyth, I., Jansen, H. et al. (1995). A lipoprotein lipase mutation (Asn291Ser) is associated with reduced HDL cholesterol levels in premature atherosclerosis. Nature Genetics , 10 , 28–34. Santamarina-Fojo, S. (1992). Genetic dyslipoproteinemias: role of lipoprotein lipase and apolipoprotein C-II. Current Opinion in Lipidology , 3 , 186–195. Schaefer, E.J., Kay, L.L., Zech, L.A. and Brewer, H.B., Jr. (1982). Tangier disease. High density lipoprotein deficiency due to defective metabolism of an abnormal apolipoprotein A-i (ApoA-ITangier). Journal of Clinical Investigation , 70 , 934–945. Schonfeld, G. (1995). The hypobetalipoproteinemias. Annual Review of Nutrition , 15 , 23–34. Scientific Steering Committee on behalf of the Simon Broome Register Group (1991). Risk of fatal coronary heart disease in familial hypercholesterolaemia. British Medical Journal , 303 , 893–896. Seidel, D., Alaupovic, P., Furman, R.H. and McConathy, W.J. (1970). A lipoprotein characterizing obstructive jaundice. II. Isolation and partial characterization of the protein moieties of low density
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lipoproteins. Journal of Clinical Investigation , 49 , 2396–2407. Serfaty Lacrosniere, C, Civeira, F., Lanzberg, A., Isaia, P., Berg, J., Janus, E.D. et al. (1994). Homozygous Tangier disease and cardiovascular disease. Atherosclerosis , 107 , 85–98. Sharp, D., Blinderman, L., Combs, K.A., Kienzle, B., Ricci, B., Wager Smith, K. et al. (1993). Cloning and gene defects in microsomal triglyceride transfer protein associated with abetalipoproteinaemia. Nature , 365 , 65–69. Slimane, M.N., Pousse, H., Maatoug, F., Hammami, M. and Ben Farhat, M.H. (1993). Phenotypic expression of familial hypercholesterolaemia in central and southern Tunisia. Atherosclerosis , 104 , 153–158. Sniderman, A., Shapiro, S., Marpole, D., Skinner, B., Teng, B. and Kwiterovich, P.O., Jr. (1980). Association of coronary atherosclerosis with hyperapobetalipoproteinemia (increased protein but normal cholesterol levels in human plasma low density (beta) lipoproteins). Proceedings of the National Academy of Sciences of the USA , 77 , 604–608. Spengel, F.A., Jadhav, A., Duffield, R.G., Wood, C.B. and Thompson, G.R. (1981). Superiority of partial ileal bypass over cholestyramine reducing cholesterol in familial hypercholesterolaemia. Lancet , 2 , 768–770. Streja, D., Steiner, G. and Kwiterovich, P.O., Jr. (1978). Plasma highdensity lipoproteins and ischemic heart disease: studies in a large kindred with familial hypercholesterolemia. Annals of Internal Medicine , 89 , 871–880. Sugrue, D.D., Thompson, G.R., Oakley, C.M., Trayner, I.M. and Steiner, R.E. (1981). Contrasting patterns of coronary atherosclerosis in normocholesterolaemic smokers and patients with familial hypercholesterolaemia. British Medical Journal (Clinical Research Ed.) , 283 , 1358–1360. Targui, P., Lonardo, A., Ballarini, G., Grisendi, A., Pulvirenti, M., Bagni, A. et al. (1996). Fatty liver in heterozygous hypobetalipoproteinemia caused by a novel truncated form of apolipoprotein B. Gastroenterology , 111 , 1125–1133. Teng, B., Thompson, G.R., Sniderman, A.D., Forte, T.M., Krauss, R.M. and Kwiterovich, P.O., Jr. (1983). Composition and distribution of low density lipoprotein fractions in hyperapobetalipoproteinemia, normolipidemia, and familial hypercholesterolemia. Proceedings of the National Academy of Sciences of the USA , 80 , 6662–6666. Thompson, G.R. and Miller, J.P. (1973). Plasma lipid and lipoprotein abnormalities in patients with malabsorption. Clinical Science and Molecular Medicine , 45 , 583–592. Tilly Kiesi, M., Zhang, Q., Ehnholm, S., Kahri, J., Lahdenpera, S., Ehnholm, C. et al. (1995). ApoA-IHelsinki (Lysl07–>0) associated with reduced HDL cholesterol and LpA-I:A-II deficiency. Arteriosclerosis, Thrombosis, and Vascular Bioliogy , 15 , 1294– 1306.
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Tonstad, S., Leren, T.P., Sivertsen, M. and Ose, L. (1995). Determinants of lipid levels among children with heterozygous familial hypercholesterolemia in Norway. Arteriosclerosis, Thrombosis, and Vascular Bioliogy , 15 , 1009–1014. Torres, A.L., Moorjani, S., Vohl, M.C., Gagne, C., Lamarche, B., Brun, L.D. et al. (1996). Heterozygous familial hypercholesterolemia in children: low-density lipoprotein receptor mutational analysis and variation in the expression of plasma lipoprotein-lipid concentrations. Atherosclerosis , 126 , 163–171. Tschope, W., Koch, M., Thomas, B. and Ritz, E. (1993). Serum lipids predict cardiac death in diabetic patients on maintenance hemodialysis. Results of a prospective study. The German Study Group Diabetes and Uremia. Nephron , 64 , 354–358. Utermann, G. (1988). Apolipoprotein polymorphism and multifactorial hyperlipidaemia. Journal of Inherited Metabolic Disease , 11 Suppl 1, 74–86. Wallace, R.B., Hoover, J., Barrett Connor, E., Rifkind, B.M., Hunninghake, D.B., Mackenthun, A. et al. (1979). Altered plasma lipid and lipoprotein levels associated with oral contraceptive and oestrogen use. Report from the Medications Working Group of the Lipid Research Clinics Program. Lancet , 2 , 112–115. Wanner, C., Rader, D., Bartens, W., Kramer, J., Brewer, H.B., Schollmeyer, P. et al. (1993). Elevated plasma lipoprotein(a) in patients with the nephrotic syndrome . Annals of Internal Medicine , 119 , 263–269. Watts, G.F., Mitchell, W.D., Bending, J.J., Reshef, A. and Leitersdorf, E. (1996). Cerebrotendinous xanthomatosis: a family study of sterol 27-hydroxylase mutations and pharmacotherapy. Quarterly Journal of Medicine , 89 , 55–63. Wiersinga, W.M., Trip, M.D., van Beeren, M.H., Plomp, T.A. and Costing, H. (1991). An increase in plasma cholesterol independent of thyroid function during long-term amiodarone therapy. A dosedependent relationship. Annals of Internal Medicine , 114 , 128–132. Williams, K.J., Petrie, K.A., Brocia, R.W. and Swenson, T.L. (1991). Lipoprotein lipase modulates net secretory output of apolipoprotein B in vitro. A possible pathophysiologic explanation for familial combined hyperlipidemia. Journal of Clinical Investigation , 88 , 1300–1306. Yamashita, S., Matsuzawa, Y., Okazaki, M., Kako, H., Yasugi, T., Akioka, H. et al. (1988). Small polydisperse low density lipoproteins in familial hyperalphalipoproteinemia with complete deficiency of cholesteryl ester transfer activity. Atherosclerosis , 70 , 7–12.
3 Epidemiological Evidence Linking Plasma Lipoprotein Disorders to Atherosclerosis and Other Diseases Jacques Genest Jr. * and Jeffrey S.Cohn Cardiovascular Genetics Laboratory and Hyperlipidaemia and Atherosclerosis Research Group, Clinical Research Institute of Montreal, Quebec, Canada
KEYWORDS: epidemiology, atherosclerosis, LDL, HDL, triglyceride, Lp(a).
INTRODUCTION It is well recognised that atherosclerosis begins early in life, even though clinical manifestations of this disease do not normally appear until well into adulthood. It was thus originally believed that atherosclerosis was a normal and hence inevitable phenomenon of aging. As infectious diseases became better controlled in the early part of this century, due to better sanitation and the efforts of public health, it became apparent that there was a rising incidence of coronary heart disease and cerebrovascular disease (Murray and Lopez, 1997). This was particularly evident in industrialised societies after World War II. Case-control studies suggested that coronary artery disease (CAD) was associated with increased serum cholesterol levels and hypertension (Steiner and Domaski, 1943; Master et al., 1939). Also of significance was the epidemiological work of Ancel Keys, showing that saturated fat intake and serum cholesterol levels were strongly related to standardised rates of coronary disease in different countries (Keys, 1980). The strength and consistency of these associations suggested a causal relationship, however, this could only be demonstrated conclusively by measuring appropriate parameters in healthy individuals, following the incidence of CAD in these individuals over a long period of time, and then relating incidence of CAD to the measured parameters (a longitudinal population study). Such a study was initiated by the United States Public Health Service Division of Chronic Diseases in 1948. The town of Framingham in Massachusetts
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was chosen to serve as a site for recruiting an appropriate study population. The Framingham Heart Study was thus launched, and this study is now in its fourth decade of investigation (Dawber, 1980). Epidemiological studies have subsequently led to the concept that the natural history of coronary heart disease is affected by various environmental and genetic *Corresponding Author: Cardiovascular Genetics Laboratory, Clinical Research Institute of Montreal, 110 Pine Avenue West, Montreal, Quebec, Canada H2W 1R7.
influences. Investigation of the occurrence and distribution of CAD in different countries, nationalities or different families has provided valuable insight into these environmental and genetic factors. Known as CAD risk factors, they include age, male gender, family history of CAD, smoking, hypertension, diabetes, left ventricular hypertrophy and elevated blood lipids (Dawber, 1980). An elevation in total plasma cholesterol concentration was one of the first CAD risk factors to be identified, however, it is now clear that there is a strong association between several plasma lipoprotein abnormalities and the incidence of CAD. The importance of these associations is born out by the very significant results of recent large-scale intervention studies in subjects with elevated plasma lipid levels and CAD, showing that intensive treatment of lipoprotein disorders leads to a decrease in cardiac morbidity and mortality, as well as a decrease in overall mortality (Scandinavian Simvastatin Survival Study Group 1994; Shepherd et al., 1995; Sacks et al., 1996). The relationship between various lipid and lipoprotein parameters and CAD will be discussed in the present chapter, in light of epidemiological data from both between- and within-population studies, and from the investigation of families with a genetic predisposition to CAD. Although, this review will focus almost entirely on lipoprotein disorders associated with atherosclerosis and CAD, there are a number of other diseases caused by abnormalities in lipid metabolism. For example, there exist several rare disorders of cellular lipid metabolism such as cholesteryl ester storage disease or Wollman’s disease (acid cholesteryl ester hydrolase deficiency) (Schmitz and Assmann, 1989), Niemann Pick disease (a genetic defect of a novel protein that shares homology with a protein (SCAP) that cleaves the sterol-regulatory-element binding protein) (Spence and Callahan, 1989), hypobetalipoproteinaemia (due to mutations of the apoB gene) (Kane and Havel, 1989), abetalipoproteinaemia (due to mutations in the microsomal transfer protein) (Wetterau et al., 1992), and chylomicron retention disease (of unknown aetiology) (Kane and Havel, 1989). The reader is referred to the aforementioned references for a detailed
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description of these abnormalities. In addition, severe hypertriglyceridaemia can cause acute as well as chronic bouts of pancreatitis (Brunzell, 1989). Pancreatitis is caused by self destruction of pancreatic tissue, which is most commonly caused by chronic alcoholism or biliary tract disease. The mechanisms leading to hypertriglyceridaemia-induced pancreatitis are not fully understood. A common pathway for the development of pancreatitis is thought to be the activation of proteolytic enzymes secreted as zymogens by pancreatic acinar cells. Acute pancreatitis of any aetiology is often a severe medical emergency with a high case fatality. Chronic bouts of pancreatitis can lead to a destruction of endocrine and exocrine pancreatic function, resulting in diabetes, in addition to malabsorption.
TOTAL CHOLESTEROL Epidemiological evidence linking elevated blood cholesterol levels to atherosclerosis is overwhelming. In the past forty years, large scale studies carried out in many countries, have shown clearly and unequivocally that total serum (or plasma) cholesterol is associated with CAD in a continuous, graded and independent fashion. This evidence has led the National Cholesterol Education Program Adult Treatment Panel (NCEP-ATP II) to consider cholesterol as a causative agent in the pathogenesis of CAD (The Expert Panel, 1993). Population Studies It has been clearly documented that the incidence of CAD varies considerably from one country to another. Northern Ireland, Scotland, Czechoslovakia and Finland have male and female CAD mortality rates which are about five times higher than those of Southern European countries (such as France and Portugal), which in turn have rates that are more than two times higher than those in Japan. According to the Seven Countries Study, these different rates of coronary mortality are related to the median plasma cholesterol level of these populations and the proportion of saturated and polyunsaturated fat in their habitual diets (Keys, 1980). As shown in Figure 3.1, a strong positive relationship (r=0.80) was observed between median serum cholesterol concentrations and 10-year CAD mortality in sixteen cohorts from 7 different countries. Although genetic (i.e. hereditary) factors are at least partly responsible for this relationship, environmental factors such as diet and life-style are also important, as demonstrated by people who have migrated from countries with a low incidence of CAD to countries with a higher incidence of CAD. For example, Japanese people living in Japan have coronary mortality rates which are 2 times lower than
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Japanese
Figure 3.1 Relationship between median serum cholesterol levels and 10-year CAD mortality in the 16 male cohorts of the Seven Countries Study (3, 4). B=Belgrade, Jug.; C=Crevalcore, D=Dalmatia, E=East Finland, Fin., G=Corfu, Gre., I=Italian Railroad, Ita.; K=Crete, Gre; M=Montegiorgio, N=Zutphen, R=American Railroad, US; S=Slavonia, Jug.; T=Tanushimaru, Jap.; U=Ushibuka, Jap.; V=Velika Krsna.; W=West Finland, Fin.; Z=Zrenjanin [3] (reproduction requested).
living in Hawaii and 3 times lower than those living in San Francisco (Marmot et al., 1975). This is associated with a progressive increase in age-adjusted mean plasma cholesterol levels (Japan: 180±38mg/dL (4.65±1.0 mmol/L), Hawaii: 218±38mg/ dL (5.64±1.0mmol/L), San Francisco: 228±42mg/dL (5.90±1.01 mmol/L)). A similar result has been obtained for different nationalities migrating to Israel (Toor et al., 1960). Conversely, groups of Irish migrating from Ireland to Boston, who do not make a major change to their dietary habits nor to their blood cholesterol levels, do not have a significant increase in their rates of CAD (Kushi et al., 1985). Results of the aforementioned between-population studies are very consistent with data derived from within-population epidemiological studies (Rosenman et al., 1964; Shekelle et al., 1981; Holme et al., 1985, Reed et al., 1986). The largest of these prospective studies, was
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the follow-up of the cohort of men who were originally screened for participation in the Multiple Risk Factor Intervention Trial (MRFIT). In more than 360,000 men, aged 35 to 57 years, baseline total cholesterol was a strong predictor of CAD mortality after 6 years. This was particularly apparent for cholesterol levels above 200mg/dL (5.2 mmol/L) (Multiple Risk Factor Intervention Trial Research Group, 1982). A similar result was obtained in the well-known Framingham Heart Study. This study was instrumental in establishing elevated blood cholesterol as a major risk factor for all clinical manifestations of CAD and demonstrated that for every 1% increase in total cholesterol there was a 2% increase in risk for CAD (Gordon et al., 1981; Castelli et al., 1986). Furthermore, in individuals under 50 years of age, total cholesterol levels were directly related to 30-year overall and CAD mortality rates. Total overall mortality increased 5% and CAD mortality increased 9% for each increment of 10mg/dL of cholesterol (Anderson et al., 1987). It is significant that diet and drug trials have consistently shown that the converse is also true, such that a decrease in total plasma cholesterol is associated with a significant reduction in morbidity and mortality (The Scandinavian Simvastatin Survival Study Group 1994; Shepherd et al., 1995; Sacks et al., 1996). Additional analyses of Framingham data have helped to define the role of different cholesterol-rich lipoproteins in the pathogenesis of atherosclerosis. Low-density lipoprotein (LDL) cholesterol levels were found to be positively related to CAD, even more strongly than total cholesterol levels (Castelli et al., 1986; Anderson et al., 1987). High-density lipoprotein (HDL) cholesterol levels were on the other hand inversely related to the development of CAD (Gordon et al., 1977). These relationships have been repeatedly supported by subsequent studies. Total cholesterol to HDL ratios, and LDL cholesterol to HDL ratios have thus been found to be useful predictors of CAD. An additional important finding from Framingham was the multiplicative nature of the interaction of risk factors. The occurrence of several risk factors in a single individual produced a greater risk than the sum of risk associated with individual factors (Kannel, 1983). This is reflected by the data in Figure 3.2, where risk of CAD due to an elevated plasma cholesterol level multiplies as additional risk factors such as diabetes and hypertension are taken into account. These results also indicate the importance of other cardiovascular risk factors in the aetiology of atherosclerosis. Total plasma levels of cholesterol reflect the sum of cholesterol contained in three major lipoprotein fractions: triglyceride-rich lipoproteins (TRL), which are made up of chylomicrons and their remnants, as well as very low density lipoproteins (VLDL) and intermediate density lipoproteins (IDL), low density lipoproteins (LDL) and high density lipoproteins (HDL) (Havel and Kane, 1995). In
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clinical medicine, the measurement of total cholesterol is used along with the measurement of total plasma triglyceride and HDL-cholesterol to calculate plasma levels of LDL-cholesterol. This calculation, known as the Friedewald formula (Friedewald et al., 1972), allows for the determination of LDL-cholesterol without cumbersome laboratory techniques such as ultracentrifugation of plasma. When expressed in mmol/L, LDL-cholesterol =total cholesterol— (triglycerides/2.19+HDL-cholesterol). This equation is accurate for triglyceride levels < 4.5 mmol/L (~400 mg/dL), and has been used in the majority of epidemiological studies to measure LDL levels. This estimation of LDL-cholesterol, however, also includes cholesterol in potentially-atherogenic IDL, and it remains unclear to what extent IDL cholesterol (representing 10% to 15% of estimated LDL) contributes to the strength of the association between estimated LDL-cholesterol concentration and CAD (Phillips et al., 1993).
Figure 3.2 Risk of cardiovascular disease according to total cholesterol at specified levels of other risk factors in the Framingham Study in men aged 35 years after 18-year follow-up [20] (reproduction requested).
LOW DENSITY LIPOPROTEINS Population Studies As described in earlier chapters, LDL are products of VLDL metabolism, through hydrolysis of triglyceride, and exchange of cholesteryl esters with HDL (Havel and Kane., 1995). There is also
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indirect evidence for direct hepatic secretion of particles in the LDL size range. Approximately two-thirds of total plasma concentration of cholesterol is due to cholesterol transported by LDL, and there is a close correlation (r>0.80) between plasma concentrations of LDL- and total cholesterol. LDL-cholesterol is often regarded as a better predictor of coronary artery disease than total cholesterol, particularly within a population. Elevations in LDL-cholesterol levels have been documented in large prospective studies to be independently associated with the presence of CAD (Gordon et al., 1981; Castelli et al., 1986). More recently, the characteristics of LDL particles, and not solely their cholesterol content, have elicited much interest. Since there is only one molecule of apoB per LDL particle, the measurement of apoB reflects the number of circulating LDL particles. Some investigators have suggested that apoB is a better measurement for the prediction of CAD than LDL-cholesterol (Sniderman et al., 1980). However, this has been a matter of debate (Sniderman and Genest, 1992), and in general, the measurement of apoB does not appear to confer additional information over the standard measurement of lipoprotein cholesterol (Wilson et al., 1997). In addition to number, the size of LDL particles is believed to be related to the development of atherosclerosis (Austin and Krauss, 1995). Patients can be broadly characterised as having a predominance of small, dense LDL, or as having a higher proportion of large more buoyant LDL particles (Krauss, 1994). Small dense LDL are particles depleted in core cholesteryl ester, and relatively enriched in apoB, and are a characteristic feature of such lipid disorders as hyperapobetalipoproteinaemia, familial combined hyperlipidaemia and atherogenic dyslipidaemia (characteristic of patients with syndrome X or the metabolic syndrome) (Grundy, 1997). The presence of small LDL in plasma has therefore been suggested to be genetically determined (Austin et al., 1993), although there is also clear evidence for the effect of environmental factors, since small LDL are a characteristic of patients with hypertriglyceridaemia and/or low levels of HDL (McNamara et al., 1987). These three lipoprotein abnormalities (hypertriglyceridaemia, low HDL level and small LDL) are metabolically related, and it has been difficult to establish their relative contributions to atherogenesis. In case-control studies, patients with CAD have consistently been found to have smaller-sized LDL (Austin et al., 1988; Tornvall et al., 1991; Campos et al., 1992). Whether the relationship between LDL size and CAD is however independent of plasma triglyceride concentration remains a subject of debate (Grundy 1997; Stampfer et al., 1996; Gardner et al., 1996). Family Studies The best known lipid disorder characterised by a pronounced elevation
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in LDL-cholesterol concentration and increased incidence of premature CAD is familial hypercholesterolaemia (FH) (Goldstein and Brown, 1995). This is a genetic disorder, transmitted in an autosomal codominant fashion (subjects with a single copy of the defective gene (heterozygotes) exhibit an elevated cholesterol level). Familial hypercholesterolaemia is caused by mutations of the gene coding the low-density lipoprotein receptor, and to date, over 200 different mutations causing FH have been identified (Hobbs et al., 1992). The prevalence of FH within the general population is unknown. It has been reported as being 1:500, based on a study of familial disorders of lipids in patients who had survived an acute myocardial infarction (Goldstein et al., 1973). In some populations with a founder effect for genetic diseases, the prevalence of FH mutations can be as high as 1:270. In two separate studies, the prevalence of heterozygous FH in premature CAD has been estimated at 3–5% (Genest et al., 1992; Weber et al., 1997). This represents a 10- to 15-fold increase in the prevalence of FH subjects in CAD subjects, compared with the general population. More recently, genetic disorders affecting the ligand for the LDL-R, namely apoB have also been identified. The first such mutation, apoB3500 results in an Arg-Gln substitution at the postulated receptor binding region of apoB; other mutations within the receptor binding region of the apoB gene can also cause defective binding to the LDL receptor (Ludwig et al., 1997). The clinical manifestations of this disorder and its association with premature CAD is similar to heterozygous FH. Many lipoprotein disorders leading to an increase in the plasma concentration of apoB-containing lipoproteins are not a result of impaired LDL clearance and catabolism, but due to hepatic oversecretion of apoB. The causes of these disorders are multiple and are explained, in part, by an increase in fatty acid availability to the liver, resulting in increased VLDL assembly and secretion. The resulting lipoprotein phenotype in this situation may be hypertriglyceridaemia alone, elevated LDL-apoB alone, or a combination of both, often associated with a reduction in HDL-cholesterol concentration. Familial lipoprotein disorders associated with CAD and characterised by an increase in LDL-cholesterol and/or LDL-apoB are shown in Table 3.1.
Table 3.1 Familial lipoprotein disorders characterised by an increase in LDL-cholesterol and/or LDL-apoB concentration
Disorder
Aetiology
Familial
Partial or
Plasma SusceptLipoprotein ibility to Characteristics CAD LDL
Increased
Evidence Linking Plasma Lipoprotein Disorders
Hypercholesterolaemia
complete absence of LDLreceptors
Familial Defective ApoB
Defective apoB causing impaired receptor recognition
LDL
Familial Combined Overproduction VLDL and Hyperlipidaemia/HyperapoB of hepatic apoB LDL particle number Metabolic Syndrome
Resistance to insulin
59
Increased
Increased
VLDL TG; Increased small-dense LDL
TOTAL PLASMA TRIGLYCERIDE Population Studies A large number of epidemiological studies have addressed the question of whether a relationship exists between elevated blood triglyceride levels and the incidence of CAD, as reviewed in a number of publications (Multiple Risk Factor Intervention Trial Research Group 1982; Gordon et al., 1981; Avins et al., 1989; Austin, 1991). This work has clearly shown that it is much more difficult to establish a statistical association between hypertriglyceridaemia and CAD than it is for total, LDL- and HDL-cholesterol. Thus, in case-control and prospective studies, patients with CAD have invariably been found to have higher levels of total plasma triglyceride, however multivariate analysis of the data (taking into account the presence of other lipid and non-lipid risk factors) has often failed to identify plasma triglyceride as a statistically significant independent risk factor. Notable exceptions have been the meta-analysis of published data (Hokanson and Austin, 1996), and analyses of patient subgroups (e.g., women) (Bass et al., 1993; Castelli, 1992) or diabetics (Fontbonne et al., 1989; Uusitupa et al., 1993; Laakso et al., 1993). Historically, this has created controversy and debate concerning the association of hypertriglyceridaemia with CAD, particularly since laboratory experiments have provided strong evidence that triglyceride-rich lipoproteins (TRL) are potentially atherogenic and thrombogenic (as reviewed in Chapter 6). A number of reasons can be put forward for the lack of an independent association between triglyceride levels and CAD. Firstly, plasma triglyceride concentration increases and decreases throughout the day in response to the ingestion of frequent meals. Even if measured after a 10- to 12-hour
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overnight fast (as is normal clinical practice), triglyceride levels vary considerably more than LDL- and HDL-cholesterol levels (Smith et al., 1993) (day-to-day biological variability is 23%, 9.5% and 7% for triglyceride, LDL- and HDL-cholesterol levels, respectively). Triglyceride levels are therefore less reliable in reflecting the basal metabolic state. Secondly, plasma triglyceride is carried in a number of different lipoprotein fractions (e.g., chylomicrons, large and small very low-density lipoproteins, partially lipolysed TRL remnants) and the ability of these lipoproteins to promote atherosclerosis is probably not the same (Bradley and Gianturco, 1994). Thus, a measurement of total triglyceride reflects the presence in plasma of both atherogenic as well as non-atherogenic lipoproteins, which weakens the ability of this parameter to predict coronary disease. Thirdly, it is apparent that patients with moderately increased levels of total triglyceride are often at risk of CAD, whereas patients with very high levels of plasma triglyceride are usually not afflicted by coronary disease (Chait and Brunzell, 1991). The relationship between total triglyceride and CAD is therefore complicated by the underlying disorder responsible for the hypertriglyceridaemia, and the type of lipoprotein responsible for transporting the excess triglyceride in the blood circulation. Fourthly, it is important to remember that there is a strong metabolic interdependence between different plasma lipids and lipoproteins. A strong relationship thus exists between different lipid and apolipoprotein parameters (e.g., the strong inverse correlation between triglyceride and HDL-cholesterol concentration). Thus when HDLcholesterol is included in the multivariate analysis models, triglyceride levels often lose their ability to predict CAD. Since people normally spend the majority of the day in the fed or postprandial state, it has been suggested that postprandial triglyceride levels could provide a better estimate of CAD risk. This concept has gained support from results showing that plasma triglyceride concentrations 6 and 8 hours after a standardised fat-rich meal were independently associated with CAD in male subjects with angio graphically documented coronary atherosclerosis (Patsch et al., 1992). Fasting plasma triglyceride concentration, in agreement with many epidemiological studies, was not an independent predictor of disease. These results are supported by the Physicians’ Health Study, whereby nonfasting triglyceride levels were a strong and independent predictor of future risk of MI during 7 years of follow-up (Stampfer et al., 1996). Postprandial triglyceridaemia has also been independently associated with the presence of carotid atherosclerosis, as determined by B-mode ultrasound measurement of intima-media thickness (Ryu et al., 1992).
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Family Studies Genetic or familial forms of hypertriglyceridaemia have provided important insights into the link between elevated levels of triglyceride in the blood and CAD. They exemplify how excess triglyceride can be transported in different types of triglyceride-rich lipoproteins (TRL), and how these lipoproteins are in turn associated with either normal or increased atherosclerosis susceptibility (Table 3.2). For example, familial chylomicronaemia is characterised by the presence in plasma of very large triglyceride-rich particles of intestinal origin. Patients with this lipid disorder have a partial or complete deficiency in lipoprotein lipase or its activator, apoC-II, and they commonly have plasma triglyceride concentrations greater than 1,000 mg/dL (>11.3 mmol/L) (Brunzell, 1989; Chait and Brunzell, 1991). Although atherosclerosis may occur in these patients in the presence of other risk factors (Benlian et al., 1996), they are not predisposed to premature CAD (Nikkilä, 1983), as is the case for patients with homozygous familial hypercholesterolaemia. Familial hypertriglyceridaemia is in turn characterised by triglyceride-enriched VLDL and a normal LDL particle number, and is thought not to be associated with increase in cardiovascular risk (Brunzell et al., 1976; Brunzell et al., 1983). A recent follow-up of families originally described with familial hypertriglyceridaemia has however suggested a slightly increased risk of CAD in these individuals (Austin et al., 1997). Familial combined hyperlipidaemia and hyperapobetalipoproteinaemia are characterised by an overproduction of VLDL, and increased risk of premature CAD is believed to be due to
Table 3.2 Familial lipoprotein disorders characterised by an increase in total plasma apoB concentration
Disorder
Aetiology
Plasma Susceptibility Lipoprotein to CAD Characteristics
Familial Hyperchylomicronaemia
Partial or complete absence of LpL or apoC-II
Familial Hypertriglyceridemia
Hypersecretion TG-enriched of VLDL VLDL triglyceride
Familial Combined
Overproduction
chylomicrons
VLDL TG;
Normal
Normal
Increased
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Hyperlipidaemia/HyperapoB of hepatic apoB small-dense LDL Familial Dysbetalipoproteinaemia
Reduced remnant lipoprotein clearance
-VLDL
Increased
the presence in plasma of an increased number of LDL particles, which are small and dense and can be readily oxidised (Chait et al., 1993). In contrast, familial dysbetalipoproteinaemia, which is also associated with increased risk of CAD, is characterised by accumulation in plasma of partially-hydrolysed TRL ( -VLDL), resulting from impaired hepatic recognition and uptake of TRL remnants (Mahley and Rall, 1989). Hence, different forms of hypertriglyceridaemia have different atherogenic potentials, mediated by: a) a direct action of TRL on lipid accumulation by cells of the artery wall; b) an indirect effect of TRL on the concentration of other lipoproteins which are pro- or antiatherogenic; or c) an effect of TRL on the coagulability of blood. In a study of 102 kindreds with CAD, a genetic plasma lipoprotein disorder was found in 54% of families (Genest et al., 1992). Prevalence of hypertriglyceridaemia was 22.5%, in comparison to a prevalence of 10.8% for hypercholesterolaemia, 39.2% for hypoalphalipoproteinaemia (reduced HDL), and 18.6% for increased Lp(a)—these groups were not mutually exclusive, such that more than one abnormality may have been present in the same proband. The combination of familial hypertriglyceridaemia with hypoalphalipoproteinaemia was one of the most common plasma lipid phenotypes, affecting 14.7% of the probands. Familial hypertriglyceridaemia without hypoalphalipoproteinaemia was found in only 1 % of CAD families, which was not a significant increase in prevalence compared to controls. This suggests that an elevated plasma triglyceride level is only important as a risk factor for CAD when associated with another lipoprotein abnormality (principally, decreased HDL).
HIGH DENSITY LIPOPROTEINS Population Studies Prospective epidemiological studies have consistently demonstrated that plasma HDL-cholesterol concentration is inversely related to the incidence of CAD (Gordon et al., 1977; Jacobs, 1985; Watkins et al., 1986; Miller et al., 1977; Goldbourt and Medalie, 1979; Enger et al.,
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1979; Assmann et al., 1996). This relationship persists until age 80 years and is also found in subjects with “normal” cholesterol levels (<5.2mmol/L or 200mg/dL). Coronary disease risk is increased by 2% in men and 3% in women for every 1 mg/dL (0.026mM) reduction in HDL-cholesterol (Gordon et al., 1989), and in the majority of studies, this relationship remains statistically significant after adjustment for other lipid and non-lipid risk factors. The ability of HDL-cholesterol to predict CAD independently of total and LDL-cholesterol is exemplified by data from the 6-year follow-up of nearly 5,000 male participants in the PROCAM study (Assmann et al., 1996), where incidence of CAD rises in relation to both decreasing HDL levels, as well as increasing LDL levels (Figure 3.3). Levels of HDL have specifically been related to an increase in coronary mortality (Wilson et al., 1988; Goldbourt et al., 1997), particularly in diabetic subjects (Fontbonne et al., 1989), and increased risk of death due to CAD has been associated with abnormally low HDL-cholesterol levels (<0.9 mmol/L), whether total cholesterol concentration is below or above 5.2mmol/L (Goldbourt et al., 1997). Case-control studies have shown that larger HDL2 rather than HDL3 are responsible for the inverse relationship between HDL levels and CAD (Miller, 1987; Robinson et al., 1987), although longitudinal data from the Physicians’ Health Study, involving nearly 15,000 men, have suggested that both HDL2 and HDL3 are protective against CAD (Stampfer et al., 1991). Despite the strong epidemiological link between reduced HDL levels and the incidence of CAD, it is unclear whether HDL play a direct or indirect role in the pathogenesis of atherosclerosis. In other words, it remains to be determined whether HDL are directly involved in the development of atherosclerotic lesions, or whether low HDL levels are simply markers of other atherogenic processes. HDL particles have been shown to promote cholesterol efflux from cultured cells, and are believed to play an important role in transporting excess cholesterol back to the liver for excretion in the bile. Although this mechanism is well documented in vitro, the importance of reverse cholesterol transport in preventing atherosclerosis in vivo has been more difficult to demonstrate. HDL particles also provide cholesterol to steroidogenic tissues through selective uptake of cholesterol by the SR-B1 receptor. Bacterial lipopolysaccharide endotoxins are adsorbed by HDL particles and may help modulate their vascular effects. HDL may also act as a preferential oxidation substrate and thus protect LDL particles from oxidation. These intrinsic antiatherogenic properties of HDL will be discussed in Chapter 5.
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Figure 3.3 Incidence of CAD per 1000 subjects after 6 years of follow-up in the PROCAM study, according to total, LDL- and HDL-cholesterol levels [77] (reproduction requested).
Considerable evidence, largely based on epidemiological data, also supports an indirect role of HDL in atherosclerosis. This evidence comes from the link between reduced HDL levels and the clustering of CAD risk factors in a single individual, now commonly referred to as the “metabolic syndrome”. This syndrome is characterised by insulin resistance, increased blood pressure, a procoagulant state, and atherogenic dyslipidaemia (or an atherogenic lipoprotein phenotype ALP) (Austin et al., 1990). The common features of the ALP (not necessarily occurring together in the same individual) are: a moderate increase in plasma triglyceride level, evidence of TRL remnant lipoprotein accumulation (e.g., increased IDL concentration), a reduced
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HDL-cholesterol concentration, and the presence of small dense LDL. It has been proposed that insulin resistance is the central cause of the metabolic syndrome, although other factors may be principally responsible, such as obesity (especially abdominal) or aging. Regardless of the aetiology of the metabolic syndrome, it is generally believed that reduced HDL levels are a result rather than a cause of this metabolic abnormality. Family Studies The investigation of families of probands with premature CAD has demonstrated that about one quarter have a familial form of HDL deficiency—familial hypoalphalipoproteinaemia (FHA) (Genest et al., 1992). Forty-four percent of these latter families had an elevation in plasma triglyceride, 40% had familial combined hyperlipidaemia and only 16% had isolated FHA. A low plasma concentration of HDL is thus, by itself, rarely associated with CAD, but is more commonly associated with disease in the presence of increased TRL or apoBcontaining lipoproteins (Genest et al., 1993). FHA is therefore most likely to be atherogenic when HDL deficiency is secondary to a perturbation in apoB metabolism (e.g., the overproduction of hepatic apoB). Primary forms of FHA, caused by a defect in genes encoding apoA-I (the major structural protein of HDL), or enzymes critical for the formation of mature HDL, and their association with premature CAD are summarised in Table 3.3. Rare genetic defects at the apoA-I gene locus cause severe reductions in HDL-cholesterol and apoA-I levels (Dammerman and Breslow, 1995; Breslow, 1995; Calabresi and Francescini, 1997). Despite markedly decreased HDL levels in affected individuals, some deficiency states are associated with CAD whereas others are not. Tangier disease is a rare disorder of HDL deficiency that presents, in classical cases, with lymphoid tissue (spleen, lymph nodes, tonsils, intestinal mucosa, liver, omentum, Schwann cells) infiltration with cholesteryl esters and a progressive neuropathy (Assman et al., 1995). Although the precise molecular defect remains unknown, the cellular defect appears to result in abnormal cellular cholesterol processing and efflux onto nascent HDL particles, which are small and rapidly catabolised. In a review of case reports on patients with Tangier disease, approximately 50% had evidence of CAD by age 45, suggesting that Tangier disease was sometimes but not always associated with premature CAD (Serfaty-Lacrosniere et al., 1994). Severe FHA is also a characteristic of two rare autosomal recessive disorders caused by mutations in LCAT, the enzyme responsible for the formation of cholesteryl esters in HDL particles. These disorders are known as familial LCAT deficiency (FLD) and fish eye disease (FED).
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More than 20 mutations in the LCAT gene have been reported. HDL particles have an abnormal composition, and the disorder may be associated with renal failure as well as corneal opacifications. The relationship between LCAT deficiency and vascular disease is also not well defined (Kuivenhoven et al., 1997). As previously discussed, genetic disorders of the TRL processing enzymes LPL or its activator CII, cause severe hypertriglyceridaemia and markedly reduced HDL-C levels (Brunzell, 1989).
Table 3.3 Familial lipoprotein disorders characterised by a decrease in plasma HDL concentration
Disorder
Aetiology
Plasma Lipoprotein Characteristics
ApoA-I gene defects
Partial or complete impairment in apoAI expression
HDL
Sometimes increased
Tangier disease
Unknown
HDL
Sometimes increased
LCAT deficiency/Fish eye disease
Absent/Impaired LCAT activity
HDL
Variable
Metabolic syndrome
Resistance to insulin
VLDL TG; small-dense LDL
Susceptibility to CAD
Increased
LIPOPROTEIN(a) Lipoprotein(a) [Lp(a)] was originally described by Kare Berg over 30 years ago, however the physiological and pathophysiological properties of this lipoprotein are still poorly understood. Lp(a) is a cholesterolrich lipoprotein composed of an LDL particle covalently linked to apo (a)—a large highly-glycosylated hydrophilic protein. Apo(a) has an amino-acid sequence similar to that of plasminogen, and due to a variable number of plasminogen-kringle IV domains, it varies considerably in size from one person to another (200 to 800 kilodaltons). Plasma concentrations of Lp(a) are genetically determined and vary from 1 mg/dL to more than 100mg/dL. In a single individual, plasma apo(a) concentration is remarkably stable over time and is not greatly influenced by age, gender, diet, or most pharmacological interventions that significantly alter the plasma concentration of other lipoproteins. The biological features of Lp(a) have been summarised in
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several reviews (Utterman, 1989; Marcovina and Morrisett, 1995). Population Studies Numerous cross-sectional and case-control studies in subjects with coronary, carotid artery or peripheral vascular disease have demonstrated that elevated Lp(a) concentrations are associated with premature atherosclerosis (as reviewed recently) (Maher and Brown, 1995). The significance of these retrospective studies has been questioned however, since it is difficult to ascertain whether increased Lp(a) levels were a cause or a consequence of disease. This is particularly relevant in the case of Lp(a), which has been identified as a positive acute phase reactant (Maeda et al., 1989). The acute phase response that occurs with acute coronary syndromes or surgical revascularisation procedures may therefore exaggerate the importance of raised Lp(a) levels. Several large prospective studies have identified Lp(a) excess as an independent predictor of CAD (Stein and Rosenson, 1997). For example, in the Lipid Research Clinics Coronary Primary Prevention Trial, involving 3806 hypercholesterolaemic men aged 35 to 59 years, Lp(a) was measured in 233 participants who developed CAD during the 7- to 10-year follow-up, and in 390 matched controls (Schaefer et al., 1994). Plasma Lp(a) concentrations were significantly higher in cases compared to controls (23.7mg/dL vs 19.5mg/dL), and this difference was statistically significant after controlling for age, body mass index, cigarette smoking, blood pressure, LDL- and HDLcholesterol. In the Göttingen Risk Incidence and Prevalence Study (GRIPS), the importance of Lp(a) level as a cardiovascular risk factor was assessed in 6002 men, aged 40–60 years, who were followed for 5 years (Cremer et al., 1994). Multivariate logistic regression analysis defined Lp(a) level as a significant predictor of disease, which ranked behind an elevated LDL-cholesterol, but was as strong as family history of disease, hyperfibrinogenaemia or decreased HDL-cholesterol in predicting CAD. Similar results have been obtained from the Framingham Study for both men and women (Bostom et al., 1996; Bostom et al., 1994), the PROCAM Study (Assmann et al., 1996), and recently, the Stanford Five-City Project (Wild et al., 1997). In this latter study, small apo(a) size isoforms were more frequent in men with CAD, though apo(a) size did not add to the predictive power of Lp(a) concentration. Several prospective studies (4 studies out of 12) have however failed to identify Lp(a) level as a predictor of disease. The most notable of these was the Helsinki Heart Study (Jauhiainen et al., 1991), in which Lp(a) levels did not differ between 138 subjects with CHD and 130 control subjects followed for 5 years. Similarly in the Physicians’ Health Study, no difference was found in Lp(a) levels
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between 296 men who suffered an acute myocardial infarction during the 5-year follow-up and paired control subjects matched for age and smoking status (Ridker et al., 1993). It has been proposed that different methods for measuring Lp(a), which have not been standardised (Marcovina et al., 1995), and the effects of variable length and temperature of sample storage (Kronenberg et al., 1996a) may explain some of these different outcomes. An additional explanation for this inconsistency, which requires confirmation, is that the pathogenecity of Lp(a) is dependent on LDL-cholesterol levels (i.e., LDL-cholesterol concentration modulates the ability of Lp(a) to promote atherosclerosis) (Maher and Brown, 1995). It is significant that abnormal levels of Lp(a) have also been associated with other metabolic abnormalities (reviewed in Kronenberg et al., 1996b). Patients with nephrotic syndrome or end-stage renal disease have significantly increased Lp(a) concentrations. Following renal transplantation, Lp(a) levels decrease to values observed in controls matched for apo(a) isoform. Patients with insulin-dependent diabetes have normal or moderately elevated levels of Lp(a), while those with insulin-independent diabetes do not have a consistent elevation in Lp(a). Despite some reports of elevated Lp(a) levels in patients with familial hypercholesterolaemia lacking functional LDLreceptors, it is now generally believed that the LDL receptor does not play a significant role in Lp(a) catabolism. The aforementioned epidemiological evidence implicating Lp(a) in the pathogenesis of atherosclerosis is supported by laboratory evidence showing that Lp(a): (a) accumulates in atherosclerotic plaques (Smith and Cochran, 1990), (b) promotes thrombosis, due to its structural similarities with plasminogen (Loscalzo et al., 1990), (c) promotes foam cell formation by stimulating cellular cholesterol accumulation (Bottalico et al., 1993), (d) stimulates smooth muscle cell proliferation (Grainger et al., 1993), (e) impairs endothelium-dependent vasodilation (Nachman, 1997) and promotes monocyte chemoattractant activity in human vascular endothelial cells (Poon et al., 1997). Lp(a) is a lipoprotein that carries cholesterol and has a propensity for sites of tissue injury. It binds to fibrin, to proteoglycans and to fibronectin. Because Lp(a) also interferes with fibrinolysis, its main role might be in enhancing tissue repair in the vasculature. Lp(a) influences plasmin generation by competing for the annexin II binding domain of plasminogen on endothelial cell surfaces. Annexin II is a large, multidomain phospholipid binding site essential for the fibrinolytic system. Lp(a) also down-regulates plasmin generation from endothelial cells and competes with tPA (tissue plasminogen activator) in the presence of fibrinogen. Oxidised Lp(a) is taken up by macrophages and may cause foam cell formation. Interestingly, oxidised Lp(a) has been shown to increase vascular endothelial cell production of plasminogen
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activator inhibitor 1 (PAI-1) (Ren et al., 1997). Plasma Lp(a) levels correlate with reduced acetylcholine-mediated coronary vasodilatation in human coronary arteries examined during diagnostic coronary angiography (Tsurumi et al., 1995). Family Studies Family studies have strongly implicated elevated Lp(a) levels in the development of CAD. Prevalence of Lp(a) excess was 18.6% in probands of families with premature CAD, compared to 14.7% for hypertriglyceridaemia with low HDL, and 13.7% for combined hyperlipidaemia (Genest et al., 1992). Similarly, in 48 patients with premature myocardial infarction below age 55, and their 78 siblings, familial excess of Lp(a) was the most frequent lipoprotein abnormality (16%), followed by familial combined hyperlipidaemia (Pay et al., 1997). Genetics of lipoprotein (a) Plasma levels of Lp(a) are strongly genetically determined. The heritability index for Lp(a) in twin studies, sib-pair analysis and family studies is the highest for any of the known lipoprotein cardiovascular risk factors (Genest et al., 1992). The main determinant of Lp(a) levels is related to the apo(a) gene. There is a strong, inverse and graded relationship between the length of the apo(a) gene (and therefore, the number of kringle 4 repeats) and plasma apo(a) levels. In a study of 25 multiplex families (De Meester et al., 1995) polymorphisms of the apo (a) gene were examined by pulse-field electrophoresis. Using quantitative sib-pair linkage analysis, the apo(a) gene was found to be the major gene controlling plasma Lp(a) levels. Evolutionarily, Lp(a) is restricted to old world monkeys and, in an example of convergent evolution, in hedgehogs (Lawn et al., 1995). At least 6 genes that are highly related to apo(a) in humans have been identified by examining yeast artificial chromosome libraries. A novel gene, apo(a) related gene (apo(a)rg) is located with plasminogen and apo(a) on chromosome 6 (Byrne and Lawn, 1994). The apo(a) gene is transcribed in the liver and the 5′ promoter region shares a high degree of homology between the 6 apo(a) related genes and pseudogenes. Of interest, the apo(a) 5′ promoter region contains functional interleukin 6 responsive elements. This is consistent with the concept that Lp(a) is an acute phase response protein (Wade et al., 1993). In addition, polymorphisms within the apo (a) gene may be independent determinants of plasma apo(a) levels (Klezovitch and Scanu, 1995). Of interest, the site of attachment of apo (a) to apoB100 resides on residue apoBcys4326 (McCormick et al., 1995).
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Clinical correlates An elevated Lp(a) level appears to be an independent risk factor for CAD in many case-control studies but controversy remains concerning the role of Lp(a) in prospective studies. Methods to lower an elevated Lp(a) level through dietary or pharmacological means have proven difficult, with the exception of niacin. Extracorporeal Lp(a) removal by selective immunoabsorption is feasible on technical grounds (Pokrovsky et al., 1994) but, because of cost considerations may not be a clinically viable option to treat patients. The question, therefore is in whom Lp(a) should be measured? Until an effective therapy is developed, and until epidemiological evidence shows that Lp(a) is a strong risk factor in prospective studies, the measurement of Lp(a) should be considered experimental.
CAROTID AND FEMORAL ARTERY ATHEROSCLEROSIS Although hypercholesterolaemia is an established risk factor for coronary artery atherosclerosis, the epidemiological data linking hypercholesterolaemia with carotid or femoral (peripheral) atherosclerosis are less conclusive. In the case of carotid artery atherosclerosis, prospective studies have demonstrated either no association between total plasma cholesterol and nonhaemorrhagic stroke (Stokes et al., 1987; Smith et al., 1992), or a significant positive association (Dyer et al., 1981; Iso et al., 1989; Stemmermann et al., 1991). Inconclusive results have also been obtained for total plasma triglyceride and HDL-cholesterol levels, as reviewed recently (Postiglione and Napoli, 1995). Increased levels of Lp(a) have however been associated with ischaemic stroke on a more consistent basis (Postiglione and Napoli, 1995). It is significant to note that the majority of drug trials with lipid-lowering agents have failed to reduce the incidence of fatal and nonfatal strokes (Atkins et al., 1993; Hebert et al., 1995), although in the recent Scandinavian Simvastatin Survival Study, in which significant (25%–35%) reductions were achieved in total and LDL-cholesterol levels, a statistically significant reduction in strokes was observed (Scandinavian Simvastatin Survival Study Group 1994). As far as femoral artery atherosclerosis is concerned, total and LDL-cholesterol levels have been related to the presence of femoral atherosclerosis in some (Vigna et al., 1992; Bergmark et al., 1995; Vitale et al., 1990) but not all recent case-control studies (Mainard et al., 1994; Valentine et al., 1994). In recent prospective studies, total cholesterol was significantly correlated with disease only in certain age groups (Kannel, 1994), or was not significantly related to disease on
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multivariate analysis (Bainton et al., 1994). More commonly, peripheral artery disease has been associated with an increase in triglycerides and a reduction in HDL levels, particularly HDL2 (Vigna and Fellin, 1996). Peripheral atherosclerosis is particularly prevalent in patients with diabetes or in those with the metabolic syndrome (Kannel and McGee, 1979; Fowkes et al., 1992), and has been associated with elevated Lp(a) levels in recent studies (Valentine et al., 1994; Cantin et al., 1995; Mölgaard et al., 1992). Furthermore, a correlation has been observed between elevated plasma IDL levels and lower limb atherosclerosis (Senti et al., 1992), which is consistent with the hypothesis that femoral atherosclerosis is strongly related to a plasma lipid profile reflecting reduced triglyceride lipolysis, remnant lipoprotein accumulation, small-dense LDL, and low HDL levels.
SUMMARY AND CONCLUSIONS The aforementioned epidemiological studies have played a central role in defining the importance of plasma lipoprotein disorders in the pathogenesis of atherosclerosis. More specifically, they have been instrumental in establishing plasma lipid and lipoprotein levels, such as the plasma concentration of triglyceride, LDL, HDL and Lp(a), as indicators of CAD risk. The challenge for future epidemiological studies will be to better define the link between CAD and a) the level of particular lipoprotein subfractions, b) factors affecting lipoprotein metabolism and c) factors affecting the atherogenicity of plasma lipoproteins. For example, it is important to define which triglyceriderich lipoprotein subfractions are most strongly associated with risk of CAD (e.g., intestinal vs hepatic TRL, large TRL vs smaller TRL remnants) or which subfractions of HDL are most protective (e.g., large vs small HDL, apoE-containing HDL vs apoAI- and AII-containing HDL). The relationship between CAD and plasma levels of lipoprotein enzymes and co-factors (e.g., cholesteryl ester transfer protein, LCAT), and also compounds that can potentially affect the oxidisability of lipoproteins (e.g., vitamins, paraoxanase) needs to be more clearly defined. Finally, future studies will need to focus not just on the relationship between plasma lipid disorders and atherosclerosis, but between plasma lipids and plaque stability and risk of thrombosis. Acknowledgements Supported by a salary award from the Fonds de la recherche en santé du Québec (JG) and by grants from the Medical Research Council of Canada and from the Heart and Stroke Foundation of Canada.
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4 Atherogenicity of Low-density Lipoproteins: Mechanisms M.John Chapman National Institute for Health and Medical Research (INSERM), Unit 321, Pavilion Benjamin Delessert, Hopital de la Pitie, 83 Boulevard de L’Hopital, 75651 Paris Cedex, 13, France
KEYWORDS: subpopulations, plaque.
Low density lipoproteins, atherogenesis, modified LDL,
INTRODUCTION The low-density lipoproteins (LDL), are pseudomicellar, quasispherical particles which constitute the major vehicle for cholesterol transport in human plasma; they consist essentially of cholesterol in both esterified and non-esterified forms, phospholipids, minor amounts of triacylglycerols, and a highly specialised protein component of hepatic origin, apolipoprotein B100 (apo.B100). A wealth of evidence presently attests to a central role of cholesterolrich LDL in the pathophysiology of atherosclerosis. Such evidence comprises data from chemical and immunological analyses of arterial wall atherosclerotic plaques, from epidemiological and genetic studies in man, from drug and dietary intervention trials in human subjects, and from animal models (Small, 1988; Harland et al., 1973; Suarna et al., 1995; Smith, 1974; Hoff et al., 1979; Nielsen, 1996; Anderson et al., 1987; Pyrola, 1987; Assmann and Schulte, 1998; Pekkanen et al., 1992; Neaton et al., 1992; Law et al., 1994; Goldstein and Brown, 1983; Schaefer et al., 1994; Brunzell et al., 1976; Consensus Conference, 1985; Frick et al., 1987; Pederson et al., 1994; Brown et al., 1990; Shepherd et al., 1995; Kane et al., 1990; Blankenhorn et al., 1990; Kromhout et al., 1995; Williams et al., 1986; Small et al., 1984; Rudel et al., 1986; Rudel et al., 1985; Schwenke and Carew, 1989a&b, Palinski et al., 1994). Elevated circulating levels of LDL have long been linked to the premature development of atherosclerotic disease. It is only recently,
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however, that the relationship of the qualitative features of LDL particles to coronary artery disease has attracted considerable interest (Krauss, 1994; Chapman et al., 1998). Indeed, a significant association has been established between the heterogeneity of plasma LDL particles and cardiovascular risk (Krauss, 1994; Chapman et al., 1998). This review will summarise our expanding knowledge of the role of distinct LDL particle subpopulations in the cellular and molecular mechanisms which underlie plaque initiation, progression and rupture. The oxidative modification of LDL within arterial tissue is now documented as a key feature of the atherogenic process (Steinberg et al., 1989; Berliner and Heinecke, 1996), and consideration will therefore be given to the susceptibility of LDL subpopulations to modification upon exposure to oxidative stress. Additional mechanisms, enzymatic or non-enzymatic in nature, may lead to modification of the particle structure and chemical composition of LDL. Indeed, such modification is a major determinant of LDL atherogenicity, leading either to extracellular cholesterol deposition and expansion of the plaque’s lipid core, or to particle uptake by macrophage scavenger receptors with formation of cholesterol-loaded foam cells, characteristic components of fragile, lipid-rich atherosclerotic plaques. LDL Particle Subpopulations: Qualitative Heterogeneity Considerable progress has been made of late in the definition of the structural, chemical and functional properties of LDL subspecies. Such advances are of immediate relevance to the potential impact of LDL particles on the arterial wall, and equally to the expression of proinflammatory and prothrombogenic activities by the cellular and non-cellular components of the plaque. LDL particles are markedly heterogeneous in their physical and chemical properties, which include size, hydrated density, molecular weight, chemical composition (lipid subclasses, lipid-soluble antioxidants, apolipoproteins and carbohydrate content), surface electric charge, apoB100 conformation and hydrodynamic behaviour (Lindgren et al., 1969; Shen et al., 1981; Krauss and Burke, 1982; Chapman et al., 1988; McNamara et al., 1987; Swinkels et al., 1989; Griffin et al., 1994; Goulinet and Chapman, 1997; Tribble et al., 1994; La Belle and Krauss, 1990; Chen et al., 1994). Indeed, multiple subpopulations of LDL particles have been identified according to their hydrated density by isopynic density gradient ultracentrifugation and to their particle size by electrophoresis in nondenaturing polyacrylamide gradient gels (Lindgren et al., 1969; Shen et al., 1981; Krauss and Burke, 1982; Chapman et al., 1988; McNamara et al., 1987; Swinkels et al., 1989; Griffin et al., 1994; Goulinet and Chapman, 1997; Tribble
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et al., 1994; La Belle and Krauss, 1990; Chen et al., 1994). On the basis of their physicochemical properties and biological activities, LDL subspecies may be grouped into three principal subclasses, i.e. light, intermediate and dense, as indicated in Figure 4.1. Plasma LDL particles are distributed as a continuum over the density range from 1.019 to 1.063g/ml, and consist of a series of discrete particle subspecies whose size, volume and lipid content decrease in parallel with increase in density (Figure 4.1). The grouping of these subspecies into three subclasses reveals that light LDL contain some 30% more cholesterol molecules (in free and esterified form) per particle than dense LDL; however, all LDL particles contain a single copy of apoB100 (Chapman et al., 1988). When considered together, the size and compositional properties of LDL subspecies are reflected in marked differences in particle molecular weights, that of dense LDL being some 23% less than that of its light counterpart. Vitamin E content diminishes in parallel with reduction in the size of the particle and of its hydrophobic core (Goulinet and Chapman, 1997): tocopherol content alone, however, does not account for the oxidative susceptibility of dense LDL (see Figure 4.1). LDL particles of the inter
Figure 4.1
mediate subclass (d 1.03–1.04 g/ml) are distinguished by an optimal configuration of apoB100 for binding to the cellular LDL receptor Lund-katz et al., 1998; by contrast, the high Kd (approximately 7nm) for dense LDL is reflected in rates of LDL degradation which are some 60% lower than those for intermediate subspecies Nigon et al., 1991. The low receptor binding affinity of both light and dense LDL is reflected in an elevated negative surface charge density relative to that
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of intermediate LDL; this latter feature almost certainly accounts for the high binding affinity of both light and dense particles to arterial matrix proteoglycans (Anber et al., 1996; Hurt-Camejo et al., 1990). In summary, dense LDL are distinct among LDL subclasses not only in exhibiting the smallest particle size, lowest vitamin E and ubiquinol-10 content Tribble et al., 1994, highest susceptibility to oxidative stress (Dejager et al., 1993; Tribble, 1995), poorest LDL receptor binding affinity and unique apoB100 conformation, but also in providing an optimal surface structure for the preferential binding and transport of platelet activating factor (Paf) acetylhydrolase (PafAH) and tissue factor pathway inhibitor (TFPI) (Tselepis et al., 1995; Lesnik et al., 1995). While PafAH exerts anti-inflammatory activity via cleavage of Paf and oxidised phospholipids with short carbon chains at the sn-2 position, TFPI exerts anti-thrombogenic activity via inhibition of Factor Xa activation of tissue factor, the key component of the extrinsic coagulation pathway. LDL Particle Subspecies and Atherosclerosis Risk Recent drug intervention trials to reduce cardiovascular events in dyslipidaemic patients at high risk (including primary and secondary prevention trials) have focussed on reduction in the circulating concentrations of LDL (Frick et al., 1987; Pederson et al., 1994; Brown et al., 1990; Shepherd et al., 1995; Kane et al., 1990). Such trials have led to an average reduction in cardiovascular events (principally myocardial infarction, angina and stroke) of approximately 35%, a decrement corresponding closely to the degree of LDL reduction obtained (30–35%) (Frick et al., 1987; Pederson et al., 1994; Brown et al., 1990; Shepherd et al., 1995; Kane et al., 1990). Circulating LDL concentrations alone have not, however, uniformly proven to be powerful predictive markers of cardiovascular risk in individual patients at high risk. Indeed, marked variation exists in the age at which coronary heart disease is clinically manifested in family members presenting familial hypercholesterolaemia; moreover, such variation is independent of LDL-cholesterol (Heiberg and Slack, 1977). Furthermore, some 20% of heterozygous subjects (in whom one of the two alleles coding for the LDL receptor gene is abnormal), do not present a myocardial infarction before their sixth decade despite marked elevation in plasma LDL (Heiberg and Slack, 1977). Such clinical findings prompted the hypothesis that the quantitative features of LDL particle profile could be significantly linked to the premature development of coronary heart disease. This relationship can now be considered as established, with abundant evidence, both retrospective and prospective in nature, substantiating a strong, independent association of small, dense LDL with premature cardiovascular disease
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in both normolipidaemic and dyslipidaemic subjects (Krauss, 1994; Sniderman et al., 1980; Austin et al., 1988; Crouse et al., 1985; Tornvall et al., 1991; Campos et al., 1992; Coresh et al., 1993; Griffin et al., 1994; Gardner et al., 1996; Stampfer et al., 1996; Lamarche et al., 1997). The above hypothesis is consistent with the notion that plasma levels of the most atherogenic LDL subspecies might be preferentially elevated (either in absolute terms or as a proportion of the total LDL fraction) in individuals with precocious disease; this is frequently the case (Sniderman et al., 1980; Austin et al., 1988; Crouse et al., 1985; Tornvall et al., 1991; Campos et al., 1992; Coresh et al., 1993; Griffin et al., 1994; Gardner et al., 1996; Stampfer et al., 1996; Lamarche et al., 1997). For example, a high proportion of normocholesterolaemic patients with angiographically-defined disease present a predominance of small LDL particles (Campos et al., 1995). Moreover, patients with atherogenic forms of dyslipidaemia typically display LDL subclass profiles in which dense LDL preponderate (“LDL phenotye B”) (Krauss, 1994; Austin et al., 1988). Thus, the asymmetric profile in combined (mixed) forms of hyperlipidaemia is dominated by a peak of small dense LDL (Dejager et al., 1993), which may attain concentrations up to fourfold higher than those in control subjects. Subjects with hypertriglyceridaemia equally display a predominance of dense LDL but, in contrast, total LDL levels in such patients are typically normal or subnormal (Eisenberg et al., 1984; Luc et al., 1988). In hypercholesterolaemic subjects, the absolute levels of dense LDL are consistently elevated (up to threefold) relative to controls; the major LDL subclass in such individuals, however, is typically light or intermediate LDL (Luc et al., 1988; Guerin et al., 1995). The intermediate subclass is equally preponderant in healthy normolipidaemic males, representing approximately 50% of total LDL mass (range approximately 120–180mg/dl). Indeed, the LDL profile is essentially symmetrical in such subjects, with dense LDL accounting for approximately 35% of total mass (average plasma concentration: 82.8±11.7mg/ml) (Nigon et al., 1991; Dejager et al., 1993; Tselepis et al., 1995). It would be misleading to leave the reader with the impression that increased cardiovascular risk is specifically and solely associated with small, dense LDL. In an investigation performed within the framework of the Montreal Heart Study, the particle size of large, buoyant LDL was significantly associated with coronary artery disease in normolipidaemic men (Campos et al., 1995). Equally, an LDL profile dominated by the light subclass was more prevalent in this latter group as compared to healthy controls (Campos et al., 1995). Light, large LDL may therefore be of elevated atherogenicity in certain cohorts. These findings may equally be indicative of the contribution of elevated amounts of atherogenic triglyceride-rich remnant lipoproteins
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to the light LDL subclass, a suggestion consistent with the recent observation that apo-E-containing triglyceride-rich particles can represent a significant component of the light LDL subclass (Barbagallo et al., 1998). A major question now arises: how are the physicochemical properties and biological activities of LDL subclasses related to the pathophysiological mechanisms implicated in the initiation, progression and rupture of lipid-rich atherosclerotic plaques? To shed light on this question, we must first consider the role of the endothelium in plaque initiation and development. Mechanisms Underlying LDL Atherogenicity Sites of predilection of plaque initiation in the arterial endothelium Endothelial cells constitute the interface between blood in the vessel lumen and the vessel wall; as such their role in vascular homeostasis is critical, as they regulate a spectrum of biological processes implicated in vascular function. Among these processes is the control of the permeability of the vessel wall to lipoproteins and other macromolecular solutes in plasma, as well as regulation of the adhesion of monocytes and other leukocytic cells. Clearly then, conditions of blood flow which perturb endothelial function could engender enhanced permeability to lipoproteins, thereby leading to enhanced lipid influx and potentially to cholesterol accumulation. Following upon the pioneering studies of Glagov and colleagues (Ku et al., 1985), it has become evident that shear stress, defined as the frictional force induced at the endothelial surface by blood flow, is a key factor in the modulation of endothelial cell function and morphology (Davies, 1995; Traub and Berk, 1998). Thus, mean positive shear stress resulting from laminar blood flow appears to assure normal endothelial cell function, thereby exerting an atheroprotective effect. On the contrary, sites at which laminar blood flow is altered, such as the carotid bifurcation and other branch points in the arterial tree, are associated with endothelial dysfunction resulting from low mean shear stress and a flow reversal phenomenon. The localised areas of the carotid, coronary and aortic endothelium at which such phenomena occur constitute sites of predilection for lesion initiation and fatty streak formation (Ku et al., 1985; Traub and Berk, 1998) and are characterised by increased permeability to LDL and other macromolecular solutes (Traub and Berk, 1988).
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Entry, Efflux and Retention of LDL in the Arterial Intima The transendothelial transport of LDL particles occurs predominantly by a receptor-independent, vesicular pathway (Vasile et al., 1983; Nielsen, 1996). This transcytotic process is bidirectional; it is mediated by low affinity binding sites confined to plasmalemmal vesicles. LDL transport is nonsaturable and energy-dependent (Vasile et al., 1983). Such transcytosis into the subendothelial space is particularly dependent on LDL concentration. It is therefore especially relevant that luminal LDL concentrations are elevated at the endothelial surface at sites of low shear stress, low blood flow velocity and high systolic pressure (Deng et al., 1995); elevated influx of LDL into the intima results. In addition to luminal concentration, particle size is equally a major determinant of the rate of arterial influx of LDL (Nielsen, 1996; Bjornheden et al., 1996; Stender and Zilversmit, 1981). Indeed, the recent investigations of Bjornheden and colleagues (1996) indicate that the rate of uptake of dense LDL particles (d 1.035–1.063 g/ml) in a rabbit aortic intima-media perfusion system is up to twofold higher than that of the light subclass. Such findings are consistent with the high binding affinity and retention of small dense LDL by arterial connective tissue proteoglycans (Hurt-Camejo et al., 1990; Anber et al., 1996) and thus provide a mechanistic basis for the specific association of this LDL subclass with increased cardiovascular risk. A critical element in atherosclerotic lesion formation is represented by the net difference between rates of LDL influx and efflux from the arterial intima, and which corresponds to the amount of which is retained (trapped) as a result either of binding to cellular and matrix components, or of degradation. In this regard, evidence to date indicates that intimal LDL uptake, concentration and residence time are all significantly increased at plaque sites (Nielsen, 1996; Schwenke and Carew, 1989 a&b; Deng et al., 1995; Bjornheden et al., 1996; Stender and Zilversmit, 1981). Even in normal arterial intima, LDL concentration is directly and proportionately related to plasma level, as demonstrated by the early studies of intimal interstitial fluid by Smith and collaborators (Smith, 1990). The penetration of these particles into the media is excluded by the internal elastic lamina, which acts as a barrier to entry of LDL into the media. This notion is supported by the observation of Bjornheden et al. (Bjornheden et al., 1996) that lipoprotein uptake into the arterial wall is characterised by two pools, a fast equilibrating subendothelial and a slow equilibrating medial pool. What is the nature then of the mechanisms which underlie the preferential retention and focal accumulation of LDL particles in arterial intima? Certainly, and as originally shown by Schwenke and Carew (1989a&b), LDL are preferentially retained at arterial sites
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characterised by their susceptibility to atherosclerosis; moreover, increased LDL retention precedes plaque development at such sites. Furthermore, the capacity of the arterial wall to degrade accumulated lipoproteins may be increased up to tenfold at sites of lesion development (Bjornheden et al., 1996). It is now established that versican chondroitin sulphate proteoglycans (CSPGs) of the arterial intima, in both pericellular and extracellular locations, bind with high affinity to basic domains of LDL apoB100 and thereby facilitate lipoprotein retention (Camejo et al., 1993). Lipoprotein lipase may enhance such interactions (Camejo et al., 1993). Further evidence for a major anchoring role of CSPGs derives from the isolation of LDL from both normal and atherosclerotic arterial tissue in the form of CSPG-lipoprotein complexes (Camejo et al., 1993). In this way lipoprotein residence time is prolonged in the intima, and with it the potential for structural and compositional modification. By contrast, the contribution of LDL receptors expressed by cells of the arterial wall appears to represent a minor component in LDL retention (Camejo et al., 1993). Intimal Metabolism of LDL, Cholesterol Accumulation and Lipid Core Formation Mechanisms of the intimal retention and modification of LDL are of direct relevance to the extracellular and intracellular forms in which lipoprotein-derived cholesterol accumulates in the developing lesion. A plethora of candidate mechanisms has been identified which may lead to structural and chemical alteration of LDL, and these are summarised in Figure 4.2; the contribution of individual mechanisms to cholesterol accumulation may differ markedly as a function of the stage of plaque development. In early fatty streak lesions, lipid accumulates in the extracellular spaces as well as within cells; intracellular cholesterol accumulates predominantly as droplets of cholesteryl ester in monocyte-derived macrophages, endowing them with a foamy appearance (Stary et al., 1994). As the lesion progresses, pools of lipid appear, giving rise to the acellular lipid-rich core characteristic of intermediate and advanced lesions (Stary et al., 1994). Debate surrounds the relative contributions of the respective pathways operative in the formation of the lipid core (Smith et al., 1968; Guyton and Klemp, 1989; Guyton and Klemp, 1994; Ball et al., 1995; Bjorkerud and Bjorkerud, 1996; Hoff and Hoppe, 1995; Guyton and Klemp, 1996; Kruth, 1997). There is however general agreement that two major pathways are predominant; (1) on one hand, LDL-cholesterol contributes to formation and growth of the lipid core as a result of the apoptosis and necrosis of cholesterolloaded macrophage foam cells and (2) on the other hand, substantial
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experimental data now support the contention that cholesteryl ester-rich particles and liposomes rich in free cholesterol, that result from the modification and degradation of LDL within lesion tissue, directly contribute to the lipid core (Smith et al., 1968; Guyton and Klemp, 1989; Guyton and Klemp, 1994; Ball et al., 1995; Bjorkerud and Bjorkerud, 1996; Hoff and Hoppe, 1995; Guyton and Klemp, 1996; Kruth, 1997). There is overwhelming evidence that the primary source of lesion cholesterol is plasma LDL. The early studies of Smith (1974) established that LDL core cholesteryl esters are primarily esterified with linoleate; by contrast, hydrolysis of LDL cholesteryl ester and subsequent cholesterol reesterification in the macrophage produces cholesteryl oleate. Clearly then, the direct accretion of LDL-derived cholesteryl ester to the lesion core represents an essential aspect of its formation and expansion. It is relevant in this context that protease modification of LDL apo-B induces particle fusion, creating large cholesteryl ester-rich particles; fused LDL bind avidly to arterial proteoglycans, thereby favouring their retention in the intima (Piha et al., 1995; Paananen et al., 1995; Pentikainen et al., 1996). Finally, the potential uptake of cholesteryl ester-rich particles and unesterified cholesterol-rich liposomes by lesion macrophages may equally represent a significant pathway of intracellular lipid accumulation (Kruth, 1997). Cholesterol in crystalline form frequently constitutes a major component of the lipid core of advanced, complex lesions (Stary et al., 1994) and reflects the existence of optimal conditions for lipid phase separation within the core (Small, 1988). Cholesterol crystals may also be formed within macrophages when solubilisation conditions (conversion to the ester) are saturated (Kruth, 1997). Mechanisms of the Proatherogenic Modification of Intimal LDL From the above and Figure 4.2, it is apparent that native LDL particles must undergo chemical modification before their capacity to enhance core lipid expansion via a non-cellular pathway is expressed. In a similar manner, native LDL do not induce significant lipid accumulation in monocyte-derived macrophages but rather must undergo prior modification (Brown and Goldstein, 1983). Mechanisms of particle modification which trigger recognition by macrophage receptors principally involve oxidation of the lipid and protein moieties of LDL, phospholipase-mediated hydrolysis of LDL phospholipids with formation of lysolecithin (secretory phospholipase A2) and ceramide (sphingomyelinase) (Romano et al., 1998; Schissel et al., 1996), cholesteryl ester hydrolysis to unesterified cholesterol by cholesterol esterases (Kruth, 1997) and proteolytic cleavage of
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apoB100 (Piha et al., 1995; Paananen et al., 1995; Pentikainen et al., 1996).
Figure 4.2
The molecular mechanisms implicated in the oxidative modification of LDL have been reviewed extensively (Navab et al., 1996; Tribble, 1995; Steinberg et al., 1989; Heinecke, 1997); their detailed discussion is beyond the scope of the present treatise. Nonetheless, the formation of oxidised, biologically active lipids and oxysterols is a key factor in the installation of the inflammatory response in the artery wall (Navab et al., 1996; Berliner and Heinecke, 1996). Moreover, oxidised bioactive lipids are now recognised as potent modulators of nuclear transcription factors and gene expression in target cells (see below). Oxidised forms of LDL may bind specifically to several distinct
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proteins at the macrophage cell surface; these include class A scavenger receptors, scavenger receptor B1, CD36, CD68 and CD32 (Fc R11) (Krieger, 1997). The in vivo relevance of certain of these proteins remains unclear: recent data do, however, support a major role for both SR-A and CD36 receptors in macrophage foam cell formation via oxidised LDL uptake (Tontonoz et al., 1998; Suzuki et al., 1997; Nozaki et al., 1995). The principal epitopes on oxLDL which define recognition and uptake by these receptors are ill-defined and await characterisation. Lysine-adducts formed in apoB100 upon reaction with aldehydic breakdown products of unsaturated fatty acids, such as malondialdehyde and hydroxynonenal, are clearly implicated (Steinberg et al., 1989; Esterbauer et al., 1987; Yla-Herttuala et al., 1989). Interestingly, scavenger receptor-mediated uptake of oxLDL by macrophages results in induction of the expression of the nuclear receptor, PPAR ; transcriptional induction of the CD36 receptor ensues (Tontonoz et al., 1998). Such activation is mediated by two of the major oxidised lipids of oxLDL, 9-HODE and 13-HODE, which are not only endogenous activators but also ligands of PPAR (Nagy et al., 1998). Moreover, high levels of expression of PPAR have been detected in lesion foam cells, thereby provoking the suggestion that these nuclear receptors play a central role in the control of macrophage lipid metabolism and thus in the pathogenesis of atherosclerosis (Tontonoz et al., 1998). LDL Subclass: Molecular and Cellular Mechanisms of Enhanced Atherogenicity In vitro studies have revealed that marked differences exist between the atherogenic potential of LDL particle subclasses (Krauss, 1994; Chapman et al., 1998). The in vivo relevance of these findings is substantiated by clinical observations in which distinct LDL subclass profiles—or phenotypes—are specifically associated with elevated cardiovascular risk (Krauss, 1994; Chapman et al., 1998). The most frequent association has been found among subjects who display an LDL profile in which the dense subclass predominates (see “LDL particle subspecies and atherosclerosis risk” above). Conversely, the greatest arteriographic benefit has been observed in coronary artery regression studies when elevated levels of LDL have been therapeutically reduced in patients exhibiting this same dense (“B”) phenotype (Superko and Krauss, 1994). Mechanistically, the available evidence reviewed herein suggests that the atherogenicity of small dense LDL is expressed as a function of its physical, chemical and biological properties (Figure 4.1) and through the sequence of events depicted in Figure 4.3. Small particle size and elevated luminal particle number (i.e. concentration) of dense LDL, together with high transmural pressure,
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facilitate enhanced arterial influx at sites at which endothelial permeability is increased due to endothelial dysfunction. This occurs at branch points in the arterial tree (e.g. carotid bifurcation) associated with low mean shear stress and flow reversal. At pre-existing plaque sites, the penetration, intimal concentration and residence time of LDL are significantly increased. Dense LDL bind with high affinity to matrix CSPGs leading to intimal retention. The deficient oxidative resistance of dense LDL predisposes them to chemical modification under conditions of oxidative stress; such oxidant stress may result from activation of the production of reactive oxygen species by endothelial cells, monocyte-derived macrophages and smooth muscle cells, or from pro-oxidant enzyme activity (lipoxygenases, myeloperoxidase) or from the presence of redox active metal ions (Heinecke, 1997). Oxidation inactivates the potential anti-inflammatory (Paf-AH) and anti-thrombogenic (TFPI) activities associated with dense LDL (Lesnik et al., 1995; Dentan et al., 1994). Receptordependent uptake of oxLDL by macrophages ensues, transforming these cells to cholesterol-loaded foam cells. Macrophage foam cells secrete numerous factors possessing proatherogenic, pro-inflammatory or pro-thrombogenic activities. Such factors, which notably include metalloproteases, favour plaque progression and matrix fragilisation, ultimately leading to plaque fissure with ensuing thrombotic complications. In the second pathway shown in Figure 4.3, dense LDL particles are highly susceptible to protease attack; proteolysed LDL fuse, leading to formation of cholesteryl ester-rich particles which may enhance expansion of the plaque’s lipid core, or alternatively, may be phagocytosed by macrophages (Kruth, 1997; Piha et al., 1995; Paananen et al., 1995; Pentikainen et al., 1996). Finally, despite advances in our knowledge of the pathophysiology underlying the elevated atherogenicity of dense LDL, the possibility that other subclasses may be highly atherogenic when their plasma particle number is markedly elevated should be considered. Indeed, intermediate LDL are typically present at fourfold higher levels in patients homozygous for familial hypercholesterolaemia (Goldstein and Brown, 1983; Luc et al., 1986) and dominate the LDL profile (Luc et al., 1986). Under these conditions, the intermediate LDL subclass is intimately implicated in the severe atherosclerosis manifest in these patients during the first decade of life.
CONCLUSION Identification of LDL subclasses of elevated atherogenicity heralds a new clinical era when therapeutic intervention may be defined as a
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direct function of the atherogenic
Figure 4.3
lipoprotein profile in any given dyslipidaemic patient at high
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cardiovascular risk. Prominent among the candidates for such targeted treatment are those who present a dense LDL “B” phenotype, potentially in association with elevated levels of triglyceride-rich remnant particles and subnormal concentrations of HDL (Superko and Krauss, 1994; Bruckert et al., 1993; Caslake et al., 1993; Gaw et al., 1994; Guerin et al., 1996). References Anber, V., Griffin, B.A., McConnell, M., Packard, C.J. and Shepherd, J. (1996). Influence of plasma lipid and LDL subfraction profile on the interaction between low density lipoprotein with human arterial wall proteoglycans. Atherosclerosis , 124 , 261–271. Andersen, K.M., Castelli, W.P. and Levy, D. (1987). Cholesterol and mortality: 30 years of follow-up from the Framingham study. JAMA , 257, 2176–2180. Assmann, G. and Schulte, H. (1998). The Prospective Cardiovascular Munster (PROCAM) Study: Prevalence of hyperlipidemia in persons with hypertension and/or diabetes mellitus and the relationship to coronary heart disease. Am. Heart. J. , 116, 1713–1724. Austin, M.A., Breslow, J.L.Hennekens, C.H., Buring, J.E., Willett, W.C. and Krauss, R.M. (1988). Low density lipoprotein subclass patterns and risk of myocardial infarction. JAMA , 260 1917–1925. Ball, R.Y., Stowers, E.C., Burton, J.H., Cary, N.R., Skepper, J.N. and Mitchinson, M.J. (1995). Evidence that the death of macrophage foam cells contributes to the lipid core of atheroma. Atherosclerosis , 114 , 45–54. Barbagallo, C.M., Levine, G.A., Blanche, P.J., Ishida, B.Y. and Krauss, R.M. (1998). Influence of apoE content on receptor binding of large, buoyant LDL in subjects with different LDL subclass phenotypes. Arterioscler. Thromb. Vasc. Biol. , 18 , 466–472. Berliner, J.A. and Heinecke, J.W. (1996). The role of oxidised lipoproteins in atherogenesis. Free Rad. Biol. Med. , 20 , 707–727. Bjorkerud, S. and Bjorkerud, B. (1996). Apoptosis is abundant in human atherosclerotic lesions, especially in inflammatory cells (macrophages and T cells) and may contribute to the accumulation of gruel and plaque instability. Am. J.Pathol. , 149 , 367–380. Bjornheden, T., Babyi, A., Bondjers, G. and Wiklund, O. (1996). Accumulation of lipoprotein fractions and subfractions in the arterial wall determined in an in vitro perfusion system. Atherosclerosis , 123, 43–56. Blankenhorn, D.H., Johnson, R.L., Mack, W.J., Zein, H.A.E. and Valais, L.I. (1990). The influence of diet on the appearance of new lesions in human coronary arteries. JAMA , 263, 1646–1652. Brown, G., Albers, J.J., Fisher, L.D., Schaefer, S.M., Lin, J.T., Kaplan, C. et al. (1990). Regression of coronary artery disease as a result of intensive lipid-lowering therapy in men with high levels of apolipoprotein B. NEJM , 323, 1289–1298.
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Brown, M.S. and Goldstein, J.L. (1983). Lipoprotein metabolism in the macrophage: implications for cholesterol deposition in atherosclerosis. Ann. Rev. Biochem. , 52, 223–261. Bruckert, E., Dejager, S. and Chapman, M.J. (1993). Ciprofibrate therapy normalises the atherogenic low density lipoprotein subspecies profile in combined hyperlipidemia. Atherosclerosis , 100 , 91–102. Brunzell, J.D., Schrott, H.G., Motulsky, A.G. and Bierman, E.L. (1976). Myocardial infarction in the familial forms of hypertriglyceridemia . Metabolism , 25, 313–320. Camejo, G., Hurt-Camejo, F., Olsson, U. and Bondjers, G. (1993). Proteoglycans and lipoproteins in atherosclerosis. Curr. Opin. Lipidol. , 4, 385–391. Campos, H., Roederer, G.P., Lussier-Cacan, S., Davignon, J. and Krauss, R.M. (1995). Predominance of large LDL and reduce HDL2 in normolipidemic men with coronary heart disease. Arterioscler. Thromb. Vasc. Biol. , 15, 1043–1048. Campos, H., Genest, J.J., Blijlevens, E., McNamara, J.R., Jenner, J.L., Ordovas, J.M. et al. (1992). Low density lipoprotein particle size and coronary artery disease. Arterioscler. Thromb. , 12, 187–195. Caslake, M.J., Packard, C.J., Gaw, A., Murray, E., Griffin, B.A., Vallance, B.P. et al. (1993). Fenofibrate and LDL metabolic heterogeneity in hypercholesterolemia. Arterioscler. Thromb. , 13, 702–711. Chapman, M.J., Guerin, M. and Bruckert, E. (1998). Atherogenic, dense low-density lipoproteins: Pathophysiology and new therapeutic approaches. Europ. Heart. J. , 19, A24–A30. Chapman, M.J., Laplaud, P.M., Luc, G., Forgez, P., Gruckert, E. et al. (1988). Further resolution of the low density lipoprotein spectrum in normal human plasma: physicochemical characteristics of discrete subspecies separated by density gradient ultracentrifugation. J.Lipid Res. , 29, 442–458. Chi Chen, G., Liu, W., Duchateau, P.Allaart, J., Hamilton, R.L., Mendel, C.M. et al. (1994). Con-formational differences in human apolipoprotein B100 among subspecies of low density lipoprotein. J.Biol. Chem. , 269, 29121–29128. Consensus Conference. Lowering blood cholesterol to prevent heart disease. (1985). JAMA , 253, 2080–2086. Coresh, J., Kwiterovich, P.O. Jr., Smith, H.H. and Bachorik, P.S. (1993). Association of plasma triglyceride concentration and LDL particle diameter, density and chemical composition with premature coronary artery disease in men and women. J.Lipid Res. , 35, 1687– 1697. Crouse, J.R., Parks, J.S., Schey, H.M. and Khol, F.R. (1985). Studies of low density lipoprotein molecular weight in human beings with coronary artery disease. J.Lipid Res. , 26, 566–573. Davies, P.F. (1995). Flow-mediated endothelial mechanotransduction. Physiol. Rev. , 75, 519–560. Dejager, S., Bruckert, E. and Chapman, M.J. (1993). Dense low density
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lipoprotein subspecies with diminished oxidative resistance predominate in combined hyperlipidemia. J.Lipid Res. , 34, 295–308. Deng, X., Marios, Y., How, T., Merhi, Y., King, M. and Guidoin, R. (1995). Luminal surface concentration of lipoprotein (LDL) and its effect on the wall uptake of cholesterol by canine carotid arteries. J.Vasc. Surg. , 21, 135–145. Dentan, C., Lesnik, P., Chapman, M.J. and Ninio, E. (1994). PAFacether-degrading acetylhydrolase in plasma low density lipoprotein is inactivated by copper and cell-mediated oxidation. Arterioscler. Thromb. , 14, 353–360. Eisenberg, S., Gavish, D., Oschry, Y., Fainaru, M. and Deckelbaum, R.G. (1984). Abnormalities in very low, low, and high density lipoproteins and hpertriglyceridemia. Reversal toward normal with bezafibrate treatment. J.Clin. Invest. , 74, 470–482. Esterbauer, H., Jurgens, G., Ouchenberger, O. and Kuller, E. (1987). Autooxidation of human low density lipoprotein: loss of polyunsaturated fatty acids and vitamin E and generation of aldehydes. J.Lipid Res. , 28, 495–509. Frick, M.H., Elo, O., Haapa, K., Heinonen, O.P., Heinsalmi, P., Helo, P. et al. (1987). Helsinki Heart Study: Primary-prevention trial with gemfibrozil in middle-aged men with dyslipidemia. Safety of treatment, changes in risk factors, and incidence of coronary heart disease. NEJM , 317, 1237–1245. Gardner, C.D., Fortmann, S.P. and Krauss, R.M. (1996). Association of small low-density lipoprotein particles with the incidence of coronary artery disease in men and women. JAMA , 276, 875–881. Gaw, A., Packard, C.J., Caslake, M.J. and Shepherd, J. (1994). Effects of ciprofibrate on LDL metabolism in man. Atherosclerosis , 108, 137–145. Goldstein, J.L. and Brown, M.S. (1983). Familial hypercholesterolemia. In The metabolic basis of inherited disease , edited by Stanbury, J.B., Syngaarden, J.B., Fredrickson, D.S., Goldstein J.L. and Brown, M.S., 5th edition. New York: McGrawHill, pp. 672–712. Goulinet, S. and Chapman, M.J. (1997). Plasma LDL and HDL subspecies are heterogeneous in particle content of tocopherols and oxygenated and hydrocarbon carotenoids. Arterioscler. Thromb. Vasc. Biol. , 17, 786–796. Griffin, B.A., Freeman, D.J., Tait, G.W., Thomson, J., Caslake, K.E., Packard, C.J. et al. (1994). Role of plasma triglyceride in the regulation of plasma low density lipoprotein subfractions: relative contribution of small dense LDL to coronary heart disease risk. Atherosclerosis , 106, 241–253. Guerin, M., Bruckert, E., Dolphin, P.J., Turpin, G. and Chapman, M.J. (1996). Fenofibrate reduces cholesteryl ester transfer from HDL to VLDL and normalises the atherogenic dense LDL profile in combined hyperlipidemia. Arterioscler. Thromb. Vasc. Biol. , 16, 763–772. Guerin, M., Dolphin, P.J., Talussot, C., Gardette, J., Berthezene, F. and
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Seven Countries Study. Preventive Med. , 24, 308–315. Kruth, H.S. (1997). The fate of lipoprotein cholesterol entering the arterial wall. Curr. Lipidol. , 8, 246–252. Ku, D.N., Giddens, D.P., Zarins, C.K. and Glagov, S. (1985). Pulsatile flow and atherosclerosis in the human carotid bifurcation: positive correlation between plaque location and low oscillatory shear stress. Arteriosclerosis , 5 , 293–302. La Belle, M. and Krauss, R.M. (1990). Differences in carbohydrate content of low density lipoprotein associated with low density lipoprotein subclass patterns. J.Lipid. Res. , 31, 1577–1588. Lamarche, B., Tehernof, A., Moorjani, S., Cantin, B., Dagenais, G.R., Lupien, P.J. et al. (1997). Small, dense low-density lipoprotein particles as a predictor of the risk of ischemic heart disease in men. Prospective results from the Quebec Cardiovascular Study . Circulation , 95, 69–75. Law, M.R., Wald, N.J, Wu, T., Hackshaw, A. and Bailey, A. (1994). Systematic underestimation of association between serum cholesterol concentration and ischemic heart disease in observational studiesdata from the BUPA study. BMJ , 308, 363–366. Lesnik, P., Dentan, D., Vonica, A., Moreau, M. and Chapman, M.J. (1995). Tissue factor pathway inhibitor activity associated with LDL is inactivated by cell and copper-mediated oxidation. Arterioscler. Thromb. Vasc. Biol , 15, 1121–1130. Lindgren, F.T., Jensen, L.C., Wills, R.D. and Freeman, N.K. (1969). Flotation rates, molecular weight and hydrated densities of the low density lipoproteins. Lipids , 4, 337–344. Luc, G., Chapman, M.J., de Gennes, J.L. and Turpin, G. (1986). A study of the structural heterogeneity of low density lipoproteins in two patients homozygous for familial hypercholesterolemia, one of phenotype E2/E2. Eur. J.Clin. Invest. , 16, 329–337. Luc, G., Chapman, M.J. and de Gennes, J.L. (1988). Further resolution and comparison of the heterogeneity of plasma low density lipoproteins in human hyperlipoproteinemias: type III hyperlipoproteinemia, hypertriglyceridemia, and familial hypercholesterolemia. Atherosclerosis , 71, 1543–1567. Lund-Katz, S., Laplaud, P.M., Phillips, M.L. and Chapman M.J. (1998). Apolipoprotein B100 conformation and particle surface charge in human LDL subspecies: implication for LDL receptor interaction. Biochemistry , 38, 1367–1378. Nagy, L., Tontonoz, P., Alvarez, J.G.A., Chen, H. and Evans, R.M. (1998). Oxidised LDL regulates macrophage gene expression through ligand activation of PPAR. Cell , 93, 229–240. McNamara, J.R., Campos, H., Ordovas, J.M, Peterson, J., Wilson, P.W.F. and Schaefer, E.J. (1987). Effect of gender, age and lipid status on LDL subfraction distribution. Results from the Framingham offspring study. Arteriosclerosis , 7 , 483–490. Navab, M., Berliner, J.A., Watson, A.D., Hama, S.Y., Territo, M.C., Lusis, A.J. et al. (1996). The Yin and Yang of oxidation in the development of the fatty streak. Arterioscler. Thromb. Vasc. Biol. ,
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5 High Density Lipoproteins: The Anti-atherogenic Fraction Philip J.Barter 1 , Moira A.Clay 2 and Kerry-Anne Rye 2 1
University Department of Medicine, Royal Adelaide Hospital, North Terrace, Adelaide, South Australia 5000, Australia 2 Lipid Research Laboratory, Hanson Centre, Frome Road, Adelaide, South Australia 5000, Australia
The main importance of high density lipoproteins (HDL) relates to the fact that they protect against the development of premature coronary heart disease. The mechanism of the protection is unknown but may be secondary to the involvement of HDL in the process of reverse cholesterol transport whereby cholesterol is transported from extrahepatic tissues to the liver for recycling or excretion from the body. The anti-inflammatory and anti-oxidant properties of HDL may also contribute to their ability to inhibit the development of atherosclerosis. The HDL in human plasma are heterogeneous, comprising a number of subpopulations of particles of distinct size, charge, composition and function. There is evidence from transgenic animal studies that some HDL subpopulations are superior to others in terms of their capacity to inhibit atherosclerosis. The situation in humans is not known. It also remains to be determined in human studies whether treatments designed to increase the concentration of HDL result in a reduced risk of coronary heart disease. KEYWORDS: HDL, coronary heart disease, atherosclerosis, reverse cholesterol transport.
INTRODUCTION The observation that high density lipoproteins (HDL) have powerful anti-atherogenic properties has stimulated extensive research into this
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lipoprotein fraction over the past two decades. There is now a substantial body of information about the structure and function of HDL. However, less is known about how HDL are regulated. There is also considerable uncertainty regarding the mechanism by which HDL inhibit the development of coronary heart disease. This chapter reviews the current state of knowledge regarding the structure of HDL, their subpopulation distribution, their metabolism and regulation, their functions, their relationship to atherosclerosis and finally, clinical states in which the concentration of HDL is abnormal.
STRUCTURE OF HDL HDL are the smallest (7.4–12 nm diameter) and densest (1.063 < d < 1.21 g/ml) of the plasma lipoproteins. Like other plasma lipoproteins, they consist of a hydrophobic core (mainly cholesteryl esters plus a small amount of triglyceride) surrounded by a surface monolayer of phospholipids, unesterified cholesterol and apolipoproteins. The main HDL apolipoproteins are apoA-I and apoA-II. They also contain small amounts of apoA-IV, the C-apolipoproteins, apoD, apoE and apoJ. Other plasma proteins involved in regulating plasma lipid transport are also associated with HDL. These include cholesteryl ester transfer protein (CETP) (Cheung et al., 1986; Francone et al., 1989; Marcel et al., 1990; Pattnaik and Zilversmit, 1979), lecithin:cholesterol acyltransferase (LCAT) (Cheung et al., 1986; Francone et al., 1989) and phospholipid transfer protein (PLTP) (Tall et al., 1983).
HDL SUBPOPULATIONS The HDL fraction in human plasma is heterogeneous (Table 5.1). When isolated on the basis of density, by ultracentrifugation, human HDL can be separated into two major subfractions: HDL2 (1.063
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while the other comprises particles which contain both apoA-I and apoA-II (A-I/A-II HDL) (Cheung and Albers, 1984). There is also a minor subpopulation of A-II HDL. ApoA-I is divided approximately equally between A-I HDL and A-I/A-II HDL in most human subjects
Table 5.1 HDL subpopulations
HDL subpopulations
Proportion of total plasma HDL a
Separated by density HDL2
1.063
HDL3
1.125
50–95%
VHDL
1.21
5%
HDL2b
mean diameter 10.6nm
0–50%
HDL2a
mean diameter 9.2 nm
0–20%
HDL3a
mean diameter 8.4 nm
50–87%
HDL3b
mean diameter 8.0 nm
10–50%
HDL3c
mean diameter 7.6nm
5–10%
Separated by size
Separated by apolipoprotein composition A-I HDL
30–60%
A-I/A-H HDL
30–60%
A-II HDL
0–10%
Separated by charge pre-
0–5% 85–95%
pre 1
1–6%
pre 2
2–3%
pre 3
<1% 0–5%
a
Proportions are estimates obtained from a variety of publications.
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(Cheung and Albers, 1984), while more than 90% of the apoA-II is in A-I/A-II HDL (Bekaert et al., 1992). Most of the A-I/A-II HDL are found in the HDL3 density range, while A-I HDL are present in both HDL2 and HDL3 (Cheung and Albers, 1982). The heterogeneity of HDL also extends to their surface charge. When subjected to agarose gel electrophoresis, HDL have either , pre, pre- or migration (Table 5.1), (Asztalos et al., 1993a; Huang et al., 1994; Kunitake et al., 1985; Sparks and Phillips, 1992). The migrating particles are spherical and account for most of HDL in plasma. They include the HDL2 and HDL3 subfractions, as well as A-I HDL and A-I/A-II HDL. Pre- HDL are discoidal particles which contain one or two molecules of apoA-I complexed with phospholipids and a small amount of unesterified cholesterol. The smallest of the pre- HDL are called pre- 1 HDL. They are 5–6 nm in diameter and consist of a single molecule of apoA-I complexed with small amounts of
Figure 5.1 HDL subpopulations separated on the basis of size and apolipoprotein composition.
phosphatidylcholine (PC), sphingomyelin and unesterified cholesterol (Fielding and Fielding, 1995). Pre- 2 HDL are larger than pre- 1 HDL and contain two molecules of apoA-I complexed with phospholipids and imesterified cholesterol. Pre- 2 HDL may represent the fusion products of pre- 1 particles (Castro and Fielding, 1988). Pre- 3 HDL are larger than either pre- 1 or pre- 2 HDL. It has been suggested that pre- 3 HDL are pre- 2 HDL complexed with LCAT (Francone et al.,
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1989). Pre- HDL also contain apoA-I (Asztalos et al., 1993a), whereas -HDL contain apoE and no apoA-I (Huang et al., 1994).
FORMATION OF HDL The rate at which HDL are formed is determined by the rates at which their main apolipoproteins are synthesised and secreted into plasma. It is also determined by factors such as LCAT which, as outlined below, contribute to the formation of mature spherical HDL particles. Formation of A-I HDL ApoA-I is synthesised in the liver and intestine and secreted into the plasma mainly as discoidal particles containing two molecules of apoA-I complexed with phospholipids and unesterified cholesterol (Hamilton et al., 1976). Discoidal HDL containing apoA-I, phospholipids and unesterified cholesterol are also generated when redundant surface components are shed from chylomicrons following hydrolysis of their triglyceride by lipoprotein lipase (Redgrave and Small, 1979; Tall et al., 1982). Irrespective of their origin, discoidal AI HDL have the capacity to acquire additional unesterified cholesterol from other lipoproteins and from cell membranes. They are also excellent substrates for LCAT, which rapidly esterifies their cholesterol. The LCAT reaction generates a core of cholesteryl esters in the discoidal particles and converts them into spherical A-I HDL (Nichols et al., 1985). The rapidity of the LCAT reaction explains why most of the HDL in plasma are spherical. Formation of A-II HDL ApoA-II is synthesised in the liver and is probably secreted into the plasma as discoidal apoA-II/phospholipid complexes which are comparable to those containing apoA-I. Unlike discoidal A-I HDL, discoidal A-II HDL do not react with LCAT and thus, are not converted into spherical particles in the usual way. The origin of the minor subpopulation of spherical A-II HDL in plasma is not known. Nor is it known why most of the apoA-II in plasma is accommodated in spherical A-I/A-II HDL, rather than in A-II HDL. Formation of A-I/A-II HDL Up to half of the HDL in human plasma are spherical A-I/A-II HDL. Their origins are uncertain, as is their relationship with A-I HDL and A-II HDL. There is little to suggest that A-I/A-II HDL are secreted as intact particles from either the liver or intestine. It is possible that they
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are assembled in the plasma from A-I HDL and A-II HDL in reactions involving plasma factors such as LCAT, CETP and PLTP.
CATABOLISM OF HDL Both the apoA-I and apoA-II in human HDL are removed from plasma with half-times of about 3–5 days (Fidge et al., 1980; Schaefer et al., 1982). Available evidence indicates that the liver is an important site of uptake of these apolipoproteins (Glass et al., 1983), although excretion through the kidney also contributes to their removal from plasma (Horowitz et al., 1992). In contrast to the apolipoproteins, the lipid constituents of HDL are either hydrolysed within the plasma by lipolytic enzymes or transferred to other lipoproteins in processes mediated by lipid transfer proteins. A proportion of the lipids in HDL are also selectively taken up by the liver and steroidogenic tissues (Glass et al., 1983; Glass et al., 1985). The net result is that HDL lipid constituents turn over much more rapidly than HDL apolipoproteins. The lipolytic and transfer reactions, as well as the cholesterol esterification catalysed by LCAT, result in extensive and continual remodelling of HDL particles during their circulation in plasma. This remodelling within the plasma compartment is, to a large extent, responsible for the size and compositional heterogeneity of HDL. It also accounts for the interconvertability of larger and smaller HDL and for the fact that the apoA-I in plasma cycles between HDL and a lipidfree state (Liang et al., 1995).
FACTORS THAT REMODEL HDL IN PLASMA Lecithin: Cholesterol Acyltransferase Lecithin: cholesterol acyltransferase (LCAT) catalyses the esterification of cholesterol on the surface of HDL (Glomset, 1968). This reaction generates cholesteryl esters which are incorporated into the HDL core and convert the discoidal HDL into spherical particles (Nichols et al., 1985). The resulting small spherical A-I HDL also interact with LCAT in processes which generate additional cholesteryl esters and increase their size. HDL can acquire additional lipid-free apoA-I as they increase in size (see below), (Liang et al., 1995; Liang et al., 1996). LCAT may also increase the apoA-I content of HDL by particle fusion (Liang et al., 1996; Nichols et al., 1985). There are marked differences in the capacity of different HDL subpopulations to act as substrates for LCAT. For example, the rate of LCAT-catalysed cholesterol esterification is greater in discoidal than in
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spherical A-I HDL (Jonas, 1991). In spherical HDL, the rate of cholesterol esterification varies inversely with particle size (Barter et al., 1985). Cholesteryl Ester Transfer Protein Cholesteryl ester transfer protein (CETP) transfers cholesteryl esters from HDL to other lipoproteins, in a process which depletes the HDL core of cholesteryl esters (Barter et al., 1982). While transfers of cholesteryl esters out of HDL are associated with transfers of triglyceride into the HDL, the cholesteryl ester transfers are greater than the triglyceride transfers. As a result there is an overall reduction in HDL core lipid content (Barter et al., 1990; Hesler et al., 1987; Rye et al., 1995). As outlined in Figure 5.2, HDL core lipids may also be depleted by hepatic lipase-mediated hydrolysis of HDL triglyceride. The reduction in HDL core lipid content which results from CETP activity decreases the size of HDL (Barter et al., 1990; Rye et al., 1995) and may be accompanied by a dissociation of lipid-free apoA-I from the HDL surface (Liang et al., 1994; Rye et al., 1995). CETP also remodels HDL by promoting particle fusion in a process which change both the size and the number of HDL particles (Rye et al., 1997). There are several potential fates for the lipid-free apoA-I which dissociates from core lipid-depleted HDL. It may be excreted through the kidney, and thus lost irreversibly from the plasma (Horowitz et al., 1992). It may enter the interstitial space and function as an acceptor of cell cholesterol. In this case HDL transports the cholesterol, via the lymphatics, back to the plasma as a component of newly assembled discoidal HDL particles (Bielecki et al., 1992; Forte et al., 1995; Hara and Yokoyama, 1992). Lipid-free apoA-I may also be formed into new discoidal HDL particles within the plasma by accepting phospholipids from other plasma lipoproteins in a process linked to the hydrolysis of triglyceride-rich lipoproteins (Redgrave and Small, 1979; Tall et al., 1982). And finally, as outlined above, it may be reincorporated into existing HDL particles which are expanding as a consequence of cholesteryl ester formation by LCAT (Liang et al., 1995; Liang et al., 1996). Hepatic Lipase The endothelial enzyme, hepatic lipase (HL), hydrolyses both triglycerides and phospholipids in HDL (Shirai et al., 1981). These processes are accompanied by a reduction in particle size and the dissociation of lipid-free apoA-I from the HDL (Clay et al., 1991; Perret et al., 1987). The size reduction is enhanced in HDL that have been enriched with triglyceride by pre-incubation with CETP and
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triglyceride-rich lipoproteins (Clay et al., 1992). Phospholipid Transfer Protein Phospholipid transfer protein (PLTP) promotes transfers of phospholipids between HDL and other plasma lipoproteins (Tollefson et al., 1988). The physiological importance of this process is uncertain. In the absence of other lipoprotein fractions, PLTP promotes extensive remodelling of HDL (Jauhiainen et al., 1993; Pussinen et al., 1995). The end result of this remodelling an increase in HDL size and the formation of very small, lipid poor, apoA-I-containing particles (Marques-Vidal et al., 1997; Pussinen et al., 1995; von Eckardstein et al., 1996). Recent studies have shown that remodelling by PLTP is enhanced significantly in triglyceride-enriched HDL (Rye et al., 1998). The precise mechanism of the PLTP-mediated remodelling of HDL is not well understood, but may involve an initial step of particle fusion (Lusa et al., 1996).
REGULATION OF APOLIPOPROTEIN-SPECIFIC HDL While there is compelling evidence showing that the size of HDL in plasma is regulated to a large extent by the plasma factors that mediate remodelling, there is less information about what regulates the distribution of apoA-I between A-I HDL and A-I/A-II HDL. Similarly, we do not know why most apoA-II is found in A-I/ A-II HDL rather than A-II HDL. In vivo kinetic studies indicate that the plasma residence time of apoA-I is significantly decreased when injected as a component of A-I HDL rather than as a component of A-I/A-II HDL (Rader et al., 1991). These studies suggest that the apoA-II in A-I/A-II HDL decreases the catabolism of the particles. They also raise the possibility that apoA-II may be important in regulating the concentration of apolipoprotein-specific HDL.
FUNCTIONS OF HDL Reverse Cholesterol Transport The best documented function of HDL relates to their involvement in reverse cholesterol transport (Figure 5.2). Reverse cholesterol transport is the term used to describe the pathway whereby cholesterol in peripheral tissues is transferred, via the plasma, to the liver from where it is either recycled or excreted from the body in bile.
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REVERSE CHOLESTEROL TRANSPORT (Transport of cholesterol from peripheral tissues to the liver) Step(i) Diffusion of unesterified cholesterol from cell membranes to HDL in th vascular space Step (ii) LCAT-mediated esterification of cholesterol in plasma HDL Step CETP-mediated transfer of cholesteryl esters from HDL to VLDL and (iii) Step Uptake of cholesteryl esters from VLDL, LDL and HDL by the liver (iv) Figure 5.2 Key steps in the pathway of reverse cholesterol transport.
This pathway involves several identifiable steps: (i) an initial step where cholesterol is transferred from cell membranes to HDL in the extracellular space, (ii) subsequent esterification of the cholesterol in the HDL by LCAT, (iii) the CETP-mediated transfer of newly formed cholesteryl esters from HDL to very low density lipoproteins (VLDL) and low density lipoproteins (LDL) and (iv) delivery of cholesteryl esters from HDL, VLDL and LDL to the liver.
Table 5.2 Extracellular acceptors of cell-derived cholesterol
Native lipoproteins
Lipid-free apolipoproteins
HDL2
apoA-I
HDL3
apoA-II
A-I HDL
apoA-IV
A-I/A-II HDL
apoC’s
Pre- HDL
apoE
Miscellaneous -cyclodextrins
-HDL
Most HDL subpopulations, some lipid-free apolipoproteins and the cyclic hepta-saccharides, -cyclodextrins, accept cholesterol from cell membranes (Table 5.2) (Barter, 1993; Pieters et al., 1994; Yancey et al., 1996). However, the preferred initial acceptor of cell cholesterol is the minor subpopulation of small, pre- -migrating, apoA-I-containing particles designated pre- 1 HDL (Castro and Fielding, 1988; Sviridov and Fidge, 1995). Pre- 1 HDL have been identified in plasma (Castro and Fielding, 1988) and interstitial fluid (Asztalos et al., 1993b;
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Lefevre et al., 1988; Reichl et al., 1991). The cell cholesterol that is incorporated into pre- 1 HDL appears subsequently in larger pre- migrating HDL (Castro and Fielding, 1988; Francone et al., 1989; Huang et al., 1993). It is uncertain whether this is a consequence of transfers of cholesterol from one HDL subpopulation to another, or the conversion of small pre- particles to larger pre- particles. The cholesterol in the larger pre- particles is esterified by LCAT (Francone et al., 1989). As the resulting cholesteryl esters are non-polar they are transferred to the hydrophobic core of the HDL, thus depleting the HDL surface of unesterified cholesterol. This generates a concentration gradient down which cholesterol flows from cells to the HDL. The cholesteryl esters which are formed by the action of LCAT on pre- migrating HDL are subsequently recovered in -migrating HDL (Castro and Fielding, 1988; Huang et al., 1993; Neary et al., 1991). This is probably a result of a conversion of the pre- particles, which do not contain cholesteryl esters into spherical, -migrating HDL which have a core of cholesteryl esters. Once in -migrating HDL, the cholesteryl esters are redistributed to other lipoprotein fractions in transfers mediated by CETP (Barter et al., 1985). This results in most of the cholesteryl esters which originate in HDL being delivered to the liver as a consequence of the receptor-mediated uptake of LDL (Brown and Goldstein, 1986) There is also evidence of direct uptake of a proportion of HDL cholesteryl esters by the liver (Glass et al., 1985). Other functions of HDL HDL have a number of functions which are unrelated to their role in plasma lipid transport (Table 5.3) but which may contribute to their antiatherogenic properties. HDL have been reported to be mitogenic (Tauber et al., 1980), to bind lipopolysaccharide (Levine et al., 1993), to stimulate endothelial cell movement (Murugesan et al., 1994), to inhibit the synthesis of platelet-activating factor by endothelial cells (Sugatani et al., 1996) and to protect erythrocytes against the generation of procoagulant activity (Epand et al., 1994). HDL also stimulate prostacyclin synthesis in endothelial cells (Fleisher et al., 1982; Tamagaki et al., 1996) by increasing the intracellular pH (Tamagaki et al., 1996). They also bind prostacyclin, thus prolonging its half-life (Yui et al., 1988). HDL also ameliorate the abnormal vasoconstriction which is a feature of early atherosclerosis, (Zeiher et al., 1994) and reduce epidermal growth factor-induced DNA synthesis in vascular smooth muscle cells (Ko et al., 1993). HDL possess antioxidant activity and have the capacity to inhibit adhesion of monocytes to endothelial cells. The last two functions may be of major importance in terms of the antiatherogenic properties of HDL.
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Table 5.3 Non-lipid transporting functions of HDL • Mitogenic • Bind lipopolysaccharide • Stimulate endothelial cell movement • Inhibit synthesis of platelet activating factor • Protect erythrocytes against procoagulant activity • Stimulate endothelial prostacyclin synthesis • Prevent abnormal vasoconstriction • Reduce epidermal growth factor synthesis in vascular smooth muscle cells • Inhibit effects of oxidized LDL (monocyte transmigration, endothelial cell death and adhesion of monocytes to endothelium) • Inhibit oxidation of LDL • Inhibit expression of endothelial cell adhesion molecules
Antioxidant Properties of HDL HDL inhibit the transmigration of monocytes and the cytotoxicity which is induced by oxidised LDL (Navab et al., 1991; Hessler et al., 1979; Suc et al., 1997). They also inhibit the adhesion of monocytes to endothelial cells which is induced by oxidised LDL (Maier et al., 1994). ApoA-I prevents the cytotoxicity which is mediated by oxidised LDL (Suc et al., 1997). It is likely that the antioxidant properties of HDL are a consequence of their ability to inhibit directly the oxidation of LDL both in vitro (Decossin et al., 1995; Kunitake et al., 1992a; Ohta et al., 1989; Mackness et al., 1993a; Mackness and Durrington 1995; Parthasarathy et al., 1990) and in vivo (Klimov et al., 1993). At present the mechanism of this inhibition is not known. One possibility is the involvement of paraoxonase, an HDL component which prevents lipoperoxide accumulation in LDL (Mackness et al., 1991; Mackness et al., 1993b; Watson et al., 1995). Inhibition of Endothelial Cell Adhesion Molecule Expression by HDL HDL inhibits in a concentration-dependent manner the cytokineinduced expression of VCAM-1, ICAM-1 and E-selectin in cultured endothelial cells (Calabresi et al., 1997; Cockerill et al., 1995). The inhibition of VCAM-I and E-selectin protein expression by HDL is paralleled by significant reductions in the steady-state mRNA levels of
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the adhesion molecules (Cockerill et al., 1995). To determine which components of HDL inhibit endothelial cell adhesion molecule expression studies have been carried out with reconstituted HDL (rHDL) containing purified apoA-I and phosphatidylcholine (Calabresi et al., 1997; Cockerill et al., 1995), as well as with the individual phosphatidylcholine and apolipoprotein constituents. Small unilamellar vesicles containing only phosphatidylcholine do not inhibit adhesion molecule expression and there is only minor inhibition with lipid-free apoA-I. However, when the apoA-I and phosphatidycholine are combined into discoidal rHDL, there is substantial inhibition. This indicates that the inhibition is not caused by contaminating factors that co-isolate with native HDL. Other studies with discoidal rHDL have also demonstrated that the inhibitory effect is not specific to apoA-I. In those experiments adhesion molecule expression was inhibited by rHDL containing apoA-II and phosphatidylcholine (Calabresi et al., 1997). The mechanism by which HDL inhibit the expression of endothelial cell adhesion molecules is uncertain. It may involve the interaction of HDL with cell surface receptors which bind both A-I HDL and A-II HDL (Graham and Oram, 1987; Tozuka and Fidge, 1989; Vadiveloo and Fidge, 1992).
HDL AND ATHEROSCLEROSIS IN EXPERIMENTAL ANIMALS The likelihood that HDL do have direct antiatherogenic properties has been greatly strengthened by recent studies of transgenic mice. Overexpression of the human apoA-I gene in transgenic mice increases the concentration of HDL cholesterol and confers dramatic protection against both diet-induced atherosclerosis (Rubin et al., 1991) and the spontaneous atherosclerosis that develops in apoE-deficient mice (Paszty et al., 1994; Plump et al., 1994). In contrast, mice overexpressing both human apoA-I and apoA-II (Schultz et al., 1993) or human apoA-II alone (Schultz and Rubin, 1994) are less protected compared to apoA-I transgenic mice, even though the different groups have comparable HDL cholesterol levels. In addition, mice overexpressing murine apoA-II develop spontaneous atherosclerosis (Warden et al., 1993). The mechanism underlying these variations is not known. Nor is it known how the transgenic animal studies relate to the situation in human subjects.
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MECHANISM OF INHIBITION OF ATHEROSCLEROSIS BY HDL Overview of Atherogenesis Several highly plausible schemes of atherogenesis have been postulated. In each case lipid deposition (both intracellular and extracellular), cell proliferation and the de
Figure 5.3 Schematic diagram showing points at which HDL may inhibit the development of atherosclerosis.
position of connective tissue are prominent features. A postulated sequence of events of the early stages of the development of atherosclerosis, and the points at which HDL may inhibit it, are presented schematically in Figure 5.3. According to this scheme, one of the earliest events in atherogenesis is the adhesion of monocytes to endothelial cells in a process involving the cell-surface expression of the adhesion molecules VCAM-1, ICAM-1 and E-selectin (Charo, 1992). Another early event is the entry of LDL into the intima. Oxidative modification of intimal LDL (Steinberg et al., 1989) sets in train several events, including the expression by endothelial cells of a monocyte chemotactic protein which attracts monocytes into the intima (Navab et al., 1991). Within the intima monocytes differentiate into macrophages which expresses a variety of factors, including growth factors (Ross, 1993) and cytokines (Nathan, 1987). One effect of the
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cytokines is to stimulate endothelial cells to express adhesion molecules (Springer, 1990). This, in turn, leads to the adherence of more blood monocytes to the endothelial cell surface. Macrophages also bind and internalise the modified LDL in a process which converts the macrophage into a foam cell (Ross and Aguis, 1992). The combination of an accumulation of foam cells and a proliferation of smooth muscle cells in response to growth factors released from macrophages results in the formation of a fatty streak. This lesion is widely regarded as the precursor of the mature atherosclerotic plaque. HDL-Mediated Cell Cholesterol Efflux There is a widespread view that HDL inhibit the development of atherosclerosis by virtue of their ability to extract cholesterol from cells in the first step of reverse cholesterol transport. According to this view, HDL counteract the atherogenic effects of LDL by removing cholesterol from cells in the arterial intima and preventing the formation of foam cells. In support of this view, it has been clearly shown in vitro that HDL promote efflux of cholesterol from cholesterol loaded cells (Fielding and Fielding, 1995) and also reduce the cholesterol content of foam cells (Miyazaki et al., 1992). Furthermore, it has been reported recently that the ability of serum to promote cholesterol efflux from a rat hepatoma cell line is related directly to the serum concentration of HDL cholesterol (de la Llera Moya et al., 1994). However, there is also evidence which does not support the point of view that a high level of HDL protects against CHD by enhancing the rate of reverse cholesterol transport. The plasma concentration of preHDL, the preferred acceptor of cell cholesterol, is elevated in subjects with hypertriglyceridaemia (Ishida et al., 1987) in whom the concentration of the total HDL fraction tends to be low. In vitro, formation of small, pre- -migrating HDL is promoted by the interaction of HDL with CETP (Kunitake et al., 1992b), in a process which also tends to reduce the concentration of HDL (Marotti et al., 1992). Thus, circumstances which increase pre- HDL levels tend to be those in which the concentration of the total HDL fraction is reduced, rather than increased. In other words, reverse cholesterol transport may be increased when the HDL level is low rather than high. Consideration of other steps of reverse cholesterol transport also raises doubts about the role of this pathway in protecting against CHD. The second major step in reverse cholesterol transport is the esterification of cholesterol on the surface of HDL in the reaction catalysed by LCAT (Glomset, 1968). The resulting cholesteryl esters move into the HDL core, leaving the surface of the particle depleted of cholesterol. This creates a concentration gradient which promotes
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further transfers of cholesterol from cell membranes to HDL. An increase in the rate of LCAT-catalysed cholesterol esterification therefore enhances the rate of reverse cholesterol transport. It is perhaps surprising, therefore, to find that the rate of the LCAT reaction in plasma correlates negatively, rather than positively, with the concentration of HDL cholesterol (Wallentin and Vikrot, 1975). Thus, subjects with low concentrations of HDL cholesterol, especially those with elevated levels of plasma triglyceride (Nestel and Monger, 1967), tend to have high rather than low rates of plasma cholesterol esterification and, by inference, high rather than low rates of reverse cholesterol transport. Once again, this observation does not support the proposition that the increased coronary risk in subjects with low levels of HDL cholesterol is secondary to a reduced rate of reverse cholesterol transport. The possibility must therefore be considered that the inverse relationship between HDL concentration and the development of CHD is mediated by a mechanism which may be independent of the involvement of HDL in reverse cholesterol transport. Antioxidant Properties of HDL There is strong circumstantial evidence that oxidative modification of LDL within the arterial intima is one of the key, early events in atherogenesis. As described above, the well documented ability of HDL to inhibit the oxidative modification of LDL has the potential to contribute to the anti-atherogenicity of these lipoproteins. The importance of this process in vivo remains to be determined. HDL-Mediated Inhibition of Adhesion Molecule Expression As outlined above, an early event in the development of atherogenesis is the adhesion of monocytes to the luminal surface of endothelial cells in a process mediated by a range of adhesion molecules (Charo, 1992). HDL have been reported to inhibit the expression of the endothelial cell adhesion molecules, VCAM-1, ICAM-1 and E-selectin (Cockerill et al., 1995; Calabresi et al., 1997). This function of HDL clearly has the potential to be highly anti-atherogenic. However, the extent to which it explains the capacity of HDL to protect against the development of CHD in human subjects remains to be determined. It also remains to be determined whether this effect of HDL on adhesion molecule expression is unrelated to lipid metabolism or whether it is secondary to an HDL-mediated alteration in membrane lipid composition.
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CONDITIONS ASSOCIATED WITH ALTERED HDL CONCENTRATION These conditions include both the HDL deficiency arising from the genetic defects which inhibit HDL apolipoprotein synthesis (Funke et al., 1991; Lackner et al., 1993; Ng et al., 1994) and deficiencies of proteins such as LCAT (Carlson and Holmquist, 1985; Funke et al., 1993; Klein et al., 1992; Wiebusch et al., 1995) as well as the elevations of HDL that are secondary to deficiencies of CETP (Akita et al., 1994; Bisgaier et al., 1991; Inazu et al., 1990; Koizumi et al., 1991). Low levels of HDL are a well recognised component of a variety of conditions in which there is a general dyslipidaemia. These include genetic conditions such as familial hypertriglyceridaemia (Brunzell et al., 1983), familial combined hyperlipidaemia (Grundy et al., 1987) and metabolic disorders such as non-insulin-dependent diabetes mellitus (Howard, 1987) and the metabolic syndrome which is characterised by truncal obesity, insulin resistence and mild hypertension. There is also a dyslipidaemia that, in addition to a low HDL level, includes hypertrigyceridaemia and an LDL fraction which tends to be smaller and denser than normal (Reaven, 1991). The level of HDL may also be reduced by drugs such as diuretics and betablockers (England et al., 1980) and the anti-oxidant, probucol (LeLorier et al., 1977). The reasons why these drugs lower HDL levels in many of these conditions is not understood. In some cases, such as hypertriglyceridaemia, it probably relates to an increased rate of transfer of cholesteryl esters from HDL to other lipoproteins (Nestel and Monger, 1967).
HDL AND ATHEROSCLEROSIS IN HUMANS Population Studies An inverse relationship between the concentration of HDL cholesterol and the development of premature coronary heart disease (CHD) has been observed in case-control studies (Enger et al., 1979; Miller et al., 1977), large-scale prospective studies (Goldbourt and Medalie, 1979; Gordon et al., 1977; Jacobs et al., 1990) and studies of subjects with existing CHD (Miller et al., 1992; Pekkanen et al., 1990). One study found that up to 40% of patients with documented CHD under the age of 60 had low levels of HDL cholesterol (Genest et al., 1992; Genest et al., 1993). In more than half of these studies the low HDL level was a component of a familial dyslipidaemia. Overall, these human studies
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suggest that coronary risk is increased by 2–3% for every 1% reduction in HDL cholesterol level. In one large study, the concentration of HDL cholesterol was also an inverse predictor of total mortality (Wilson et al., 1988). While human population studies have consistently demonstrated that a low concentration of HDL cholesterol is associated with an increased risk of CHD, studies of patients with genetic conditions in which the concentration of HDL cholesterol is either very low or undetectable have been less clear. Some of these genetic disorders are associated with the expected increase in the incidence of premature CHD (Goldstein et al., 1973; Hargreaves et al., 1992; Kannel, 1985); some, however, are not (Assmann et al., 1990; Schaefer, 1984; SerfatyLacrosneire et al., 1994). The explanation for the varying susceptibility is not known but may relate to differences in the kinetics of specific HDL subpopulations (Rader et al., 1994). A further element of confusion has been introduced by the observation that mice genetically engineered to lack apoA-I, and are therefore deficient in HDL, do not have an increased susceptibility to atherosclerosis (Li et al., 1993). These inconsistencies have raised the possibility that a low HDL cholesterol level in patients with premature CHD may be an epiphenomenon, rather than a cause of the disease. In other words, the CHD may be caused by some other factor which happens to correlate inversely with the level of HDL cholesterol. For example, a low concentration of HDL cholesterol is common in subjects in whom hypertriglyceridaemia and small, dense LDL are present (Austin, 1991). Since both triglyceride-rich lipoproteins (Austin, 1991) and small, dense LDL (Austin et al., 1988) may be atherogenic, an inverse relationship between the concentration of HDL cholesterol and the development CHD may reflect no more than an increase in the levels of triglyceride-rich lipoproteins or small dense LDL. This proposition has received support from observations that the presence (Steiner et al., 1987), severity (Reardon et al., 1985; Tatami et al., 1981) and rate of progression (Krauss et al., 1987) of CHD correlate positively with the concentration of intermediate density lipoproteins which, in turn, correlate inversely with the concentration of HDL cholesterol (Krauss et al., 1987). Thus, the human population studies of the relationship between HDL and CHD do not prove cause and effect. Nor, however, do they exclude such a possibility. Evidence from human population studies and from transgenic animal studies (see below) have raised the possibility that A-I HDL may be superior to both A-I/A-II HDL and A-II HDL in their ability to protect against atherosclerosis (Amouyel et al., 1993; Schultz et al., 1993). It has also been suggested that populations of larger HDL may be more protective than smaller HDL (Miller, 1987; Robinson et al., 1987). More recently, the possibility has been raised that minor
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subpopulations of discoidal, pre- -migrating HDL may be superior to spherical, -migrating HDL in their ability to inhibit atherosclerosis. This proposition is based on the observation that pre- -migrating A-I HDL are preferred over -migrating A-I HDL as the initial acceptors of cell cholesterol (Castro and Fielding, 1988). While superficially appealing, this view has serious flaws, as discussed above. For example, the concentration of pre- HDL is increased and that of HDL is decreased in patients with elevations of plasma triglyceride and reduced levels of HDL cholesterol. This profile is associated with an increased, rather than a decreased, CHD risk and implies that -, rather than pre- HDL, have the superior ability to inhibit atherosclerosis. The potential importance of apolipoprotein-specific subpopulations of HDL as predictors of CHD has been addressed in a study of adolescents with parents who suffered a myocardial infarction under the age of 55 (Amouyel et al., 1993). When compared with matched controls who did not have a family history of premature CHD, those with a positive family history had lower concentrations of HDL cholesterol, apoA-I and A-I HDL than the controls. The observation that there was no difference in concentrations of A-I/A-II HDL between the two groups raised the possibility that the A-I HDL subpopulation may represent the fraction which is cardioprotective. It should be emphasised, however, that there have been no prospective studies of the relationship between apolipoprotein-specific subpopulations of HDL and CHD. Nor are there any intervention studies assessing the impact of changing apolipoprotein-specific subpopulations of HDL in human subjects. Such intervention studies are currently not feasible in humans because there is no known therapy which selectively increases the concentrations of apolipoproteinspecific HDL subpopulations. Overall, the evidence linking protection against CHD to apolipoprotein-specific HDL subpopulations in humans is conflicting. The relationship between HDL size and protection against CHD is also unclear. Human case control studies have shown that the inverse relationship between HDL cholesterol concentration and CHD is a function of the concentration of the HDL2 subfraction (Miller, 1987; Robinson et al., 1987). In another study which looked at the relationship of HDL subpopulations to CHD, both the severity and rate of progression of coronary lesions correlated significantly and inversely with the concentration of HDL2b in patients with normal triglyceride levels, although there was no such significant correlation in hypertriglyceridaemic patients (Johansson et al., 1991). However, other studies have found that the development of CHD correlates significantly and inversely with the concentrations of both HDL2 and HDL3 (Buring et al., 1992; Miller, 1987; Stampfer et al., 1991). It is unclear whether the sie or density of HDL per se influence their antiatherogenic properties.
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Intervention Studies There have been no reports of clinical trials designed specifically to determine whether raising the level of the total HDL fraction (let alone specific HDL sub-populations) translates into a reduced incidence of CHD. However, many intervention studies have addressed more general questions relating to plasma lipids and CHD. In several of these studies, treatment-induced elevations of HDL cholesterol were found to be independently associated with both a reduction in CHD events (Gordon et al., 1986; Manninen et al., 1988) and decreased progression of coronary atherosclerosis (Brown et al., 1990; Mack and Blankenhorn, 1991). The Lipid Research Clinics Primary Prevention Trial was a doubleblind, placebo-controlled study of hypercholesterolaemic men in which cholestyramine was used as the active agent (Gordon et al., 1986). A reduction in CHD events correlated positively with changes in LDL cholesterol levels and negatively with changes in HDL cholesterol. For every 1 % increase in the concentration of HDL cholesterol there was an 0.6% reduction in CHD events which was independent of the changes in LDL levels (Gordon et al., 1986). An even more powerful result was observed in the Helsinki Heart Study which used gemfibrozil as the active agent. In this study, a 1 % increase in HDL cholesterol was associated with a 2–3% decrease in CHD events. This result was again independent of changes in LDL levels (Manninen et al., 1988). Subgroup analysis of the Helsinki Heart Study revealed that the beneficial effect of gemfibrozil was greatest in subjects with initially low levels of HDL cholesterol, especially those in whom the ratio of LDL/HDL cholesterol was>5 in combination with a plasma triglyceride level>2.3mmol/L (Manninen et al., 1992). The benefit of raising HDL levels has also been observed in secondary prevention studies, although the numbers of subjects studied is small and conclusions are limited. There are, however, two angiographic studies in which an increase in HDL concentration is associated with a reduced rate of lesion progression (Brown et al., 1990; Mack and Blankenhorn, 1991) or even regression (Brown et al., 1990). There is an obvious need for prospective primary and secondary prevention studies in subjects with low levels of HDL cholesterol. Such studies should be designed specifically to test the hypothesis that raising the level of HDL cholesterol translates into a reduction in CHD events. If possible, they should also address the issue of the relationship between CHD and specific HDL subpopulations.
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CONCLUSION There is now a large body of information about the structure and function of HDL. However, understanding of how HDL and their subpopulations are regulated is still limited. There is clear evidence that HDL protect against coronary disease, although the mechanism of the protection is not known. While transgenic animal studies have shown that a primary increase in HDL inhibits the development of atherosclerosis, it remains to be proven that increasing HDL levels in humans has a comparable effect. Similarly, while transgenic animal studies suggest that A-I HDL are superior to A-I/ A-II HDL as inhibitors of atherosclerosis, direct evidence of this is not yet available in human subjects. Thus, there are several challenges ahead: (i) there is a need to determine whether there are differences in the anti-atherogenicity of specific HDL subpopulations; (ii) there is a need to understand how the specific subpopulations are regulated so that strategies can be devised to increase their concentrations selectively; (iii) there is a need to understand the precise mechanism by which HDL inhibit the development of atherosclerosis; (iv) there is a need for intervention studies to determine whether, in human subjects, a primary increase in the concentration of the total HDL fraction or any of its subpopulations inhibits atherogenesis and translates into a reduction in the development of clinical coronary heart disease. References Akita, H., Chiba, H., Tsuchihashi, K., Tsuji, M., Kumagai, M., Matsuno, K. et al. (1994). Cholesteryl ester transfer protein gene: two common mutations and their effect on plasma high-density lipoprotein cholesterol content. J.Clin. Endocr. Metab. , 79 , 1615– 1618 Amouyel, P., Isorez, D., Bard, J.-M., Goldman, M., Level, P., Zylberg, G. et al. (1993). Parenteral history of early myocardial infarction is associated with decreased levels of lipoparticle AI in adolescents. Arterioscler. Thromb. , 13 , 1640–1644 Assmann, G., Schmitz, G., Funke, H. and von Eckardstein, A. (1990). Apolipoprotein A-I and HDL deficiency. Curr. Opin. Lipidol. , 1 , 110–115. Asztalos, B.F., Sloop, C.H., Wong, L. and Roheim, P.S. (1993a). Twodimensional electrophoresis of plasma lipoproteins: recognition of new apoA-I-containing subpopulations. Biochim. Biophys. Acta. , 1169 , 291–300. Asztalos, B.F., Sloop, C.H., Wong, L. and Roheim, P.S. (1993b). Comparison of apoA-I-containing subpopulations of dog plasma and
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prenodal peripheral lymph: evidence for alteration in subpopulations in the interstitial space. Biochim. Biophys. Acta. , 1169 , 301–304. Austin, M.A. (1991). Plasma triglyceride and coronary heart disease. Arterioscler. Thromb. , 11 , 2–14. Austin, M.A., Breslow, J.L., Hennekens, C.H., Buring, I.E., Willett, W.C. and Krauss, R.M. (1988). Low-density lipoprotein subclass patterns and risk of myocardial infarction. J.Amer. Med. Assoc. , 260 , 1917–1921. Barter, P.J. (1993). High-density lipoproteins and reverse cholesterol transport. Curr. Opin. Lipidol. , 4 , 210–217. Barter, P.J., Chang, L.B.F., Newnham, H.H., Rye, K.-A. and Rajaram, O.V. (1990). The interaction of cholesteryl ester transfer protein and unesterified fatty acids promotes a reduction in the particle size of high density lipoproteins. Biochim. Biophys. Acta. , 1045 , 81–89. Barter, P.J., Hopkins, G.J. and Calvert, G.D. (1982). Transfers and exchanges of esterified cholesterol between plasma lipoproteins. Biochem. J. , 208 , 1–7. Barter, P.J., Hopkins, G.J. and Gorjatschko, L. (1985). Lipoprotein substrates for plasma cholesterol esterification: influence of particle size and the high density lipoprotein subfraction-3. Atherosclerosis , 58 , 97–107. Bekaert, E.D., Alaupovic, P., Knight-Gibson, C., Norum, R.A., Laux, M.J. and Ayrault-Jarrier, M. (1992). Isolation and partial characterization of lipoprotein A-II (LP-A-II) particles of human plasma. Biochim. Biophys. Acta. , 1126 , 105–113. Bielecki, J.K., Johnson, W.J., Weinberg, R.B., Glick, J.M. and Rothblat, G.H. (1992). Efflux of lipid from fibroblasts to apolipoproteins: dependence on elevated levels of cellular unesterified cholesterol. J.Lipid Res. , 33 , 1699–1709. Bisgaier, C.L., Siebenkas, M.V., Brown, M.L., Inazu, A., Koizumi, J., Mobuchi, H. 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 , 21–33. Blanche, P.J., Gong, E.L., Forte, T.M. and Nichols, A.V. (1981). Characterization of human high-density lipoproteins by gradient gel electrophoresis. Biochim. Biophys. Acta. , 665 , 408–419. Brown, G., Albers, J.J., Fisher, L.D., Schaeffer, S.M., Lin, J.-T., Kaplan, C. et al.. (1990). Regression of coronary artery disease as a result of intensive lipid-lowering therapy in men with high levels of apolipoprotein B . N.Engl. J.Med. , 323 , 1289–1298. Brown, M.S. and Goldstein, J.L. (1986). A receptor-mediated pathway for cholesterol homeostasis. Science , 232 , 34–47. Brunzell, J.D., Albers, J.J., Chait, A., Grundy, S.M., Groszek, E. and McDonald, G.B. (1983). Plasma lipoproteins in familial combined hyperlipidaemia and monogenic familial hypertriglyceridemia. J.Lipid Res. , 24 , 147–155. Buring, J.E., O’Connor, G.T., Goldhaber, S.Z., Rosner, B., Herbert, P.N., Blum, C.B. et al. (1992). Decreased HDL2 and HDL3
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Salvayre, A. (1997). HDL and apoA prevent cell death of endothelial cells induced by oxidized LDL. Arterioscler. Thromb. Vasc. Biol. , 17 , 2158–2166. Sugatani, J., Miwa, M., Komiyama, Y. and Ito, S. (1996). High-density lipoprotein inhibits the synthesis of platelet-activating factor in human vascular endothelial cells. J.Lipid Mediat. Cell. Signal. , 13 , 73–88. Sviridov, D. and Fidge, N. (1995). Pathways of cholesterol efflux from human hepatoma cells. Biochim. Biophys. Acta. , 1256 , 210–220. Tall, A.R., Abreu, E. and Shuman, J. (1983). Separation of a plasma phospholipid transfer protein from cholesteryl ester/phospholipid exchange protein. J.Biol. Chem. , 258 , 2174–2180. Tall, A.R., Blum, C.B., Forester, G.P. and Nelson, C.A. (1982). Changes in the distribution and composition of plasma high density lipoproteins after ingestion of fat. J.Biol. Chem. , 257 , 198–207. Tamagaki, T., Sawada, S., Imamura, H., Tada, Y., Yamasaki, S., Toratani, A. et al. (1996). Effects of high-density lipoprotein on intracellular pH and proliferation of human vascular endothelial cells. Atherosclerosis , 123 , 73–82. Tatami, R., Mabuchi, H., Ueda, K., Haba, T., Kametani, T., Ito, S. et al. (1981). Intermediate-density lipoprotein and cholesterol-rich very low density lipoprotein in angiographically determined coronary artery disease. Circulation , 64 , 1174–1184. Tauber, J.-P., Cheng, J. and Gospodarowicz, D. (1980). Effect of high and low densty lipoproteins on proliferation of cultures bovine vascular endothelial cells. J.Clin. Invest. , 66 , 696–708. Tollefson, J.H., Ravnik, S. and Albers, J.J. (1988). Isolation and characterization of a phospholipid transfer protein (LTP-II) from human plasma. J.Lipid Res. , 29 , 1593–1602. Tozuka, M. and Fidge, N. (1989). Purification and characterization of two high density lipoprotein binding proteins from rat and human liver. Biochem. J. , 261 , 239–244. Vadiveloo, P.K. and Fidge, N.H. (1992). The roles of apoproteins AI and AII in binding of high-density lipoprotein3 to membranes derived from bovine aortic endothelial cells. Biochem. J. , 284 , 145– 151. von Eckardstein, A., Jauhiainen, M., Huang, Y., Metso, J., Langer, C, Pussinen, P. et al. (1996). Phospholipid transfer protein mediated conversion of high density lipoproteins generates pre- 1 HDL. Biochim. Biophys. Acta. , 1301 , 255–262. Wallentin, L. and Vikrot, O. (1975). Lecithin: cholesterol acyl transfer in plasma of normal persons in relation to lipid and lipoprotein concentration. Scand. J.Clin. Lab. Invest. , 35 , 669–676. Warden, C.H., Hedrick, C.C., Qiao, J.-H., Castellani, L.W. and Lusis, A.J. (1993). Atherosclerosis in transgenic mice overexpressing apolipoprotein A-II. Science , 261 , 469–471. Watson, A.D., Berliner, J.A., Hama, S.Y., La-Du, B.N., Faull, K.F., Fogelman, A.M. et al. (1995). Protective effect of high density
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lipoprotein associated paraoxonase. Inhibition of the biological activity of minimally oxidized low density lipoprotein. J.Clin. Invest. , 96 , 2882–2891. Wiebusch, H., Cullen, P., Owen, J.S., Collins, D., Sharp, P.S., Funke, H. et al. (1995). Deficiency of lecithin: cholesterol acyltransferase due to compound heterozygosity of two novel mutations (Gly33Arg and 30 bp ins) in the LCAT gene. Hum. Mol. Genetics , 4 , 143–145. Wilson, P.W.F., Abbott, R.D. and Castelli, W.P. (1988). High density lipoprotein cholesterol and mortality. The Framingham Heart Study. Arteriosclerosis , 8 , 737–741. Yancey, P.G., Rodrigueza, W.V., Kilsdonk, E.P.C., Stoudt, G.W., Johnson, W.J., Phillips, M.C. et al., (1996). Cellular cholesterol efflux mediated by cyclodextrins: demonstration of kinetic pools and mechanism of efflux. J.Biol. Chem. , 271 , 16026–16034. Yui, Y., Aoyama, T., Morishita, H., Takahashi, M., Takatsu, Y., Kawai, C. et al. (1988). Serum prostacyclin stabilising factor is identical to apolipoprotein A-I (apoA-I). J.Clin. Invest. , 82 , 803– 807. Zeiher, A.M., Schachinger, V.and Hohnloser, S.H. (1994). Coronary atherosclerotic wall thickening and vascular reactivity in humans. Elevated high-density lipoprotein levels ameliorate abnormal vasoconstriction in early atherosclerosis. Circulation , 89 , 2525– 2532.
6 Chylomicron Remnants and Atherosclerosis John C.L.Mamo and Spencer D.Proctor University Department of Medicine, Medical Research Foundation Building, Rear of 50 Murray Street, Perth WA 6000, Australia
Atherosclerosis is a complex, progressive disease characterised by the deposition of lipid derived from plasma lipoproteins. Epidemiological studies suggest that dietary fats can accelerate vascular disease. However, the mechanism was thought to be indirect, because chylomicrons were considered too large to penetrate arterial tissue. Recent studies have found that arterial influx of chylomicrons occurs once the particles are converted to smaller cholesterol rich remnants, supporting a causal role in atherogenesis. Chylomicron remnants become trapped within the subendothelial space when their concentration in plasma is elevated and arterial delivery exceeds efflux. Initiation of an inflammatory response may lead to unabated uptake by macrophages of chylomicron remnants via phagocytic like pathways, which result in lipid accumulation, generation of free radicals and decreased cell viability. Clinical studies have shown that plasma chylomicron remnant concentration is increased in a number of primary and secondary dyslipidaemic states. Furthermore, strong evidence that chylomicron remnants are atherogenic comes from recent studies which have identified postprandial dyslipidaemia in normolipidaemic subjects with coronary artery disease. Less clear is whether lipid lowering strategies following dietary and/or drug intervention will reduce chylomicron remnant concentration sufficiently, to attenuate or regress atherosclerosis. Further studies to determine the key regulatory sites of chylomicron assembly and secretion are required
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to compliment strategies aimed at increasing their clearance from plasma. Ingested fats and lipophilic substances can influence chylomicron synthesis, clearance, arterial retention and inflammation. It may be that targeting specific dietary habits could lead to further reductions in morbidity and mortality associated with atherosclerosis. KEYWORDS: Chylomicron, postprandial, atherosclerosis, metabolism.
INTRODUCTION The aqueous environment of plasma dictates that hydrophobic lipids are transported in complex macromolecules referred to as lipoproteins. All lipoproteins are comprised of a core of hydrophobic lipids, predominantly esterified cholesterol and triglyceride, encompassed by a monolayer of amphiphylic lipids including unesterified cholesterol and phospholipids. A series of proteins containing lipid binding domains (referred to as apolipoproteins) bind to the lipoprotein complex and essentially dictate the catabolism of the particle by acting as co-factors for enzymes, or ligands for cell membrane binding sites (Mahley et al., 1984). The simplistic definition of lipoproteins suggests homogeneity, however, of the spectrum of plasma lipoproteins, chylomicrons are perhaps uniquely heterogeneic, because unlike other lipoproteins they are the vehicle by which dietary hydrophobic substances are transported into circulation. In addition to ingested fats, chylomicrons mediate the transport of fat soluble vitamins, hydrophobic antioxidants such as atocopherol and beta carotene (Traber et al., 1990) and lipophylic chemicals including therapeutic compounds (Cheema et al., 1987; Mamo et al., 1993; Roth et al., 1993). In fact chylomicrons might even be a vehicle for delivery of microbial lipopolysaccharides (Read et al., 1993). Chylomicron size and lipid composition will also vary widely depending on the nature of lipids ingested (Redgrave, 1983). Therefore, it would seem reasonable to suggest that the putative atherogenicity of chylomicrons may to some extent be dependent on the compliment of lipophylic substances which could influence their catabolism. There are many confounding factors that may impact on chylomicron atherogenicity. Collectively, one might suspect that chylomicrons contribute to atherosclerosis when arterial retention is increased as a consequence of overproduction or delayed clearance or when the chylomicrons possess pro-inflammatory properties. Presently, there are insufficient data to enable the unequivocal identification of chylomicron particles that are proatherogenic. Hence, articles which have reviewed the metabolism of chylomicrons in relation to vascular disease, have by
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necessity had to consider these lipoproteins as a singular entity. Despite these significant shortcomings, there is now an accumulating body of evidence suggesting that chylomicrons, or at least their remnants, are likely to play a significant role in atherogenesis (Karpe et al., 1994; Mamo, 1995; Steiner, 1993). There is general consensus amongst lipoprotein physiologists that chylomicron remnants are atherogenic, although many believe that this is a consequence of modulating the metabolism of other pro- or antiatherogenic lipoproteins and not because the particles are atherogenic per se. While this is a possibility, this review will be limited to a consideration of whether chylomicrons directly contribute to arterial cholesterol accumulation and vascular dysfunction.
CHYLOMICRONS: ORIGIN AND SECRETION Chylomicrons are synthesised exclusively in the epithelial cells of the small intestine (Redgrave, 1988). Chylomicrons are differentiated from hepatically derived lipoproteins based on the isoform of one of the proteins which associates with these particles. Apolipoprotein B is a structural protein found with larger lipoproteins synthesised by the intestine and liver. The two isoforms of apolipoprotein B, namely B48 and B100, are the products of the same gene. In the human intestine, epithelial cells possess an mRNA editing step, which inserts a premature stop codon approximately halfway through translation of the apoB100 gene (Chan et al., 1997). The abridged version of apoB100, known as apoB48, serves as a useful marker for distinguishing intestinal lipoproteins from hepatically derived particles. Character isation of nascent chylomicrons isolated from lymph suggest that apolipoprotein B48 is the predominant form of the protein synthesised by the intestine, although apoB100 containing lipoproteins can also be found in significant quantities in lymph. Moreover, in cultures of immortalised human intestinal cells, the predominant isoform of apoB secreted can be readily changed by modulating the culturing conditions or substrate supplied (van Greevenbroek et al., 1996). Therefore, the possibility exists that in vivo, certain dietary components or genotypes may lend to the preferential production of intestinal lipoproteins containing apoB100. Although greater production of intestinal B100 relative to B48 remains to be demonstrated in man, such a possibility needs to be fully explored if we are to delineate the putative role of chylomicrons in atherogenesis. Several studies have now suggested that each chylomicron particle contains a copy of apoB48 and that the protein is an obligatory component for secretion (Martins et al., 1994). Consistent with this hypothesis is evidence of substantial enterocyte steatosis in subjects
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with abetalipoproteinaemia, where a defect in apoB biogenesis occurs (Schonfeld, 1995). On the other hand, the intestine appears to have the capacity to also synthesise small dense lipoproteins not unlike high density lipoproteins (HDL) and which probably don’t contain apoB48. The accumulation of lipid within enterocytes of subjects with abetalipoproteinaemia suggest that apoB containing lipoproteins are quantitatively the most important intestinally derived lipoprotein responsible for the transport of dietary derived fat. However, basal rates of synthesis of non-apoB containing lipoproteins by enterocytes may over a period of time equate to significant delivery of endogenous lipid. Clearly, chylomicrons synthesised without apoB, or with apoB100, would significantly confound our interpretation of the atherogenicity of hepatic versus intestinal derived lipoproteins. In fact, either scenario would require a re-definition of what we mean by the term “chylomicron”. Limited studies suggest that chylomicrons synthesised from different regions of the intestine may possess different metabolic properties (Caspary, 1992). Heterogeneity, of chylomicrons synthesised from different regions of the intestine may simply reflect the nature of substrate available for incorporation into the lipoprotein complex. Critically, the characterisation of lymph chylomicrons may represent a number of specific sub-population of particles, some of which may be particularly atherogenic and others which may be relatively “inert” (Karpe, 1997). Chylomicron metabolism may be influenced as a consequence of genetic and environmental factors. There is evidence that gender and metabolic status significantly influence chylomicron metabolism (Chen et al., 1996). The impact of ethnicity is presently not known. Greater rates of coronary disease in some populations may reflect a predisposition to over-secrete or poorly clear chylomicrons (Mamo et al., 1997; Zilversmit, 1995). Obviously, this paucity of knowledge necessitates a number of assumptions when considering the relative importance of chylomicrons as vehicles of circulating lipid compared to other lipoproteins and when reviewing their potential role in coronary disease. On a typical Western diet man consumes approximately 250–500mg of cholesterol per day (Schaefer et al., 1995). Bile sterols reabsorbed following emulsification of dietary lipid might provide an additional 1000mg of chylomicron cholesterol daily. When bile and dietary cholesterol are combined, the mass of sterols transported in the circulation associated with chylomicrons appears to be insignificant compared to that of other lipoproteins. However, studies in animal models suggest that chylomicrons are synthesised continuously, even in the absence of dietary fat (Martins et al., 1994; Redgrave, 1983). The basal secretion of chylomicron cholesterol in man is not known,
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although an estimate can be derived based on the fractional rate of clearance from plasma under “steady state” conditions and the distributional analysis of plasma cholesterol. In the fasted state (assumed to be at steady state), 2–5% of total plasma cholesterol is associated with intestinally derived lipoproteins (Thompson, 1990). Hence, the entire plasma pool would be approximately 0.15g (given a plasma cholesterol concentration of 250mg/dL and plasma volume of three litres). With a turnover rate of at least 100 pools per day, the mass of cholesterol transported via these particles would be substantial (approximately 15g) and would exceed that of other plasma lipoproteins over the same period. For example, the mass of cholesterol metabolised via LDL over a 24h period would approach 4–5g in a normolipidaemic subject with average body weight (Mamo, 1995; Soutar et al., 1977; Thompson, 1990). Man is normally in a postprandial and post-absorptive state. Therefore, in response to increased rates of biosynthesis following fat intake, larger quantities of intestinal chylomicron sterols may be secreted. We would suggest that it is erroneous to consider chylomicrons as not being significantly involved in cholesterol transport. Moreover, it seems inappropriate to describe chylomicrons as “exogenous” lipoproteins, because the majority of sterols secreted by the intestine are probably not dietary derived. The regulation of apoB48 biosynthesis and by extension chylomicron secretion by the intestine has not been studied extensively, but is probably similar to secretion of hepatic apoB100 containing lipoproteins. Chylomicron biosynthesis in the enterocyte involves the intracellular packaging of lipids with apolipoprotein B48. Triglyceride is added to the protein during translation and translocation within the endoplasmic reticulum (Gordon et al., 1996). Maturation (addition of lipids) probably occurs within the Golgi apparatus prior to exocytotic secretion into mesenteric lymph. Nascent lymph chylomicrons enter the circulation via the thoracic duct which drains into the jugular vein. Chylomicron synthesis and secretion is probably governed at several stages. Some studies have shown that fatty acids can have differential effects on apoB and triglyceride secretion, with fish oils being inhibitory in comparison to mono-unsaturates (Murthy et al., 1992; Wang et al., 1993). Inhibition of cholesterol esterification also appears to slow chylomicron secretion (Martins et al., 1994). Microsomal transfer protein modulates the rate of lipid transfer to newly synthesised apoB (Gordon et al., 1996; Gordon et al., 1993). In the liver, lipid associated with apoB protects the protein from intracellular degradation and hence microsomal transfer protein may be a critical regulatory site for secretion. It seems reasonable to suggest that the synthesis of apolipoproteins B100 and B48 share similar regulatory mechanisms, although evidence
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suggests that there may be some important differences. For example, there appears to be variance between the promoter region of apoB genes of hepatic and intestinal cells (Paulweber and Levy-Wilson, 1991). It may be that different transcription factors and/or ratio of enhancers/suppressors confer differential regulation of apoB48. Altering the mRNA sequence and the 3′ untranslated regions can also have a gross effect on mRNA stability and hence translation of the gene product (Jarzembowski and Malter, 1997; Mcleod et al., 1994). Evidence for this come from studies with primary cultures of rat hepatocytes, where it was found that stability of apoB48 mRNA was greater than for apoB100 mRNA (Pullinger et al., 1989) (note that in rodents, hepatocytes produce both isoforms of apoB). Indirect evidence that chylomicrons are secreted in the nonabsorptive state is the observation that in human liver tissue apoB100 gene expression is constitutive (Pullinger et al., 1989). ApoB100 secretion by the liver appears to be primarily dependent on posttranslational events, that is the fraction of newly synthesised apoB100 that escapes intracellular degradation. It is the co-translational addition of lipids to apoB100 that is critical for transfer of apoB100 to the ER lumen and subsequent secretion and it’s probable that the same is true for apolipoprotein B48 (Boren et al., 1992; Gordon et al., 1996; Gordon et al., 1993). Several studies suggest that apoB synthesis may be under endocrine control with both thyroid and insulin hormones being inhibitory (Levy et al., 1996; Sparks and Sparks, 1990; Theriault et al., 1992). The latter observations raise the intriguing question as to whether chylomicron over-secretion occurs in diabetes or hypothyroidism, conditions which have significantly greater rates of atherogenesis.
THE METABOLISM OF CHYLOMICRONS: RELEVANCE TO ATHEROGENESIS We discussed earlier the likelihood that chylomicrons might be the primary cholesterol carrying lipoprotein in plasma. Nonetheless, chylomicrons are usually considered as “triglyceride-rich” lipoproteins, because on a molar basis they are normally made up predominantly of this lipid. Chylomicrons often contain substantial amounts of triglyceride as a consequence of the composition of ingested lipids. Unfortunately, the lipid “description” of chylomicrons infers that chylomicrons are cholesterol “poor”, yet one single particle is estimated to contain more than 40 times more cholesterol than a low density lipoprotein (Fielding, 1992). Furthermore in man and several other species, certain fatty diets can induce intestinal biosynthesis of chylomicrons which are predominantly comprised of cholesterol
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(Crook et al., 1990). The mass of lipid packaged within chylomicrons can differ significantly. In the fasted state, chylomicron size is approximately 40– 100nm, however during the postabsorptive state chylomicrons as large as 500nm may be isolated. There is considerable debate as to how size and to a lesser extent composition influences the catabolism of chylomicrons although both these factors may impact on potential atherogenicity. The general metabolism of chylomicrons has been elegantly reviewed on a number of occasions (Mamo, 1995; Redgrave, 1983; Redgrave, 1988) and will not be discussed in detail here (see Figure 6.1). Briefly, once within the circulation chylomicrons interact with lipolytic enzymes located on the capillary surface of several tissues.
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Figure 6.1 Metabolism of chylomicrons. Chylomicrons are synthesised exclusively and constitutively by intestinal epithelial cells. They are secreted into lymph and contain a number of proteins including apolipoprotein B48, which is obligatory for secretion. Their predominant lipid component is usually triglyceride. Chylomicrons vary significantly in size from 40 nm in the fasted state to more than 500 nm in the postabsorptive state. They enter plasma by way of the lymphatics. In plasma they are hydrolysed by lipases that are located on the endothelial surface of a number of tissues, including adipose, muscle and liver. During the course of this hydrolysis, chylomicrons become smaller, more dense, exchange apolipoproteins and are referred to as remnants. Chylomicron remnants are normally removed rapidly from circulation, primarily by the liver, via high affinity uptake pathways. The LDL-receptor is thought to contribute significantly to this process.
Lipoprotein lipase (LPL) is found on the intimal endothelial membranes of muscle and adipose tissue and is responsible for the hydrolysis of chylomicron (and VLDL) triglyceride (BlanchetteMackie, 1991). Hydrolysis by LPL occurs at sequential sites and 70– 80% of triglyceride mass can be removed within minutes of entry into the circulation. Lipoprotein lipase requires apolipoprotein CII as an obligatory co-factor for full expression of activity (Fojo and Brewer, 1992). Chylomicrons containing inadequate apoCII or alternatively, excessive quantities of apolipoproteins which inhibit lipolysis, for example apoCIII, may be converted to the remnant form at a slower rate. Curiously several studies have recently found that subjects with elevated apoCIII have higher rates of atherogenesis (Fruchart and Duriez, 1995). Although causality has never been established one might speculate whether the elevated apoCIII phenotype is indicative of impaired chylomicron remnant metabolism. Chylomicrons which have become depleted in triglyceride may interact with a second hydrolytic enzyme associated with the liver and other steroidogenic organs (hepatic lipase) (Griglio et al., 1992; Shafi et al., 1994). Hepatic lipase is a potent phospholipase in addition to hydrolysing triglyceride, therefore excess surface phospholipids (generated as a consequence of the depletion of core triglyceride) can be simultaneously removed. During the lipolytic cascade, Chylomicrons become smaller (40–50nm) and increase in density. Whilst it is the triglyceride depleted remnant form of Chylomicrons which are considered to be atherogenic, the
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shedding of surface material during hydrolysis results in the generation of nascent high density lipoprotein (HDL) particles which may also influence atherogenesis. High density lipoproteins have the potential to remove sterols from arterial tissue (Barter and Rye, 1996) and may possibly inhibit inflammation by modulating the cytokine induced expression of leucocyte adhesion molecules (Vadas et al., 1997). Chylomicron remnants are usually cleared rapidly from the circulation following interaction with high affinity binding sites (Havel, 1995). Clearly, if expression of the removal sites is compromised, the residency time of chylomicron remnants will be increased and arterial cholesterol deposition may be exacerbated. In dyslipidaemic states where there is accumulation of chylomicron remnants as a consequence of impaired clearance, the frequency of, or rate of progression of atherogenesis is greatly enhanced, consistent with a causal role (Mamo, 1995; Steiner, 1993; Zilversmit, 1995). There has been considerable and ongoing debate as to which receptor (s) mediate clearance of chylomicron remnants (Havel, 1995; Mamo, 1995). Studies in the early 1980’s in LDL receptor deficient rabbits (Watanabe rabbits) suggested that chylomicrons are cleared by LDLreceptor independent means (Kita et al., 1982). In LDL-receptor knockout mice similar conclusions were drawn, based on the observation that clearance from plasma of radiolabelled Chylomicrons was not dissimilar to wild type mice possessing normal LDL-receptor expression (Ishibashi et al., 1994). In addition, the accumulation of apoB48 in knockout mice appeared somewhat insignificant compared to apoB100 (used as a marker of LDL). Moreover, in man oral fat tolerance tests in subjects with familial hypercholesterolaemia, a condition in which expression of the LDL-receptor is seriously compromised, there was no significant difference in the postprandial lipid response (Rubinsztein et al., 1990). The studies in Watanabe rabbits and particularly in FH were critical because they provided pivotal evidence that low density lipoproteins are independently atherogenic. However, contradictory studies have now been published. We recently studied chylomicron kinetics in Japanese subjects who inherited two mutant alleles coding for the LDL-receptor, that is homozygous FH. In contrast to the conclusions drawn by Rubinsztein et al. (1990) we found utilising several markers of chylomicron metabolism, a substantial impairment in the clearance of chylomicron remnants (Mamo et al., 1998). Moreover, the fasting concentration of apoB48 in FH subjects was five fold greater than controls, suggestive of massive accumulation of small dense cholesterol enriched remnants. The paradoxical observations of our studies and Rubinsztein’s may be explained by the possibility that in the South African cohort, chylomicrons were not completely recovered from plasma under the centrifugation conditions described (see section Monitoring
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chylomicron metabolism in vivo). Critically, identification of a chylomicron remnant metabolic disorder in FH raises the intriguing question of whether premature atherogenesis in this syndrome is in part a consequence of disturbed postprandial lipoprotein kinetics. Contradictory findings were also found in studies with Watanabe rabbits and LDL receptor knockout mice. Kita et al. (1982) reported normal clearance of chylomicrons in homozygote Watanabe rabbits, but there have since been three studies which found significant delays in metabolism (Assadollahi et al., 1994; Bowler et al., 1991; Hussain et al., 1995), observations possibly explained by methodological differences. In LDL receptor knockout mice Mortimer et al., (1995) reported that remnants were rapidly “captured” within the hepatic sinusoid, possibly associated with heparin proteoglycans, but internalised slowly by low affinity pathways. Hence, while clearance from plasma appeared normal, internalization by hepatocytes was markedly impaired. Work by Ishibashi et al., (1996) also suggested that remnant clearance was significantly impaired in LDL-receptor knockout mice. Nowadays, most lipoprotein physiologists would agree that the LDLreceptor is in part responsible for clearance of postprandial lipoproteins, although there remains widespread disagreement as to the quantitative significance of this pathway (Assadollahi et al., 1994; Biesiegel et al., 1994; Bowler et al., 1991; Choi et al., 1993; Havel, 1995; Hussain et al., 1991; Hussain et al., 1995; Ishibashi et al., 1996; Mamo 1995; Mamo et al., 1997; Mamo et al., 1998; Mortimer et al., 1995; Rubinsztein et al., 1990). One study has suggested that there is no evidence for high affinity mechanisms other than the LDL-receptor (Bowler et al., 1991). Given that FH, and hence the “LDL-receptor deficient” genotype is the most common genetic abnormality in man, it is important to unequivocally determine the role of the LDL-receptor in clearance of chylomicron remnants, as this would probably improve the development of therapeutic strategies aimed specifically at promoting the clearance of intestinally derived lipoproteins. The studies of Brown and Goldstein (1986) demonstrated the elegant mechanisms by which cells generally maintain cholesterol homeostasis. Put simply, when the availability of sterols exceeds that required by the cell, LDL-receptor expression is down regulated. Epidemiological studies which link dietary patterns with impaired clearance of LDL as a consequence of decreased LDL-receptor expression, may also impact on the clearance of postprandial lipoprotein remnants. Diets rich in saturated fats or cholesterol may have multi-factorial effects by altering chylomicron production/composition, receptor expression and influencing hepatic production of lipoproteins. It is difficult to distinguish if any one, or a combination of these possibilities are responsible for the progression of atherogenesis which occur as a
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consequence of poor dietary habits, but clearly disturbed chylomicron metabolism must be a contributing factor. Chylomicron remnants interact with the LDL-receptor utilising apolipoprotein E as the ligand (Innerarity et al., 1978; Mahley et al., 1984). In contrast, hepatically derived lipoproteins and particularly LDL bind to the LDL-receptor via apolipoprotein B100. Lipoproteins which utilise apoE to bind to the LDL-receptor appear to require a cluster of four receptors to enable internalization, which in part explains the greater affinity compared to lipoproteins which bind via apoB100 (Innerarity et al., 1978; Mahley and Innerarity, 1983). LDL are believed to interact with the receptor on a one ligand (particle)/one receptor basis (Innerarity et al., 1978; Mahley and Innerarity, 1983). Therefore, given a similar number of particles, chylomicron remnants would require greater receptor expression than LDL for efficient clearance. Consider the model depicted in Figure 6.2. Frame A shows a cell being presented with either two chylomicron remnant particles or two LDL particles. If the cell was to express a full complement of receptors (in this example eight) clearance of both lipoprotein types would be normal. In frame B, if the cell can only express half the normal number of receptors due to a genetic abnormality (in this case four), the receptor deficient cell would in theory have impaired clearance of chylomicron remnants, but still be able to clear LDL efficiently. Extrapolation of this simplistic model in vivo would suggest that there may be individuals with moderate LDL-receptor deficiency who have abnormal clearance of chylomicron remnants, but normal clearance of LDL. Consistent with this hypothesis, in heterozygote Watanabe rabbits possessing 50% of normal LDL receptor expression, LDL clearance is normal, while clearance of chylomicron remnants is significantly delayed (Bowler et al., 1991). Importantly, heterozygote Watanabe rabbits are normolipidaemic in the fasting state. Therefore, if a similar situation occurred in man, there may be individuals undiagnosed for LDL-receptor expression deficiency but with postprandial dyslipidaemia. It is interesting to note that two studies have now reported postprandial dyslipidaemia in normolipidaemic subjects with coronary artery disease (Groot et al., 1991; Weintraub et al., 1996). However, in each of these studies the reason(s) responsible for postprandial dyslipidaemia were not determined. We have previously suggested that the large number of subjects who develop coronary disease in the absence of classical lipid risk factors may have postprandial dyslipidaemia as a consequence of decreased LDL-receptor expression (Mamo, 1995; Mamo et al., 1997). The use of diet and therapeutic strategies in this population to promote clearance of chylomicron remnants will ultimately be dependent on sufficient receptor expression.
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Chylomicron remnants normally clear quite quickly from plasma probably as a consequence of their high affinity for the LDL-receptor. One interpretation is that clearance is too rapid to indicate causality in atherogenesis. Alternatively, rapid clearance is consistent with a disease that normally takes many years to become clinically evident. A
Figure 6.2 A model of the effects of moderate LDLreceptor deficiency on LDL and chylomicron remnant (Cm-Rm) metabolism. Frame A shows “normal” cells with full expression of LDL receptors. Sufficient receptors are expressed to clear LDL and chylomicron remnants. LDL interact with the LDL receptor on a one ligand/one receptor basis, whereas chylomicron remnants may require four receptors for proper uptake. In frame B, the model cells express only half the normal complement of receptors. Sufficient receptors are expressed in these cells for proper removal of LDL; however, according to this model, uptake of chylomicron remnants is delayed. Extrapolation of this model to what might occur in vivo, would predict that subjects with moderate LDL-receptor deficiency can have normal clearance of LDL but abnormal clearance of chylomicron remnants.
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fundamental question is what impact does receptor expression have on arterial exposure to chylomicron remnant cholesterol. Based on the flux of cholesterol suggested in this chapter a 20% increase in chylomicron remnant concentration at basal rates would equate to an increased in arterial exposure of around 3g, or 75% of the entire LDL-cholesterol pool. In ongoing studies in our laboratory, we have found the basal fasting apoB48 concentration to be doubled in normolipidaemic subjects with CAD and five times greater in subjects with familial combined hyperlipidaemia, compared to age and weight matched controls free of significant coronary disease (Mamo unpublished). One must keep in mind that the residency time of chylomicron remnant lipoproteins and indeed all atherogenic particles will impact directly on the rate of cholesterol flux across the arterial wall and presumably the rate of cholesterol deposition.
ARTERIAL DELIVERY AND RETENTION OF POSTPRANDIAL LIPOPROTEINS The primary strategy for preventing and regressing atherogenesis is based on the hypothesis that decreasing arterial exposure to proatherogenic lipoproteins will reduce arterial retention of lipid and attenuate the pro-inflammatory cascade (Figure 6.3). Intervention studies to reduce the concentration of LDL cholesterol indicate that this approach can be effective, although arguably not as significant as we might hope for (Mamo et al., 1997; Watts and Burke, 1996). Presently, therapeutic strategies are primarily aimed at reducing the plasma concentration of hepatically derived cholesterol and triglyceride associated with LDL and intermediate density lipoproteins respectively. Perhaps greater reductions in atherosclerotic disease might be achieved if production of chylomicrons is decreased and clearance is enhanced. This notional strategy would only be effective if there was evidence that cholesterol in arterial lesions is to some extent derived from intestinally derived lipoproteins (Mamo, 1995). The suggestion that cholesterol in lesions might be derived from ingested fats was conceived in the 1950’s following the observation that patients with coronary heart disease have increased post-prandial turbidity in their plasma (Albrink and Man, 1959). However, direct involvement of chylomicrons in arterial cholesterol deposition was subsequently excluded based on studies which suggested that these particles were too large to penetrate arterial tissue (Nordestgaard and Tybjaerg-Hanson, 1992). Consequently, chylomicrons were suggested to be “indirectly” atherogenic, that is as a consequence of modulating LDL-cholesterol metabolism. Indeed, postprandial lipaemia can stimulate the production of LDL precursor lipoproteins and delay
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clearance of LDL (Hennessy et al., 1992; Lairon, 1996).
Figure 6.3 Chylomicrons were labelled with rhodamine and then injected into eviscerated rabbits to generate remnants. Chylomicron remnants were perfused in situ through a carotid vessel of a New Zealand White rabbit for 15 min and under physiological conditions of pressure, temperature and oxygen tension. The vessel was then perfused with buffer alone for 20 min and the retention of remnants determined by digital confocal microscopy. Shown here is a typical focal zone of chylomicron remnant accumulation within the subendothelial space, with little uptake associated within the medial regions of the vessel.
Support that chylomicrons might be atherogenic in their own right came from studies where it was found that following hydrolysis to their remnant form, chylomicrons readily penetrate arterial tissue (Mamo and Wheeler, 1994; Proctor et al., 1996). The conversion of chylomicrons to remnants is a requisite for intimal delivery of lipoproteins because large macromolecules are transported to the
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subendothelial in transcytotic vesicles which only have a diameter of approximately 70 nm (Simionescu et al., 1991). Lipoproteins larger than 70 nm such as chylomicrons and VLDL remain on the luminal surface of arterial vessels (Nordestgaard and Zilversmit, 1988; Nordestgaard, 1996). It is reasonable to suggest that cholesterol in early atherosclerotic lesions may in part be derived from postprandial lipoproteins, specifically chylomicron remnants. Consistent with this were the findings that arterial fatty lesions in cholesterol fed and Watanabe rabbits had greater uptake of chylomicron remnants but not of LDL (Proctor et al., 1996), although a putative causal role in atherogenesis was confounded because the majority of association appeared to be within the tunica media and not the intimal region of the tissue. Moreover, this and other studies (Nicoll et al., 1981; Nordestgaard and Zilversmit, 1988; Nordestgaard, 1996; Pittman et al., 1983; Stender and Hjelms, 1988) employed the use of radioisotopic tracers to monitor arterial uptake of lipoproteins, which provide a limited distinction between lipoprotein delivery versus retention. It is the latter of these two processes, which is considered to be the initiating event of atherogenesis. Recently, visualisation of arterial chylomicron delivery and retention was achieved by confocal imaging of fluorescent labelled chylomicron remnants (Proctor et al., 1998). Utilising an in situ perfusion system of rabbit carotid vessels we found that chylomicron remnants penetrate arterial tissue rapidly, in a uniform manner and via the luminal surface. Critically, although most particles were found to passage through the tissue, we found that arterial efflux of chylomicron remnants was often not complete and that significant sporadic focal accumulation of remnants occurred within the subendothelial space (Figure 6.4). We were unable to determine why chylomicron remnants only accumulated within certain regions of the subendothelial space although very recent work by Chait et al. (1997) suggests that the local extracellular matrix environment may be critical. The plasma concentration of chylomicron remnants will vary depending on the nature and frequency of an individuals eating habits. Given that the arterial retention of chylomicron remnants is positively related to the degree of exposure, it might be that cyclical acute exposure to increased concentrations of chylomicron remnants following a fatty meal, exacerbates arterial retention of these particles. Certainly, this possibility is consistent with epidemiological data linking poor dietary habits with increased atherogenesis (Keys, 1988; McGee et al., 1984) and provides an alternate explanation to “dietary induced” atherogenesis rather than as a consequence of disturbed LDL metabolism.
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Figure 6.4 The putative sequence of events by which chylomicron remnants induce atherosclerosis. When the plasma concentration of remnants is increased as a consequence of overproduction or decreased clearance, transcytotic delivery of remnants to the subendothelial space occurs. If delivery exceeds efflux through arterial tissue, chylomicron remnants may become trapped within extracellular matrices. The accumulation of remnants stimulates production of cytokines and chemokines which inturn induce endothelial expression of adhesion molecules, extravasation of circulating monocytes and transformation into macrophages. The scavenging leukocytes degrade chylomicron remnants via a phagocyticlike mechanisms which is not sensitive to the intracellular concentration of sterols. Uptake of remnants also triggers a respiratory burst which can compromise cell viability and in turn lead to plaque instability.
CHYLOMICRON REMNANTS, INFLAMMATION AND METABOLISM BY MACROPHAGES The focal accumulation of lipid within the subendothelial space of arterial vessels is thought to initiate the inflammatory cascade observed in atherosclerotic lesions (Ross, 1993; Seifert et al., 1990).
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Cyto/chemokines released from endothelial and smooth muscle cells in response to excessive arterial retention of lipoproteins induces endothelial expression of leucocyte adhesion molecules which encourage extravasation of circulating monocytes through the intima (Fuster et al., 1992). Under the influence of growth factors, monocytes transform into macrophages which then avidly degrade lipoproteins trapped within the subendothelial space. (Gown et al., 1986; Watanabe et al., 1985). Thereafter, excessive accumulation of sterols by arterial macrophages occurs when uptake of lipoprotein lipid exceeds efflux. Lipid laden macrophages (foam cells) are a predominant feature of early atherogenesis and are found in numerous quantities in arterial “fatty streaks” (Gown et al., 1986; Watanabe et al., 1985). In advanced lesions, foam cells are not a requisite feature, however, the necrotic lipid core which is derived following macrophage death has important implications. Cytotoxic substances released from foam cells are thought to contribute to plaque destabilisation which could then result in a clinical event such as an heart attack or stroke (Gown et al., 1986; Guyton and Klemp, 1996; Watanabe et al., 1985). The source of sterols in foam cells and subsequent cause of death has not been unequivocally established. Lipid oxidation products associated with LDL are thought to contribute based on observations that macrophages have unabated uptake of oxidatively modified LDL via scavenger receptors which is consistent with the finding of oxysterols in arterial lesions (Carpenter et al., 1995; Steinberg et al., 1989). Scavenger receptors, unlike the LDL-receptor are not sensitive to the intracellular concentration of cholesterol. Oxidised LDL are also cytotoxic towards monocyte-derived macrophages (Morel et al., 1983) and other vascular cells (Hessler et al., 1979; Peng et al., 1979) and may contribute to vascular dysfunction. The myeloperoxidase system of scavenging leucocytes suggests that macrophages are the principal mechanism by which LDL become modified in vivo, although arterial endothelial and smooth muscle cells can also contribute to this process. Evidence that postprandial lipoproteins could also induce macrophage sterol loading came with studies using -VLDL, a cholesterol rich lipoprotein of intestinal and hepatic origin found in mammals on high cholesterol diets. Beta-VLDL can induce substantial lipid loading of macrophages in the absence of lipoprotein modification (Goldstein et al., 1980). To investigate whether chylomicron remnants induce macrophage accumulation of sterols (from donors not given cholesterol), chylomicrons were isolated from donor animals given a commonly used lipid supplement free of sterols (Intralipid). Chylomicron remnants were generated in eviscerated rabbits and a highly purified remnant fraction free of apoB100-containing lipoproteins was isolated (Mamo et al., 1996). The remnant preparations were found to be free of lipid and protein oxidation
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products and were not aggregated, yet massive lipid loading was still observed within hours of incubation at concentrations of remnant cholesterol which would normally be found in plasma (Figure 6.5). Chylomicron remnants bind to a cell surface protein which initiates a “phagocytic-like” process which does not appear to be regulated by the intracellular concentration of sterols (Mamo et al., 1996). Presently, the exact mechanism of chylomicron remnant uptake by macrophages has not been unequivocally determined although studies by Ramprasad et al., (1995) have identified macrophage proteins which bind remnants of triglyceride rich proteins. The observations that chylomicron remnants induce macrophage lipid loading, coupled with the findings of focal arterial accumulation of these lipoproteins in early fatty lesions, provides strong evidence for a causal role in atherogenesis.
Figure 6.5 A highly purified fraction of chylomicron remnants was generated in eviscerated rabbits. Human monocyte macrophages were incubated for six hours with chylomicron remnants at a concentration of 25 g cholesterol/ml. Substantial lipid loading (shown in red) was observed. Chylomicron remnants had no detectable levels of oxysterols and were not aggregated.
Several studies have suggested that remnants of triglyceride-rich
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lipoproteins may adversely alter vascular function as a consequence of cytotoxic fatty acids which remain with the particles during the lipolytic cascade (Chung et al., 1995). Indeed, macrophages have substantial expression of lipoprotein lipase (Koo et al., 1981), suggesting that these cells might exacerbate remnant fatty acid accumulation. However, studies from our laboratory have found macrophage cell viability to be rapidly compromised by remnants in the absence of unesterified fatty acids (Yu and Mamo, 1996). Our observations suggest that additional mechanisms contribute to remnant induced macrophage cytotoxicity. Phagocytotic uptake by scavenging leucocytes normally initiates activation of the respiratory burst resulting in the generation of oxygen free radicals as well as other microbiocidal agents. It seems plausible to suggest that products of the respiratory burst contribute to macrophage cell death given that the process of degradation shares similarities to phagocytosis. Indeed chylomicron remnant internalization by macrophages need not be a requisite for induction of the respiratory cascade. NADPH oxidase, which is a key enzyme involved in the respiratory burst, is membraneassociated and can be activated by receptor-mediated and receptorindependent means (Robinson and Badway, 1995). Continuous or even cyclical contact between the cell and the stimulus, in this case, chylomicron remnants, might be sufficient to induce production of oxygen free radicals. Interestingly, although sterol accumulation and the inflammatory cascade might be exacerbated if lipoproteins become oxidatively modified, the proposed model of remnant “derived” macrophage cholesterol does not predict oxidative modification as a requisite for uptake (as is the case for LDL). One might ask are oxysterols associated with arterial lesions derived from oxidatively modified lipoproteins or are they a consequence of remnant uptake by scavenging leukocytes. Ongoing studies in our laboratory have found that macrophage cell viability in the presence of chylomicrons is inversely related to the production of superoxide (Yu and Mamo, 1997a). The respiratory burst in macrophages is part of an elaborate pathway which consists of myeloperoxide, H2O2 and a halide, i.e., the MPO-H2O2-halide system (Klebanoff, 1980). Superoxide is extremely toxic but has a short halflife because it is unstable. Myeloperoxide is present in the azurophile granules of macrophages and is discharged into the phagosome during phagocytosis, where it interacts with H2O2 and Cl− to form hypochlorous acid (HOCl) (see Figure 6.6). We found that combined incubation of macrophages with superoxide dismutase and catalase could significantly attenuate cell death (Yu and Mamo, 1997a). The release of intracellular enzymes such as phospholipases, proteinases and lysozymes might exacerbate cell viability and in vivo, plaque stability.
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CHYLOMICRON REMNANTS AND VASCULAR SMOOTH MUSCLE CELLS The interplay between arterial smooth muscle cells, lipoproteins and atherogenesis is not well understood although the involvement of this cell type in the disease is considerable. A small but significant number of arterial foam cells appear to be derived from smooth muscle cells which have lost their proliferative nature and migrated to the subendothelial space (Campbell et al., 1985). Contractile smooth muscle cells may form the bulk of the fibromuscular cap (frequently observed in mature lesions) and may be critical in isolating necrotic debris following excessive uptake of lipid by scavenging leucocytes (Gown et al., 1986). Furthermore, smooth muscle cells may be proinflammatory as a consequence of secreting cytokines, chemokines and growth factors in response to retention of lipid. There is evidence to suggest that smooth muscle cells dictate the type of extracellular matrices deposited and degree of matrix sulphation (Hedin, 1994; Libby and Tamaka, 1997), which in turn may impact on lipoprotein retention and the phenotypic characteristics of the smooth muscle cells, i.e. contractile versus proliferative. Arterial delivery studies have found that significant quantities of chylomicron remnants can associate within the tunica media of normal healthy arteries (Mamo and Wheeler, 1994; Proctor et al., 1996). In cell culture studies of arterial smooth muscle cells in the contractile state, chylomicron remnants are avidly degraded, primarily via the LDLreceptor, which accounts for approximately 90% of uptake (Yu and Mamo, 1997b). Unlike macrophages, incubation of arterial smooth muscle cells with remnants does not normally induce sterol loading, consistent with the normal axis of cholesterol homeostasis which operates via the LDL-receptor mechanism of uptake (Yu and Mamo, 1997a). Less clear is if and how metabolism proceeds when LDLreceptor expression is compromised. Diminished uptake of chylomicron remnants by medial smooth muscle cells we have suggested would increase retention within extracellular matrices (Yu and Mamo, 1997a) and presumably encourage secretion of proinflammatory cytokines (Campbell et al., 1985). Low affinity uptake pathways by arterial smooth muscle cells may become critical as a consequence of decreased LDL-receptor expression. Extracellular accumulation may lead to the generation of pro-inflammatory proteins, although one might conceive a more serious consequence if non-contractile smooth muscle cells located on the luminal surface of arterial vessels become compromised. Uptake of chylomicron remnants by smooth muscle cells via non-LDL receptor
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Figure 6.6 A model for chylomicron remnant induced macrophage cytotoxicity. Chylomicron remnants are taken up via a phagocytic like mechanism which elicits a respiratory response. The respiratory burst in macrophages is part of an elaborate microbiocidal mechanism which consists of MPO, H2O2 and a halide. Superoxide is extremely toxic but has a short half-life because it is unstable. Myeloperoxide is present in the azurophile granules of macrophages and is discharged into the phagosome during phagocytosis, where it interacts with H2O2 and and Cl− to form hypochlorous acid (HOCl). H2O2 can be scavenged by the enzymes, SOD and catalase respectively. Excessive uptake of remnants in the absence of insufficient scavengers of the oxygen radicals which are generated may compromise cell viability. A combination of superoxide dismutase and catalase can significantly attenuate chylomicron remnant induced cytotoxicity of macrophages.
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pathways can lead to significant changes to cell morphology in vitro and even death (Yu and Mamo, 1997a). Smooth muscle cell viability is maintained in the presence of antioxidant enzymes suggesting a mechanism of uptake not unlike that described for macrophages. We would suggest that when retention of chylomicron remnants in vivo exceeds arterial smooth muscle cell uptake via the LDL-receptor, cell viability may be jeopardised as a consequence of phagocytic degradation of these particles.
CHYLOMICRON REMNANTS AND VASODILATATION Nitric oxide is a potent vasodilator produced principally by the arterial endothelium (Marin and Rodriguezmartinez, 1997). Decreased vasodilatory responses is considered the first indication of atherogenesis and may precede significant arterial lipid deposition or involvement of inflammatory cells. There is some evidence to suggest that chylomicron remnants impair the physiological actions of nitric oxide, compatible with recent evidence demonstrating that a high fat meal diminishes post-ischaemic flow-mediated dilatation of the brachial artery (Vogel et al., 1997). Therefore, by implication excessive arterial retention of chylomicron remnants might induce endothelial dysfunction in the coronary circulation.
CHYLOMICRON METABOLISM IN DYSLIPIDAEMIC STATES AND IN NORMOLIPIDAEMIC SUBJECTS WITH CORONARY ARTERY DISEASE: STRATEGIES FOR TREATMENT Epidemiological data, turnover and cell culture studies implicate a role of postprandial lipoproteins in coronary disease (Cabezas et al., 1993; Hodis and Mack, 1995; Mamo, 1995; Mamo et al., 1997; Slyper, 1993; Steiner, 1993; Zilversmit, 1995). However, atherogenesis is a complex disease which occurs not only in individuals with raised plasma lipids but also in subjects who are normolipidaemic. Presently, there appears to be no common “denominator” which might be described as the primary atherogenic risk factor, although LDL-cholesterol has often incorrectly assumed this role. There is increasing evidence that chylomicron remnant metabolism may be disturbed in most categories of high atherogenic risk including subjects who have coronary artery disease in the absence of dyslipidaemia (Groot et al., 1991; Weintraub et al., 1996). It is critical that we verify whether remnant dyslipidaemia is common in subjects with atherogenesis in order to develop strategies aimed specifically at reducing their concentration in plasma.
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Fortuitously, some dietary and therapeutic interventions already utilised may be of some benefit in accelerating chylomicron remnant clearance. When considering chylomicrons as an atherogenic risk factor one needs to clearly distinguish between the nascent triglyceride rich particles, versus the triglyceride depleted remnant, because ironically it was the observation in type I hyperlipoproteinaemia which suggested that chylomicrons per se did not contribute to arterial cholesterol deposition. Subjects with the type I dyslipidaemic syndrome have little or no functioning lipoprotein lipase and therefore have extremely high levels of circulating chylomicron triglycerides (Thompson, 1990). Apparently, it is the inability to convert chylomicrons to the remnant form which protects individuals from atherogenesis, because the particles are too large to be delivered by transcytosis to the subendothelial space. Type II Hyperlipoproteinaemia (Familial Hypercholesterolemia) and Familial Combined Hyperlipidaemia Considered to be the most common monogenic abnormality in man, familial hypercholesterolaemia (FH) has an estimated frequency of 1 in 500 when subjects inherit one mutant allele coding for the LDLreceptor (Thompson, 1990). In homozygote subjects there is significant postprandial hyperlipidaemia (Mamo et al., 1998). Evidence in animal models and cell culture studies suggest that heterozygote FH subjects are also likely to have delayed clearance of remnants, however this is yet to be demonstrated in human studies. Familial combined hyperlipidaemia may be of polygenic origin but collectively one may describe that in addition to a defect in the clearance of LDL there is overproduction of hepatic LDL precursor lipoproteins (VLDL) and/or defective hydrolysis of triglyceride rich lipoproteins (Thompson, 1990). In analogy to subjects with FH, it is likely that LDL-receptor expression is compromised in FCH and therefore, chylomicron remnant clearance would also be delayed. Alternatively, competition for high affinity clearance pathways might impede chylomicron remnant clearance. Several groups have reported exaggerated postprandial lipaemia in FCH subjects (Cabezas et al., 1993). Hypertriglyceridaemia which accompanies FCH might reflect reduced lipolysis of chylomicrons and VLDL, overproduction of either of these lipoproteins and/or decreased removal of remnants which still contain significant levels of triglyceride. Presently, there is no evidence to suggest that the hydrolytic enzyme activities are compromised in subjects with FCH although this possibility cannot be ruled out. A widely used therapy to treat FH and FCH is to use one of the
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“statin” group of compounds. Statins are thought to lower LDLcholesterol by promoting clearance through enhanced LDL-receptor expression and by lowering hepatic production of LDL precursor lipoproteins (Watts and Burke, 1996). However, the efficacy of statins in promoting clearance of postprandial lipoproteins will be dependent on the level of receptor upregulation achieved. Whilst the fasting concentration of plasma cholesterol and triglyceride are often dramatically lowered following statin therapy, caution needs to be exercised as to whether the cholesterol rich remnants of chylomicrons are also reduced. In an ongoing study, cholesterol and triglyceride targets have been achieved in subjects with combined hyperlipidaemia without significant reduction in the concentration of postprandial remnants as indicated by the fasting concentration of apoB48. Studies in Watanabe rabbits with 50% of normal LDL receptor expression (model for heterozygous FH), combined treatment with cholestyramine (a bile binding resin) and Pravastatin failed to ameliorate slow clearance of chylomicrons (Mamo et al., 1994), suggesting that very aggressive therapy is required to attain a sufficient level of receptor expression which will promote chylomicron remnant clearance. Type III Hyperlipoproteinaemia In type III hyperlipoproteinaemia remnants of triglyceride rich lipoproteins accumulate in plasma leading to moderate hypertriglyceridaemia and sometimes concomitant hypercholesterolaemia (Breslow et al., 1982). Subjects with type III dyslipidaemia are often characterised by aggressive coronary heart disease, perhaps testimony to the putative atherogenicity of remnant lipoproteins. The clearance of chylomicron remnants is often compromised in the type III syndrome because of the isoform of apolipoprotein E expressed, the protein responsible for docking to the LDL-receptor. In man three isoforms of apoE (apoE2, E3 and E4) can lead to one of six possible genotypes. In type III dyslipidaemia, a large number of subjects express the apolipoprotein E2/E2 genotype, suggesting that this isoform of the protein impairs chylomicron remnant metabolism (Breslow et al., 1982). Not all subjects with the type III phenotype are homozygous for apoE2/E2, which means that other causes can result in this phenotype. Clearly, it is critical to delineate the basis for the type III profile because of the aggressive nature of this phenotype. The apoE2/E2 syndrome is difficult to treat in the sense that one cannot correct for the nature of the ligand. Reducing secretion of intestinal and hepatic lipoproteins by dietary intervention and pharmacotherapy with fibrates or statins is normally advised. In type III subjects, there is no evidence
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to suggest that LDL-receptor expression is compromised. Type IV and Type V Hyperlipoproteinaemia Type IV dyslipidemia is usually indicative of hepatic overproduction of VLDL. It is unclear whether intestinal secretion of chylomicrons is also enhanced in the type IV syndrome. The type V syndrome is similar to type IV, however in addition to oversecretion, there is impaired conversion of triglyceride rich lipoproteins to the remnant form, i.e., a decreased rate of lipase mediated hydrolysis. It is not clear whether the metabolism of postprandial lipoproteins is impaired in all types of the IV and V syndromes. An exaggerated pool of hepatic lipoprotein remnants which utilise the LDL receptor might result in competition for receptor binding sites and therefore increased residency time of chylomicron remnants in plasma. Whether receptor expression per se is compromised in type IV or V hyperlipoproteinaemia might also depend on the metabolic basis for this disorder (Kwiterovich and Margolis, 1973; Thompson, 1990). The type IV and V dyslipidaemic profiles are often a consequence of endocrine disorders. Two of the more prevalent forms which often feature type IV or type V phenotype are diabetes and hypothyroidism. Both insulin and thyroid hormone can have profound effects on lipid metabolism including postprandial lipoproteins (Redgrave and Callow, 1990; Redgrave et al., 1991; Zerbinatti et al., 1991). In the insulin resistant state, normal or elevated levels of insulin in the presence of hyperglycaemia and increased plasma non-esterified fatty acids lead to increased lipogenesis and secretion of VLDL (Lewis and Steiner, 1996). Furthermore, peripheral insulin resistance has the potential to limit conversion of triglyceride rich lipoproteins, including chylomicrons to remnants, due to the loss of insulin stimulated synthesis of lipoprotein lipase. Insulin strongly stimulates LDLreceptor biosynthesis in adipocytes (Decingolani et al., 1996) and it is feasible that in the insulin resistant (or deficient) state, LDL-receptor expression is also reduced (Wade et al., 1988). The effects of thyroxine are qualitatively similar to the actions of insulin. Several groups have shown postprandial lipaemia to be impaired in hypothyroidic subjects and in animal models of thyroid deficiency (Redgrave and Callow, 1990; Redgrave et al., 1991; Zerbinatti et al., 1991). Thyroid hormones probably regulate postprandial lipaemia primarily via regulation of receptor expression, rather than as a consequence of modulating the lipolytic cascade. Clinical evidence suggests that type IV and type V phenotypes secondary to other metabolic disorders are attenuated as a consequence of good metabolic control. However, presently it is less clear as to whether hypoglycaemic compounds, insulin or thyroid replacement
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therapy, or insulin sensitising compounds restore postprandial dyslipidaemia. Chylomicron Remnants Clearance in Normolipidaemic Subjects with Coronary Artery Disease Dyslipidaemia is often considered a requisite feature for disturbed lipoprotein metabolism. However, chylomicrons are unique in the sense that because at any one time they represent a small fraction of the plasma lipid pool, a significant delay in their clearance will not substantially alter the concentration of triglyceride or cholesterol in plasma. Subtle defects in chylomicron metabolism are difficult to identify based on routine lipid screening of individuals in the fasted state. Several groups have shown using fat challenge tests that in normolipidemic subjects with coronary artery disease, postprandial lipaemia is exaggerated compared to controls (Albrink and Man, 1959; Groot et al., 1991; Weintraub et al., 1996). We have previously suggested that such individuals may be undiagnosed for decreased LDL-receptor expression (Mamo and Wheeler, 1994; Mamo, 1995; Mamo et al., 1997) although this remains to be confirmed. Of relevance to this population is whether pharmacotherapy in otherwise normolipidemic subjects should be advocated without evidence of a reduction in an identified risk factor. Presently, one might argue that strategies aimed at reducing the concentration of LDL cholesterol might benefit chylomicron metabolism by promoting the LDL-receptor and reducing competition for limited receptor sites, however this remains to be demonstrated.
MONITORING CHYLOMICRON METABOLISM IN VIVO Postprandial lipaemia may be quantitated by measuring the response of plasma triglycerides, vitamin-A esters or apoB-48 to a standardised oral fat load (Fîger and Patsch, 1993; Karpe et al., 1995). The response, which is invariably monophasic is normally expressed as the incremental change in plasma concentration following the meal, that is the “area under the curve”. A number of laboratories utilise triglyceride alone as a marker for chylomicron kinetics. However, triglycerides are primarily cleared as a consequence of lipolysis and therefore provides little information with respect to metabolism of the atherogenic remnant form of postprandial lipoproteins. Inclusion of retinyl esters (Vitamin A) in the test meal and subsequent measurement provides a more specific test than monitoring triglycerides alone because retinyl
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esters are not removed during particle lipolysis (Cohn, 1994; Fîger and Patsch, 1993; Karpe et al., 1995). Most laboratories measure retinyl esters in a density range of less than 1.006g/ml because at greater densities the vitamin is suggested to represent transfer to more dense lipoproteins and not indicative of chylomicron remnant concentration. However, more recent studies in vitro and in vivo suggest that esterified vitamin A is not subject to transfer between lipoproteins and that its isolation at densities greater than 1.006 g/ml represents the generation of small dense postprandial remnants (Martins et al., 1991; Mamo et al., 1998). In fact in situations where high affinity uptake is compromised one would predict increased appearance of remnants in more dense plasma fractions as a consequence of increased interaction with lipolytic enzymes. Given that it is the small cholesterol rich remnants of chylomicrons which are considered to be most atherogenic, it is important to ensure their complete recovery by isolating lipoproteins at sufficiently dense gradients. Considerable discussion has been provided previously and in this chapter. Presently the only exclusive marker which can be utilised to assess chylomicron metabolism is apolipoprotein B48, a protein which fortuitously, is produced exclusively by the intestine in man (Chen et al., 1987). ApoB48 is considered to reflect the particle concentration of chylomicrons and their remnants. However, the quantitation of apolipoprotein B48 at present remains difficult and unfortunately, not standardised. There are no commercially available specific antibodies for apoB48, because of the overlapping homology with apolipoprotein B100. Some laboratories have developed what appear to be potentially specific antibodies (Isherwood et al., 1995; Peel et al., 1993), however, the limited epitopes to which the antibodies are generated raise concerns about their suitability to accurately quantitate lipoproteins of different size and composition (where the critical epitopes may be masked). Other techniques are designed to remove cholesterol carrying lipoproteins other than chylomicrons, and to subsequently derive a value for “remnant like protein” cholesterol (Nakajima et al., 1993). However, the specificity, sensitivity and relevance of these fractions remains to be elucidated. Several laboratories including ours quantitate apoB48 using an immunodetection technique (Smith et al., 1997). However, because separation of apoB48 from B100 is required prior to quantitation, the procedure is labour intensive, costly and therefore not ideally suited to for routine clinical screening. Despite the drawbacks in quantifying apoB48, presently it is arguably the most appropriate test to assess for the presence of remnant dyslipidaemia. In an ongoing study we have found that fasting apoB48 has been able to accurately identify exaggerated postprandial lipaemia in eight out of ten subjects. Confirmation of our preliminary observations in a larger number of subjects would greatly enhance our ability to assess individuals for
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remnant dyslipidaemia and limit the necessity to do oral fat tolerance tests. It is important to note that significant quantities of apoB48 can be found at densities which encompass the LDL range, (consistent with the appearance of vitamin A esters at these greater densities) and therefore we recommend isolation at densities≤1.063 g/ml. Given that chylomicron remnant concentration is invariably elevated in hypertriglyceridaemic individuals, apoB48 probably only needs to be measured in individuals at risk of coronary disease with fasting concentrations of triglyceride less than <2.0mmol/L.
THERAPEUTIC OPPORTUNITIES Several diet and lifestyle changes are likely to positively influence chylomicron metabolism. An adjunct to these changes should aim to reduce abdominal obesity leading to increased insulin sensitivity and improved chylomicron metabolism. The intake of saturated, animal and dairy fat should be reduced or replaced with polyunsaturated fat, specifically n-3 fatty acids in fish. Regular physical activity probably encourages clearance of postprandial remnants. Whilst there is limited evidence to suggest that moderate alcohol intake can promote clearance of chylomicrons, this needs to be considered with respect to the possibilities of aggravating central obesity, insulin resistance and other factors likely to adversely influence postprandial lipoprotein metabolism. Cessation of cigarette smoking may also improve postprandial dyslipidaemia, in addition to reducing coronary risk in its own right. The pharmacotherapies available include fibrates, resins, statins, niacin and omega-3 fatty acid supplements. Fibrates are effective in lowering triglyceride, however, their effect on clearance of remnants is less clear. In fact in hypertriglyceridaemic subjects fibrates may increase the production of LDL which may compete for remnant clearance via receptor pathways. In this situation combination therapy may be necessary. The relative efficacy of statins in stimulating the clearance of chylomicron remnants remains to be further evaluated but would seem promising given that these particles share the same mechanism of clearance as for LDL. Probucol is another drug option, given experimental evidence that this agent stimulates hepatic uptake of chylomicron remnants (Mamo et al., 1993), but confirmatory work in humans is required.
SUMMARY AND CONCLUSIONS The conventional strategy for managing subjects with atherogenesis presently relies on screening for fasting hypercholesterolaemia and
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targeting therapy to optimising plasma LDL cholesterol concentration and, in certain instances, increasing HDL cholesterol (Vos et al., 1993). Our standard clinical approach to patients does not account for the notion that chylomicron remnants are causally related to the development of atherosclerosis. Recognition of this concept is especially important, since humans spend most of their lifetime in the postprandial state. Chylomicrons are a major cholesterol transport vehicle which appear to be selectively retained in early atherosclerotic lesions. Chylomicron remnants appear to be pro-inflammatory and induce lipid loading in scavenging macrophages, a hallmark feature of atherogenesis. Chylomicron remnants may consequently be a primary atherogenic lipoprotein. There is an accumulating body of evidence which suggests that chylomicron remnants (even in the absence of a meal) may be an independent atherogenic lipoprotein. We have provided a brief discussion which suggests that chylomicron remnant metabolism is disturbed in primary and secondary dyslipidaemic states prone to premature atherosclerosis and more importantly in normolipidemic subjects with coronary artery disease. Although assessment of chylomicron metabolism remains a challenge to lipoprotein physiologists, an awareness of their potential atherogenicity will no doubt lead to better management of dyslipidemia and a new focus on strategies which might be used to attenuate atherosclerosis. Much is known of dietary lipids which enhance and delay postprandial lipaemia. Current lipid lowering regimes might also enhance chylomicron metabolism but we should be aware that whilst their efficacy may appear substantial based on fasting plasma lipids, the effects on chylomicron remnant clearance may be more modest. References Albrink, M.J. and Man, E.B. (1959). Serum triglycerides in coronary artery disease. Archives of International Medicine , 103 , 4–8. Assadollahi, F., Cavallero, E., Buxtorf, J.C., Jacotot, B. and Beaumont J.L. (1994). Familial xanthomatous hypercholesterolemia— Abnormal exogenous lipid metabolism evidenced by the vitamin A test. Annual of Nutrition and Metabolism , 38 , 307–312. Barter, P.J. and Rye, K.A. (1996). Molecular mechanisms of reverse cholesterol transport. Current Opinion in Lipidology , 7 , 82–87. Biesiegel, U., Krapp, A., Weber, W. and Olivecrona, G. (1994). The role of alpha 2m/LRP in chylomicron remnant metabolism. Annual N.Y.Academy of Science , 10 , 737–769. Blanchette-Mackie, E.J. (1991). Lipoprotein lipase and fatty acid transport in heart, adipose tissue and mammary gland: immuno and cytochemistry. Endocrine Regulations , 25 , 63–69.
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7 High Density Lipoprotein Receptors Noel H.Fidge Baker Medical Research Institute, PO Box 6492, St Kilda Road Central, Melbourne 8008, Australia
Identification of putative high density lipoprotein receptors has been difficult, probably due to the complex nature of the ligand, HDL. Several HDL binding proteins, quite disparate in structure, have been cloned and their role in HDL metabolism is currently being assessed. High density lipoprotein binding protein, HBP, was found to lack a transmembrane domain and was assumed to be anchored to the cell surface. Although responsive to cell cholesterol levels, the physiological significance of HBP has not been established. SR-B1, a member of the class B scavenger receptors is the most studied HDL receptor. The level of SR-B1 expression correlates with both cholesterol efflux from cells and the selective transfer into cells of cholesteryl ester. Its mechanism probably involves a docking process whereby HDL is anchored at the cell surface for lipid exchanges. SR-B1, like all scavenger receptors, exhibits broad ligand specificity. However it appears to be regulated by the action of pituitary hormones that stimulate steroidogenesis, and may play an important role in supplying precursor cholesterol for steroid hormone production. HB2, one of a pair of liver HDL binding proteins has been cloned. It shows high sequence homology with adhesion molecules, particularly ALCAM. When HB2 is overexpressed in cells, HDL binding increases. In macrophages, HB2 expression is down regulated by cholesterol loading. The nature of the ligands recognised by the HDL receptors remains controversial, particularly their affinity for apoAI versus apoAI/AII rich HDL particles. Identification of receptor binding domains in apoAI and the involvement of repeated
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amphipathic -helices in cell binding is also discussed. More recent evidence for post-receptor mediated cell signalling pathways offers alternative functions for HDL, some of which may not be primarily related to lipid transport. Growing evidence for the involvement of lipid free apoAI as a mediator of such pathways is also considered in this chapter. KEYWORDS: High density lipoproteins, HDL receptors, apolipoprotein AI, apolipoprotein AII, cholesterol efflux.
INTRODUCTION The complexity of HDL provides a clue to the diverse nature of the biological events with which it is associated and at least an expectation that it is likely to be involved in many functions rather than just one physiological process. The degree of complexity of this lipoprotein class has been addressed in detail in another chapter; suffice it to remind the reader that HDL comprises several subclasses varying in size and composition and that the existence of these various particles is inextricably related to an organised lipid transport system. A key feature of the transport system is remodelling and probable recycling of the macromolecules in response to the action of lipolytic enzymes and lipid transfer proteins. These plasma factors orchestrate an elaborate exchange and transfer of both lipid and apolipoprotein moieties producing a number of identifiable subclasses of HDL particles that appear to have different functions in the body. The rearrangement of HDL molecules occurs concomitantly with changes in the composition of the other lipoprotein classes resulting from an exchange or transfer of components between VLDL, LDL or chylomicrons: these interactions are integral to the transport of lipid in the blood, producing and maintaining a balance between a supply of energy to all tissues, and the removal of excess fat from the body. The plasma factors referred to above play a major role in modulating these pathways, and much information is now available about their regulation by hormones and diet. Implicit in the above consideration of the lipid transport system is the existence of biological systems that regulate the entry and exit of metabolites (that comprise the circulating lipoproteins) into and out of the circulation. Apart from some notable exceptions (e.g., the LDL receptor) there is much less certainty about the nature of the cellular as opposed to the intraplasma processing of lipoproteins, a statement particularly relevant to our understanding of HDL metabolism. Not surprisingly, this has been the subject of considerable interest to
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researchers for some time, although when compared with other biochemical mechanisms, advancement has been slow. This chapter is concerned principally with the HDL receptor, although such a discussion would be incomplete without addressing briefly the involvement and nature of the ligands that bind to the putative receptors. Similarly, it is important to consider the range of biochemical events that are influenced as a consequence of the ligand (HDL)-receptor and for these reasons, the chapter is divided into 3 sections— • Identification, cloning and properties of candidate HDL receptors • Nature of the ligands recognised by HDL receptors • Biochemical processes affected by the HDL receptor interaction
IDENTIFICATION, CLONING AND PROPERTIES OF CANDIDATE HDL RECEPTORS Many cell proteins that bind HDL have been proposed as putative HDL receptors but definitive evidence supporting such claims remains unresolved. The search for cell surface receptors recognising HDL was motivated by earlier reports of specific HDL binding sites on different cultured cells including intestinal mucosal cells (Suzuki et al., 1983), fibroblasts (Biesbroeck et al., 1983), hepatocytes (Bachorik et al., 1982), adipocytes (Barbaras et al., 1986) and steroidogenic tissues (Chen et al., 1980). In most of these studies, it was apparent that a substantial proportion of the observed total binding was attributable to non-specific binding sites, despite the fact that sufficient quantities of unlabelled HDL were included in incubations designed to estimate the non-specific component of the interaction. This feature would provide a clue to the problems later encountered in attempts at purifying and cloning the elusive receptor. As shown in subsequent experiments, binding sites for HDL or its major apolipoprotein, apoAI, with parameters more convincing of the features of a physiological receptor were identified in liver plasma membranes (Morrison et al., 1992) and in hepatocytes (Barbaras et al., 1994). These sites, which are characterised by rapid dissociation of the ligand, binding HDL with high affinity (3×10−9 M), are only revealed by swift washing and removal of adsorbed HDL. Under these circumstances both the high affinity binding site and another of lower affinity (approx. 2×10−7 M), the latter possibly reflecting weaker (but not necessarily less physiologically significant) lipid-lipid or protein-lipid associations, are identified. The non-specific binding between cell membranes and HDL produces false positives to confound the interpretation of both attempted purification of the putative receptors and cloning strategies which depend on binding assays to identify only specific interaction.
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This feature has frustrated many previous attempts at identification and has contributed to the elusiveness of receptor characterisation. Nevertheless, as mentioned above, several candidate HDL receptors, initially identified as binding proteins have been isolated. Some have been purified and others, which have been cloned will be considered in detail in this first section. Earliest reports of candidate receptors were based on the use of ligand binding techniques to identify the presence of binding proteins in tissues or cultured cells, the assumption being that electrophoretic separation of partially purified membrane proteins would facilitate the isolation of specific binding proteins and provide a useful assay to monitor the progress of their purification. A binding protein of Mr. 78,000 was isolated from adrenal cortical membranes (Fidge et al., 1985), and another from sheep kidneys (Fidge, 1986). Although further attempts at purification were hampered by loss of activity, the size of this protein, its presence in steroidogenic tissue and the demonstration that binding activity was weakened following reduction, suggest in hindsight that this protein was SR-B1. Other HDL binding proteins of larger size, ranging between 100–120 kDa, were found in various cells including fibroblasts and endothelium, (Graham and Oram, 1987) adipocytes (Barbaras et al., 1990), and hepatocytes (Tozuka and Fidge, 1989). Some laboratories identified two binding proteins in the same cells (Bond et al., 1991) raising the possibility that more than one class of HDL receptors exists. The fact that candidate receptors were identified in a variety of cells, particularly cells from tissues known to process HDL in the body, was consistent with the theory of reverse cholesterol transport, in which it was anticipated that cholesterol transfer to HDL from peripheral cells would involve receptor recognition of HDL, a preferred acceptor of cell cholesterol. Cholesterol associated with HDL would then be delivered to the liver through a mechanism that most probably also depends on specific recognition through the same or a different receptor. Evidence was produced to support the suggestion that these membrane proteins were acting beyond the limits of mere binding agents, exhibiting some of the properties expected of physiological receptors. Graham and Oram (1987) found that the “binding activity” of an HDL binding protein identified in fibroblasts, bovine aortic endothelial cells and hepatocytes was enhanced several-fold when human arterial smooth muscle cells were pre-treated with cholesterol; the expression of this 110 kDa protein appeared to be stimulated in a dose-dependent manner by cholesterol loading. That a protein was responsible for the recognition of HDL was strengthened by the observation that trypsin treatment of the cells abolished binding activity. To test the hypothesis that this protein was a candidate HDL receptor, the laboratory (McKnight et al., 1992) isolated a cDNA clone encoding the novel 110 kDa binding protein, designated HBP, and
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demonstrated that HBP mRNA expression in cultured cells increased in response to cholesterol loading, (see Table 7.1 ). The authors noted that the predicted structure of HBP revealed neither the presence of a classic hydrophobic transmembrane spanning sequence nor clearly defined cytoplasmic or extracellular domains that characterise known receptors. Seemingly attached to the cell surface, it most likely
Figure 7.1 Schematic representation of three HDL receptors which have been cloned and sequenced. HBP (vigilin) is most likely attached to the cell surface. It binds HDL and is upregulated by cholesterol loading of cells. SR-B1 most likely traverses the membrane twice, the NH2- and COOH-termini both within the cell. It acts as a docking protein and mediates both selective transfer of cholesteryl ester into cells and cholesterol efflux from cells in the presence of HDL. HB2 (ALCAM) has one transmembrane region and a short cytoplasmic tail, the larger NH2-terminal domain being extracellular. HB2 binds HDL and is down regulated when cells are cholesterol loaded. Due to homo-or heterotypic interactions this adhesion molecule possibly increases macrophage migration into the vessel wall which, as suggested in the model shown, is reduced as a result of binding HDL. Like other adhesion molecules it may also activate cell signalling pathways.
performed as an anchor for HDL, enabling exchange of lipid to occur.
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(Figure 7.1) If anchored through glycosylphosphatidylinositol enriched domains there is a possibility for interaction with signalling molecules or caveolae, known to be sensitive to cell cholesterol content. Future investigations may reveal the existence of such a mechanism.
Table 7.1 Summary of the characteristics of HDL binding proteins that have been cloned
Receptor Reference Homologues Structure
Distribution Ligands Functio (Major) (e)
HBP(a)
Fibroblasts, HDL, endothelial apoAI cells, chondrocytes
McKnight Vigilin et al. (1992)
SR-BI(b) Acton et al. (1996)
HB2(c)
150 kDa,(d) 110 kDa, no transmembrane domain not glycosylated
CD36, CLA-I 82kDa, Steroidogenic HDL, Docking uptake; glycosylated, tissue liver LDL, acylated, modified efflux colocalised LDL with caveolae, apoAI, two ApoAII, transmembrane apoC domains
Matsumoto ALCAM, et al. BEN (1997)
100 kDa, glycosylated, one transmembrane domain
Liver, brain, HDL, intestine, apoAI, lung, apoAII macrophages
a HBP=HDL binding protein; b SR-B , scavenger receptor, class B ; 1 1 c HB ; HDL binding protein 2 2 d unprocessed form; e
Unknow Choleste
tissue distribution of immunoreactive proteins.
The authors were also cautious about the interpretation of experiments testing the function of the protein when overexpressed in cells. There was some concern that several HBP products lacked HDL binding activity although it was suggested that cell processing may have produced different forms of HBP, not all of which are capable of binding HDL. It was also difficult to explain why HBP-specific antibodies were unable to block HDL binding to isolated cell proteins, or to immunoprecipitate binding activity from isolated membranes. They concluded that there was insufficient data to either support or
Unknow Choleste Adhesio
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exclude the possibility that HBP functions as an HDL receptor and that more studies were needed to assess its involvement in cell cholesterol metabolism. The group (Chiu et al., 1998) recently detected HBP (vigilin) in human atherosclerotic plaques but not in non-diseased coronary intima, consistent with earlier observations that HBP (vigilin) is upregulated by cholesterol loading of cells. This cautionary approach to the findings related to the function of HBP were understandable in view of the range of physiological outcomes that may have been expected to follow the interaction between HDL and its receptor. Oram et al., (1983) had in previous experiments, found strong evidence supporting a relationship between cholesterol loading of cells and a corresponding increase in HDLbinding sites that appeared to be “specific” as assessed by the customary binding experiments. Further, there was good reason to believe that a specific metabolic response was triggered by the interaction because HDL binding was accompanied by an apparent translocation of intracellular cholesterol to the plasma membrane (Oram et al., 1991). It was also speculated that cell signalling was involved in the process, because the investigators had obtained preliminary data implicating the activation of protein kinase C (Mendez et al., 1991), an enzyme known to initiate many signalling pathways following receptor-ligand association. Not inconsistent with a physiological role for the HDL-cell interaction in reverse cholesterol transport, this proposal broadened prevailing opinion that HDL acted merely as a passive acceptor of cholesterol which diffused into the aqueous extracellular fluid, functioning independent of specific processes or of direct contact between the lipoprotein and the cell membrane. What needs to be understood however is that the characteristics of the high affinity binding sites for HDL revealed by binding studies is clearly distinct from the lower affinity interactions that are associated with apoprotein-lipid interactions such as occur between apoAI and the phospholipid present in cell membranes. As will be discussed in following sections some of these uncertainties are approaching a stage of understanding following identification of other cell proteins that recognise HDL. Several lipoprotein binding sites with broad lipoprotein specificity, that include recognition of HDL have been reported in the literature. A putative receptor in pig hepatocytes was unmasked in binding studies performed by Bachorik et al. (1982) which appeared to mediate the uptake and degradation of apo-E free HDL as well as LDL. Since HDL is present at much higher concentrations than LDL in pig plasma, it was conceivable that this “lipoprotein binding site, LBS” may have acted as a functional HDL receptor despite its inability to distinguish the two lipoproteins. The affinity of the site(s) for binding either HDL
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or LDL was in the order of 2×10−8 M, similar to that observed for HDL binding sites in other tissues. Another report of a site which appeared to interact with all lipoproteins (Brissette and Noel, 1986) thus resembling this unspecified lipoprotein binding domain in terms of broad ligand specificity was found in rat liver membranes, although the authors considered this to represent lower affinity sites according to their binding parameters. The identity of the site(s) was not pursued and therefore the properties of this membrane component, presumably a protein, including its molecular size, remain unknown. Possibly however, these laboratories had made the first observation of the site subsequently identified as SR-B1, which as described below, appears to possess similar properties in terms of broad ligand specificity to those described for LBS. SR-B1 belongs to the class B scavenger receptor family of proteins which are characterised by an immunodominant ligand binding domain (residues 155–183; Puente et al., 1996) as in CD36, compared to the charged collagenous structure that typifies the class A type I and II macrophage scavenger receptors. Class B also differ from class A scavenger receptors in terms of binding specificity, the main difference being that the large number of polyanionic ligand binding sites in class A proteins are not present in class B receptors. CD36 was isolated as a result of expression cloning of oxidised LDL receptors (Endemann et al., 1993) but like other scavenger receptors, exhibited broad specificity and was expressed in a variety of tissues. While investigating the physiological properties of CD36, in a variant CHO cell line using acetylated LDL as ligand Acton et al. (1996) by expression cloning, isolated the cDNA for a new member of the CD36 family of membrane proteins, named SR-B1. Initially, it was found to bind native and modified lipoproteins, including LDL, but as reported in a subsequent study, in which SR-B1 was transfected into LDL receptor-negative CHO cells, this scavenger receptor bound HDL with “high affinity” and mediated the selective transfer of cholesteryl esters into the cells (see Table 7.1). Whereas approximately 20% of the HDL cholesterol added to the medium was taken up by SR-B1 transfected cells, less than 0.5% of the apolipoprotein was internalised, indicating that whole HDL particles were not endocytosed following receptor recognition. These findings are essentially similar to those first reported by Pittman et al. (1987) who demonstrated that the selective uptake of cholesteryl esters is evident in a variety of cultured cells, is not endocytotic and represents a net mass transfer and not mere exchange of lipid components. Pittman et al. (1987) made no attempt to identify or isolate the membrane component responsible for the selective mode of HDL metabolism. Similarly, Brissette et al. (1996) demonstrated that the site previously termed “LBS” (see above) appeared to be responsible for the selective uptake of LDL cholesteryl esters by
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HepG2 cells and since 45% of the total cholesteryl ester taken up was hydrolysed, LBS was clearly physiologically significant. Attempts to isolate LBS or estimate its molecular mass by ligand-blotting were unsuccessful, but the exceptional similarities in properties suggest that LBS and SR-B1 are identical or close members of the same family of scavenger receptors. The structure of CD36 has been somewhat controversial although recent studies have strengthened the notion that both the N-terminus and the C-terminus are cytosolic. Confirmation of this structural model followed observations of Tao et al. (1996) who found two sites of palmitoylation at cysteine residues at the extreme of the N-terminus (residues 3 and 7) and the C-terminus (residues 464 and 466). These termini flank the two hydrophobic domains of CD36 and it is well established that the bulk of the segment between these two hydrophobic (transmembrane) regions is extracellular. The role of palmitoylation in the function of CD36 is not known but the attachment of palmitate may assist in targeting proteins to membranes. SR-B1, like CD36 is also fatty acylated at Cys 462 and Cys 470 in the C-terminal cytoplasmic domain and colocalises with plasma membrane caveolae and is copurified with caveolin (Babitt et al., 1997). These membrane domains may play an important role in SR-B1 mediated lipid transfer between cells and lipoproteins, although new studies show that cells transfected with SR-B1 but not CD36 efficiently transfer lipid, a finding attributed to the presence of specific extracellular domains in SR-BI, not present in CD36 (Gu et al., 1998). The identification of SR-B1 as a candidate HDL receptor was followed rapidly by a series of reports establishing its receptor status and providing clues to its role in HDL metabolism. An analysis of mouse tissues by immunoblotting showed that SR-B1 was highly expressed in liver, ovary and adrenal gland with lower expression in the testis and mammary gland and only a trace amount in the heart (Acton et al., 1996). This distribution is consistent with the proposal that the main function of SR-B1 is to mediate delivery of cholesterol to steroidogenic tissues (as a precursor of steroid hormones) and to the liver as a final process of reverse cholesterol transport, a proposal supported by subsequent findings that treating rats in vivo with oestrogen increased expression of the receptor in the adrenal gland and corpus luteum but decreased it in the liver (Landschultz et al., 1996). Independent confirmation for the presence of SR-B1 in the rat was reported by Mizutani et al. (1997) who used a subtraction cloning procedure to isolate a gonadotropin inducible gene from rat ovaries. The cDNA was shown to be homologous to SR-B1. As with other steroid responding tissues, ovarian SR-B1 was strongly induced by the corresponding pituitary hormone. Since in the study of Landschultz et al. (1996) administration of human chorionic gonadotropin also
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induced a dramatic increase in SR-B1 in the Leydig cells of the testis, these reports taken together provide strong evidence for an association between SR-B1 regulation and fluctuations in steroid hormone production. Further confirmation of its role in steroidogenesis came from immunohistochemical localisation of SR-B1 in murine adrenal cortex to the surface of cortical cells: the authors (Rigotti et al., 1996) also demonstrated that the receptor expression was induced in vivo in the adrenal cortex after the administration of ACTH to mice. The initial observations on the tissue distribution of SR-B1 predicted that this HDL receptor would play a significant role in steroidogenic tissue and the studies summarised above have served to consolidate this position by providing strong evidence for the regulation of SR-B1 by key steroid hormones. Upregulation in expression of the receptor makes provision for extra supplies of precursor cholesterol by tissues challenged to synthesise additional steroid hormones. Sometimes overlooked is the fact that in rodents, HDL carries most of the circulating cholesterol. This contrasts with humans where LDL is the chief sterol transporter and where most tissues synthesising steroids obtain supplies additional to de novo synthesis through regulation of the LDL receptor. A number of laboratories have demonstrated in vitro and in vivo that LDL and not HDL is the preferred source of cholesterol by steroidogenic tissue. With cultured adrenal cells, LDL uptake and catabolism was stimulated 5–6 fold by the addition of ACTH to the medium while the uptake and degradation of HDL was not influenced. Cholesterol from HDL3 (devoid of apoE) was not used for adrenal steroid hormone production (Carr et al., 1980). To test the role of lipoproteins in steroidogenesis in humans, Illingworth et al. (1982b) studied corticosteroid synthesis in patients with phenotypic abetalipoproteinaemia, a condition characterised by extremely low or by absent plasma LDL. Although cortisol production appeared normal in these patients, possibly as a result of increased de novo synthesis or uptake of HDL, chronic stimulation of adrenal hormone production by ACTH resulted in subnormal production of corticosteroids. In humans therefore, there seemed to be little evidence for a pituitary stimulated upregulation of a pathway, that under these chronic conditions, provided extra precursor sterol via HDL. A female patient with hypobetalipoproteinaemia, also showed impaired steroid synthesis (Illingworth et al., 1982a). Under normal conditions, the corpus luteum produces copious quantities of hormone, reaching approximately 30–40 mg progesterone/day from 1 g of tissue, during the midluteal phase of the menstrual cycle. She had a marked reduction in progesterone levels despite normal levels of circulating HDL providing a useful model for investigating the relationship between lipoprotein cholesterol supplies and steroidogenesis. Taken together, all of these studies in humans provide little evidence for the utilisation of HDL cholesterol by
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steroidogenic tissue. By default, it seems as though HDL cholesterol may substitute for LDL in cases of LDL deficiency but this may not be adequate for maintaining steroidogenesis under conditions of sustained stimulation by pituitary hormones such as ACTH. To evaluate the precise role of SR-B1 in delivering HDL cholesterol to tissue in human subjects will require further investigation. New studies have provided evidence of another role for SR-B1 in reverse cholesterol transport, possibly relevant to the initial phase of cholesterol entry into the pathway. Ji et al. (1997) found that cholesterol efflux was stimulated 3–4 fold in CHO cells stably transfected with murine SR-B1 and that the efflux rates correlated with the degree of overexpression of the receptor and the HDL concentration in the medium. The functional significance of this finding was underscored by the additional observation that the rate of cholesterol efflux to HDL was positively correlated (r=0.859) with SR-B1 expression in six different cell types, ranging from peritoneal macrophages to fibroblasts, hepatoma and adrenal cells. These results, suggestive of the influence of SR-B1 in removing cholesterol deposits from tissues such as the arterial wall, received support from in situ hybridisation experiments demonstrating the presence of SR-B1 in macrophage foam cells present in atheromatous lesions of apoE knockout mice. That SR-B1 can apparently mediate both cholesterol efflux from cells, such as foam cells, while also promoting selective transfer of cholesteryl esters into cells of other tissues, would endorse the proposal that this receptor protein alone can facilitate all the cellular phases involved in reverse cholesterol transport with the caveat that its action in humans mirrors its performance in rodents. In this respect, the model proposed by Steinberg (1996) of a docking action for SR-B1 that results in the anchoring of HDL to cells receives an experimental imprimatur (Figure 7.1). By a mechanism yet to be identified, it appears that HDL docks onto SR-B1, and, so captured, either donates or accepts cholesterol from cells, the direction of movement of sterol presumably determined by a concentration gradient: such a chemical gradient may not be limited to sterol alone but may include other membrane components such as phospholipid. Ensuing changes in ratios of cholesterol: phospholipid, and alterations in fatty acid composition that affects spatial distribution of cholesterol in cell membranes (or of corresponding alterations in HDL composition), may act as signals to change bi-directional diffusion to a more productive net movement of sterol between cells and HDL. The quantitative net movement of lipid will also depend on the level of SR-B1 expression which in turn will undoubtedly be dependent on gene regulation including that already observed with steroid hormones. Some information supporting this concept has already been reported.
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Wang et al. (1996) have found in vivo evidence that SR-B1 is a functional receptor under feedback control. Using apoAI or hepatic lipase knockout (KO) mice as models of cholesterol depletion, they measured adrenal SR-B1 mRNA and protein levels using an SR-B1 antibody and found a substantial increase in expression of the receptor in apoAI-KO but not in apoAII, apoE or LDL receptor KO mice. No differences were seen in SR-B1 expression in the liver, presumably because depletion of cholesterol in liver is less severe than in adrenals or else SR-B1 is not such an important determinant of sterol levels in liver. A similar level of up-regulation (approximately 3–4 fold) of SRB1 expression was observed in female but not male hepatic lipase KO mice, in which adrenal stores of free and esterified cholesterol were significantly depressed. Hepatic lipase also mediates the uptake of cholesterol and the selective transfer of cholesteryl esters into tissues, and not surprisingly the absence of this enzyme depletes adrenal sterol stores, also resulting in increased expression of SR-B1 to compensate for the loss. Further evidence that SR-B1 expression is under feedback control was obtained when mice were stressed by cold swimming, a challenge which is known to stimulate ACTH release and corticosteroid synthesis, with a substantial depletion of adrenal cholesterol stores. The challenged mice showed a two-fold increase in SR-B1 mRNA over untreated mice. Using a different approach, in which SR-B1 was overexpressed in mice, Kozarsky et al. (1997) found that increased levels of this receptor resulted in a dramatic decrease in both HDL cholesterol and apoAI levels and a substantial increase in biliary cholesterol. These studies were performed in mice transiently overexpressing SR-B1 following infection with a recombinant, replication defective adenovirus, in which >99% of transgene expression is localised to the liver. In view of this non-physiological mode of expression (exclusive to the liver) and considering the experiments described above where cholesterol depletion in apoAI KO mice had no effect on liver expression of SRB1, further data on regulation of liver SR-B1 is needed to assess its role. Nevertheless, these studies have highlighted another potentially important function for SR-B1 which involves controlling cholesterol concentrations in the bile, with potentially important consequences in the pathogenesis of atherosclerosis and gallstone formation if applicable to humans. CLA-1, a human homologue of rodent SR-B1 has recently been cloned and sequenced (Murao et al., 1997). Sharing 81% homology with SR-B1, CLA-1 also mediates uptake of cholesteryl esters and is abundant in the adrenal gland, liver and testis. In addition, CLA-1 is also present in monocytes and it recognises apoptotic thymocytes, suggesting it may play an additional role in processing damaged cells. Alternative splicing of SR-BI precursor transcripts produces a variant receptor, (SR-BII) which also mediates selective
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lipid transfers but at lower effeciency (Webb et al., 1998). Two other membrane proteins that bind HDL have been isolated and purified from rat liver (Tozuka and Fidge, 1989; Hidaka and Fidge, 1992). They were named HB1 and HB2 with Mr. of 120 and 100 kDa respectively and since the expression of both proteins was down regulated by 50–60% following administration of simvastatin to rats (Mathai et al., 1990), further investigations were undertaken to explore their role in HDL metabolism. Although HBl, present in low amounts and possibly unstable to extensive purification procedures has not been sequenced, HB2, the more abundant of the pair has now been cloned and sequenced (Matsumoto et al., 1997). It is not structurally related to either of the HDL receptors described above, viz., HBP or SR-B1, but belongs to the immunoglobulin superfamily and bears striking resemblance to the adhesion molecules ALCAM (93% homology) and BEN (70% homology) (see Table 7.1). Apparently these proteins, originally identified as adhesion molecules have alternative functions in cell physiology. ALCAM was isolated by immunoabsorbance (Bowen et al., 1995) from a breast carcinoma cell line (HBL-100) that expressed high levels of both a CD6 ligand and an antigen recognised by the antibody J4–81. Finding significant homology in this protein with BEN, a chicken neural adhesion molecule (Pourquie et al., 1992). Bowen et al. (1995) used BEN DNA fragments to screen a PHAactivated human T-cell cDNA library which resulted in the cloning and sequencing of ALCAM. To determine whether HB2 remained a candidate for an HDL receptor, COS, CHO or HepG2 cells were transfected with HB2. Cells that expressed HB2 showed 80–100% increases in HDL binding and ligand blots of membranes isolated from these cells confirmed the findings by revealing substantially enhanced HDL interaction with a band corresponding to HB2. A more clinically interesting observation was made when THP-1 cells, a monocytic cell line, were induced to differentiate by treatment with PMA. HB2 mRNA, hardly detectable in undifferentiated cells, was strikingly elevated following transformation of THP-1 cells into macrophages, and ligand blots of membranes taken from these transformed cells showed considerably higher association of HDL to both HB1 and HB2 than with untreated cells. Furthermore, when these transformed cells were incubated with acetylated LDL, thereby increasing their cholesterol content, a dose-dependent decrease in HB2 mRNA was observed, (Matsumoto et al., 1997) suggesting some association, either direct or indirect, between HB2 expression and cholesterol metabolism. This suggestion strengthened our previous observation that simvastatin treatment caused the downregulation of HB1 and HB2 in rats and another more recent demonstration of a similar finding in simvastatin treated rabbits (Fujiwara et al., unpublished). Further work aimed at identifying biochemical pathways
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that may be connected with these observed changes in HB2 expression and cholesterol metabolism is planned but preliminary studies appear to exclude selective uptake of cholesteryl ester from HDL as a process mediated by HB2 (Matsumoto et al., 1997). HB2 like ALCAM is widely distributed amongst many tissues in the body (ibid.). HB2 was detected in the lung, liver, intestine and in smaller amounts in the kidney, ovaries and testis, but the HB2 antibody did not detect this antigen in heart, spleen, thymus or brain. HB1 on the other hand appeared to differ slightly in tissue distribution because strongest signals (on Western blots) were observed in membranes isolated from the spleen, but weaker although detectable quantities were seen in the liver, intestine and lung. This difference in tissue distribution together with previous findings that HB1 and HB2 do not immunochemically cross-react (Tozuka and Fidge, 1989) indicates that the two proteins have different functions, possibly involving distinct roles in HDL metabolism. The distribution of HB2 mRNA, although similar, was not identical to HB2 protein distribution, since in the rat it was strongly expressed in the brain, possibly representing a rat homologue of BEN, known to be present in neuronal tissue. In fact human HB2 mRNA was expressed most strongly in the brain, then prostate, small intestine and liver with low levels in the lung. The structural features of HB2 are consistent with a “receptor” role for this protein, based on available structural information on this group of membrane proteins of the IgG superfamily. They are characterised by the presence of a 32-amino acid cytoplasmic tail (C-terminus), a 24amino acid hydrophobic transmembrane domain, and approximately 500 residues of an extracellular domain terminating in the NH2 residue. There are eight potential N-glycosylation sites, all extracellular, at residues 95, 167, 265, 306, 361, 457, 480, and 499. Although most of these sites in isolated HB2 appear to be glycosylated, their function remains unclear, since deglycosylation did not appear to affect HDL binding according to previous ligand blot studies (Hidaka and Fidge, 1992). Provisional PKC phosphorylation sites were also found at residues 8, 73, 74, 209, and 421 but since these are extracellular, their signalling potential is unknown, although they may become active if HB2 is internalised. The increase in expression of HB2 following PMA treatment of THP-1 cells raises the consideration of another role for this protein in the context of HDL function and its apparent protection against heart disease. HB2/ALCAM is an adhesion molecule and if it is upregulated following, for example, cytokine induced differentiation of monocytes into macrophages, it probably contributes, with other known adhesion molecules, to vascular remodelling and an initiation of atherosclerosis. HDL, or more likely, particular subclasses of apoAI-rich particles considered to be antiatherogenic, may compete with adhesion sites by
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binding to HB2 with high affinity thereby reducing the migration of macrophages into the arterial wall (Figure 7.1). The influence of HDL in this capacity could be quite pronounced considering the tendency of these adhesion molecules to self-associate. HB2/ALCAM is known to form multimers in solution (Bowen et al., 1996) so presumably forms oligomers on cell surfaces providing an amplification of reactive sites that would increase the effficiency of homo- or heterotypic cell adhesion. HDL binding would, by disrupting oligomerisation, decrease the high avidity interactions between neighbouring cells and if this involved migration of macrophages into the intima, reduced foam cell formation would achieve some protection against damage to the vessel wall. This alternative scenario is entirely consistent with the observed antiatherogenic properties of HDL and not mutually exclusive to its other defences that include an active participation in reverse cholesterol transport. Together with the observation that HDL inhibits the expression of soluble adhesion molecules such as VCAM and ICAM (Cockerill et al., 1995), a combined reduction in cell adhesion due to HDL actions may provide a formidable offensive against arterial wall disease. In a later section of this chapter, reference will be made to new and emerging evidence that the interaction between the ligand, HDL or its apolipoprotein moiety, and its candidate receptors, may stimulate biological events through biochemical signalling. In this context, it is useful to point out that some members of the adhesion molecule family act as signalling receptors: possibly, HDL interaction with these receptors promotes transduction pathways that have ultimate antiatherogenic effects or inhibits others which tend to produce atherogenic metabolites. Future research should unravel speculative from valid biological events involved in these pathways.
NATURE OF THE LIGANDS RECOGNISED BY HDL RECEPTORS In writing about HDL receptors, it would be incomplete not to consider the nature of the molecules that bind to them, a consideration that particularly applies to HDL due to its complex nature. And while there is a consensus that apoAI binds to candidate receptors, the extent to which other apolipoproteins participate in binding or share common receptor binding domains remains controversial. That fact that subclasses of HDL (see other chapters) varying in size and composition exist, encourages investigators to consider the biophysical influences of shape and composition on protein conformation that ultimately affects receptor binding domains, pre-cedents for which exist with other receptor-ligand systems. Moreover, the metabolic destinies of HDL
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subpopulations differ, their fate probably determined in part at least by the cells, and their receptors, to which they bind. The origin or formation of some HDL particles may also depend on cell processing and the presence of receptors or docking proteins to anchor them, enabling the necessary exchange or transfer of components between tissues and nascent HDL. Early suspicions that HDL2 and HDL3, the most conspicuously different HDL subclasses present in plasma in terms of size and composition, would most probably interact with different receptor sites on cells, was not supported by laboratory experiments. There was some justification for the belief that HDL3 or subpopulations within this subclass, which are smaller, depleted of lipid including cholesterol and consequently better acceptors of cell cholesterol, would participate in the reverse cholesterol process at a different level than HDL2. These latter particles are larger, lipid-rich particularly in cholesteryl ester following LCAT reaction, less efficient in vitro acceptors of cholesterol and presumably destined for the final stages of reverse cholesterol transport which would involve discharging their overloaded sterol cargo in the liver for eventual excretion from the body. Thus the role of one as an acceptor and the other as a donor could be fulfilled by the action of different receptors, where one would anchor the particle while it loads cholesterol, but a different receptor may initiate unloading at the other end by a process involving uptake and endocytosis of the particle. However, early studies on HDL binding to cells demonstrated that unlabelled HDL2 and HDL3 competed equally for binding of 125I-labelled HDL3 despite the fact that HDL2 is far less efficient than HDL3 at promoting cholesterol efflux from cells (Brinton et al., 1986). It appears therefore that moieties shared in common between the two particles, such as the apolipoproteins, may be the most significant determinants of binding, possibly to the same sites and that the differences in sterol content determine the direction of the diffusion gradient and whether or not particles act as acceptors or donors of cell cholesterol. The recent demonstration that SR-B1 can mediate both cholesterol efflux as well as selective transfer of cholesteryl esters into cells, together with the fact that this receptor exhibits broad ligand specificity (Xu et al., 1997) provides a plausible explanation for those earlier observations. Alternatively the apparent similarity in binding of HDL2 and HDL3 to cell sites may simply demonstrate that the apolipoproteins interact with receptors that are not determinants of cholesterol flux but of other biochemical events, a proposal that is discussed in some detail in following sections of this chapter. Early attempts to identify which HDL apolipoprotein served as ligand for the HDL receptor(s) (Fidge and Nestel, 1985), revealed that both AI and AII apolipoproteins appeared to be involved. Some laboratories reported sites that specifically recognised only apoAI
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(Rifici et al., 1984 Schmitz et al., 1985; Fong et al., 1987) while others found broader specificity since apolipoproteins AI, AII and C appeared to be recognised by HDL “receptors” (Hwang and Menon, 1985). These data, however, must be interpreted cautiously because of the instability of the ligand preparations in some experiments and the uncertainties inherent in methods for comparing binding parameters in others. For example, where artificial lipid-protein complexes were used as competitors for radiolabelled ligands, a fairly rapid exchange of apolipoproteins or phospholipid that is known to occur between these complexes may have resulted in the formation of mixed, undefined lipoproteins precluding a definitive interpretation of the results. The alternative approach of probing suspected ligand sites with antibodies has pitfalls also, including possible steric hindrance if IgG is used, and the uncertainty even with polyclonal preparations, that epitopes associated with binding sites are represented in the antibody mixture. Recognising these constraints, Vadiveloo and Fidge (1992) revisited the problem using ligands that were considered to be more physiologically and structurally valid for use in experiments designed to compare binding parameters between different HDL particles. Particles containing only apoAI or AII were prepared that in size, protein mass and lipid composition resembled HDL3. It was reasoned that these particles overcame all the shortcomings referred to above and in addition, by minimising variation in size and composition and therefore the shape of the particles, that apolipoprotein conformational changes, known to be influenced by the above parameters would be minimally affected. With this approach it was found that HDL3 containing only apoAI, or only apoAII, both bound to putative receptor sites on bovine aortic endothelial cells and isolated membranes, but with different characteristics. ApoAI-HDL3 bound with higher capacity (3–4 fold) than apoAII-HDL3, but with only half the affinity of apoAIIHDL3. Since ligand blots indicated that similar membrane binding sites were involved, a model was constructed which fitted the binding parameters and indicates that apoAII-containing particles can occupy up to four times the number of receptors as apoAI-only particles, and consequently fewer apoAII-containing particles are required to saturate the receptors as reflected in the experiments which showed their lower binding capacity. These biochemical findings lend some support to clinical/epidemiological observations that were reported at about the same time. Puchois et al. (1987) observed that elevated levels of plasma HDL3 containing mainly apoAI protect against heart disease and others found in vitro evidence that apoAII richer HDL are less effective at promoting cholesterol efflux than particles rich in apoAI. Since apoAII appeared to act as an antagonist for apoAI particles, and apoAI rich HDL seem to be more efficient acceptors of cell cholesterol,
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the model (Vadiveloo and Fidge, 1992) may contain the key to the problem. It is important to note that not all investigators have demonstrated marked differences in the ability of apoAI or apoAII rich HDL particles to mediate cholesterol efflux. Johnson et al. (1991) and von Hodenburg et al. (1991) reported that both were equally effective in transferring cholesterol from Fu5AH hepatoma cells or macrophages. However, another line of evidence exists which makes it important to distinguish between efflux from plasma membrane or from intracellular sources (Oram et al., 1991; Sviridov and Fidge, 1995), one (plasma membrane), being less dependent on specific interactions than mobilisation from intracellular pools which appears to involve apolipoprotein binding to initiate the process. Whether or not the mechanisms concerned with cholesterol efflux depend on apolipoprotein-receptor interactions, work carried out in transgenic mice overexpressing human apoAI showed that the animals were protected against the atherogenic effects of lipid rich diets (Rubin et al., 1991) whereas apoAII transgenic animals were not spared from the development of atherosclerosis (Warden et al., 1993). Further, it was found that amongst groups of animals in which LpA-I: LpAII ratios varied, atherosclerosis was least evident in mice where LpA-I rich HDL particles predominated (Schultz et al., 1993). If both apolipoproteins equally promote cholesterol efflux from cells, then it follows that other biochemical pathways, which distinguish between the actions of AI and AII apolipoproteins, are regulated to some extent by apoAI, achieving the antiatherogenic effects observed in the in vivo experiments described above. However, attempts to assign specific ligand properties to any one apolipoprotein have not yet been successful. One explanation for this is that all potential candidate HDL receptors exhibit a very broad specificity for apolipoproteins, or, that most of the physiological events associated with lipid exchanges or transfers between HDL and cells do not involve receptor interaction, or at least are not triggered by a specific amino acid sequence of a particular apolipoprotein, a concept proposed by Segrest et al. (1992), Mendez et al. (1994), Yancey et al. (1995) and Leblond and Marcel, (1991). The view shared between most of these investigators is that cooperativity between class A amphipathic helices, possibly requiring at least four tandem repeat helices, is structurally all that is needed to achieve their lipid efflux properties. At issue however is not whether the amphipathic repeated structures shared in common between some apolipoproteins can bind lipid or interact with membrane lipid or protein (or both) but whether the relatively low affinity (classified as “high affinity” in some reports) sites identified by the helices present in the model synthetic or native peptides used in these studies, represent physiological sites capable of stimulating downstream cellular events.
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Other investigators have reported the existence of two HDL binding sites on cell membranes. In one study, time course experiments were performed to measure association and dissociation rate constants to determine whether the high affinity site displayed mass action kinetics, a requirement for calculating affinity constants for putative receptor sites. This approach revealed the presence of two distinct sites (Morrison et al., 1992), one with a Kd of 0.94 ug/ml (3.1×10−9 M) and another low affinity site with a Kd=33ug/ml, the latter being in the range of published values for HDL binding sites. Proteolysis of HDL with trypsin abolished the high affinity but not the low affinity site suggesting the requirement for an intact peptide sequence. These studies were essentially confirmed by Barbaras et al. (1994) who further showed that this high affinity site recognised apoAI either in a lipid poor form or when present in HDL, while another lower affinity site was also revealed for HDL. The non specific characteristics of amphipathic helices that are commonly shared between apolipoproteins are unlikely ligands for this putative apoAI receptor. Biological events following binding, including the processing of the ligand (either HDL or apoAI) remain poorly understood and controversial. For every report claiming evidence of internalisation by endocytosis or alternative uptake mechanisms, there is one denying the existence of such pathways. A paucity of morphological data exists, presumably because HDL, unlike the more stable and larger LDL, is difficult to visualise, and resistant to complexing with electron opaque elements such as gold. Despite early claims to the contrary, no electronmicrographic evidence of gold-labelled HDL exists whereas LDL-gold complexes, easy to prepare, appear in many reports (Handley et al., 1981; Paavola et al., 1985). Any post-receptor events that follow HDL binding must therefore be detected by biochemical assays, immunochemical identification or attachment of fluorescent probes to trace movement of the ligand. Furthermore, the rapid dissociation kinetics characteristic of HDL high affinity binding and uptake require sensitive assays to follow its cellular localisation. With these constraints in mind, Garcia et al. (1996) using radiolabelled HDL recently observed a high level of HDL internalisation (100ng/mg cell protein) by HepG2 cells which corresponded to 45% of the total HDL associated with the cells within 15 min. and found that most of the radiolabelled apoA-1 of the HDL was associated with clathrin-coated vesicles. There was little evidence for the involvement of caveolae in the process. Competition by ligands of SR-B1 did not inhibit HDL binding, suggesting that at least in hepatocytes, sites other than SR-B1 mediate the uptake of HDL. Although there is much disagreement about the specificity and identity of the ligand(s) that contribute to HDL receptor processing by cells, there is a consensus that, even if not exclusively involved, apoAI
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mediates some of the action and is therefore a justifiable target for investigation of structural domains recognised by the receptor(s). Several groups have probed these potential apoAI sites with antibodies, or peptides of apoAI hoping to reveal active binding domains. Morrison et al. (1991), by direct binding studies with different cyanogen bromide fragments of apoAI incorporated into apolipoprotein-phospholipid complexes, found that the carboxyl terminal fragment bound with the highest affinity, the binding parameters resembling those of the intact apoAI molecule. Importantly, only the C-terminal fragment bound to HDL binding proteins on ligand blots thus eliminating proteinlipid association from the interaction. These findings were supported by other investigators (Dalton and Swaney, 1993) who fragmented apoAI by proteolysis and found that carboxyl terminal residues 149–219 were essential for cell membrane interaction, as well as lipid binding. Subsequent work showed that HDL binding to cell membranes could be inhibited by monoclonal antibodies that recognised epitopes positioned between residues 150 and 243 (Allan et al., 1993) and ligand blotting revealed that the same antibodies inhibited binding to the putative HDL receptors HB1 and HB2. In similarly designed studies however, Leblond and Marcel (1991) were not able to delineate a special cell binding domain using their panel of nine monoclonal antibodies which reacted against epitopes widely distributed along the AI apolipoprotein. Rather, they interpreted their results as being compatible with the concept of HDL binding to the cells via a nonexclusive interaction of each of the amphipathic -helical repeats of apoAI. Their monoclonal antibodies similarly affected the association of cholesteryl ester-labelled HDL with cells. While clearly different to the results reported above (Allan et al., 1993), it is possible that their source of receptor (cells) may have contributed to their observations. The interaction between HDL and whole cells presents a wider opportunity for diverse interactions to occur, particularly less specific lipid-lipid or lipid-protein associations, than those available in purified plasma membranes. Also the authors (Leblond and Marcel) did not test the inhibitory properties of their antibodies with purified or isolated putative receptors as was included in the studies of Allan et al. (1993). Several laboratories have probed apoAI with antibodies to gain information about sites that may regulate cholesterol efflux, the assumption being that HDL-receptor association precedes cholesterol acceptance by the lipoprotein. As we gain new knowledge about the events involved in the exchange or transfer of sterol between cells and plasma, it is clear that such an experimental approach can offer only a limited view of the process. As pointed out by Oram et al. (1991) and Sviridov and Fidge (1995), it is important to distinguish between cellular pools of cholesterol since not all are controlled by biochemical
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signalling pathways. Clearly, the movement of sterol from the cell surface is a passive process, influenced by the rate of diffusion into the aqueous phase (Phillips et al., 1980) whereas cell regulatory factors are involved in the mobilisation of intracellular cholesterol to prevent uncontrolled fluctuations in sterol concentration and accompanying changes in composition of cell membranes. Therefore, when attempting the identification of structural sites, such as receptor binding domains of apoAI in experiments where the activity is related to rates of cholesterol flux, cellular pools must be distinguished so that nonspecific processes, that may outweigh specific events, do not confound the result. This point is apparently not appreciated by many investigators who proceed with measure of total cholesterol efflux alone. Recognising these differences, Sviridov et al. (1996b) probed with monoclonal antibodies, sites of apoAI that may specifically mediate mobilisation of intracellular cholesterol, and found evidence that a central region involving residues 140–150, when masked by antibodies, inhibited the efflux of intracellular sterol to serum in a dose-dependent manner. None of the antibodies affected transfer of plasma membrane cholesterol. The region identified in these experiments was close to, or overlapped sites identified in other reports, namely apo AI137–44 and apoAI141–148 (Fielding et al., 1994) and apoAI135–148 (Luchoomun et al., 1994) or apoAI96–111 (Banka et al., 1994). The inhibiting antibodies used by Sviridov et al. (1996b) recognised epitopes within one amphipathic -helix, but it is unlikely that disruption of only one helical region would specifically affect this type of apoAI-membrane association, since many lipid binding helices remain intact and dispersed throughout the entire molecule. According to Mendez et al. (1994) or Davidson et al. (1994) one or two amphipathic domains are sufficient to promote cholesterol efflux. Amphipathic -helices, by virtue of their capacity for phospholipid, and the formation of apolipoprotein-phospholipid complexes which are essential prerequisites for sequestering membrane cholesterol (Forte et al., 1993), will most likely have influenced plasma membrane cholesterol, whereas a biologically active motif within the central apoAI region appears to influence intracellular cholesterol mobilisation. The fact that both the carboxyl terminus and a central region of apoAI could be involved in cholesterol efflux was confirmed by Sviridov et al. (1996a) using apo AI that had been systematically truncated to eliminate sequentially C-terminal portions of the molecule. The residues between 222–243 of apoAI were clearly important in phospholipid binding but truncation to either residues 135 or 150 completely restored cholesterol and phospholipid efflux suggesting that another cryptic binding domain had been exposed. It is clear, as with other examples of searches for active motifs in putative ligands that a
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series of point mutations needs to be systematically produced to identify active domains in apo AI. Another important consideration is the likelihood that domains of apo AI involved with cholesterol efflux may not coincide with those that bind to candidate receptors which signal biochemical pathways unrelated to cholesterol metabolism, or at least cholesterol transport. Now that some HDL receptors have been cloned, sufficient quantities of these proteins can be expressed to allow structural studies of interactive sites and precise identification of binding domains particularly those present in the ligand, apoAI
BIOCHEMICAL PROCESSES AFFECTED BY HDL RECEPTORS The least understood area in the field of HDL receptors is the nature of the post receptor events which occur following HDL binding. Apart from the observation that the degree of expression of SR-B1 correlates positively with cholesterol efflux and its transient overexpression in mice depletes plasma of HDL cholesteryl ester there is little other data showing cause and effect following interaction between HDL and candidate receptors. Even with SR-B1, there are no clues to the mechanisms involved in these sterol transfers, except that the membrane protein putatively acts as a docking region for the lipoprotein, providing an opportunity for cholesterol exchanges to occur; furthermore the direction of movement appears not to be regulated by receptor driven events but by a gradient that is dependent on the relative concentration of cholesterol between the ligand and cell membrane. HB2, a member of the immunoglobulin superfamily of membrane proteins that binds HDL, appears to be regulated by a number of biochemical events, some associated with cholesterol synthetic pathways or others related to cell differentiation (Matsumoto et al., 1997). Like many other adhesion molecules, HB2 (almost identical to ALCAM) may also trigger signalling pathways, but whether HDL binding initiates signal transduction is not yet known. However, there is a growing body of evidence for the activation of known signalling pathways following the interaction between HDL and cells, observations inevitably focusing attention on receptor activity. Since the receptors that have been cloned and sequenced and discussed in the previous section have as yet, not been attributed with signalling properties, there is further speculation that signalling receptors responding to HDL moieties exist but have not yet been identified. In this context it is intriguing to reconsider the association between putative receptors and apoAI. As discussed, a site in cell membranes exhibits high affinity for apoAI, characterised by fast associationdissociation constants (Morrison et al., 1992). Despite this binding site
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having all the hallmarks of a physiological receptor, and potentially being an important determinant of biochemical events concerned in lipid metabolism, scepticism prevails because even in some HDL deficient states, the circulating levels of HDL would fully saturate the receptors at all times, resulting in perpetual up- (or down) regulation of downstream events. However, apoAI, if it existed in a form dissociated from HDL, could satisfy the credentials of a ligand in many ways. First, it would be present at variable concentrations depending on the parent HDL, the state of which is influenced by the interactive processes of many factors (lipolytic enzymes, plasma factors like CETP, PLTP, diet etc.). In this context the concentration of lipid poor apoAI may reflect normal or dysfunctional lipoprotein states and through negative feedback control, influence pathways that restore (or maintain) balances in lipid metabolism. Evidence is also emerging that apoAI does exist in a lipid “free” or at least lipid poor form in low concentrations in human plasma (Neary and Gowland, 1987). These concentrations could fluctuate widely, depending on lipase activities and fat absorption (Liang et al., 1994). At the lower levels of apoAI in extracellular fluids than in blood or in trace amounts if apoAI is transiently dissociated and then rapidly reassociated with HDL, the high affinity sites recognising apoAI would not be “turned on” continuously. Instead, they would be activated at a critical mass of the ligand or by changes in its conformation that accompany the removal or addition of lipid, exposing receptor binding domains. (Figure 7.2). In fact changes in the net charge and structure of apoAI do occur in response to increases or decreases in cholesterol content of artificial “HDL” particles, the magnitude of these changes being considered sufficient to affect the interaction between HDL and cell surfaces (Sparks et al., 1993). Interaction between free apolipoprotein and cells is considered to be one mechanism for the formation of HDL (Hara and Yokoyama, 1991) although not all cells participate in this process (Komaba et al., 1992). The authors assume that the generation of HDL from extracellular free apolipoproteins occurs through non-specific processes but concede that protein-protein interactions may be involved (Hara and Yokoyama, 1991). In our laboratory, we have demonstrated that 14C-labelled apoAI is internalised by HepG2 cells (unpublished observations) in a manner suggestive of a regulated rather than a passive process. Holian et al. (1991) found that lipid-free apoAI, but not HDL-bound apoAI was phosphorylated by protein kinase C (PKC) in vitro, independent of calcium or lipids, suggesting that apoAI is a
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Figure 7.2 Scheme of a possible role for apoAI in cell lipid metabolism, based on separate, published data. During HDL remodelling by hepatic lipase or CETP action (a), apoAI is dissociated from the particle. (Liang et al., 1994). In the “free” form apoAI can bind to cell membranes and isolated candidate HDL receptors (Tozuka and Fidge, 1989). At least two sites have been implicated in apoAI-cell interactions (b), one at the COOH terminus (Allan et al., 1993) and others in a central region (Sviridov et al., 1996; Fielding et al., 1994). One, or more, receptors may be involved, and interaction between apoAI and HDL receptors may initiate changes in cholesterol metabolism. Speculation increases about an unidentified apoAI receptor (c) since an unidentified high affinity site for apoAI exists in cell membranes (Morrison et al., 1992; Barbaras et al., 1994). Sites at the COOH-terminus of apoAI are susceptible to proteolytic cleavage which may affect conformation and the binding capacity of apoAI. ApoAI is internalised by cells and in the lipid poor form, apoAI can be phosphorylated (Holian et al., 1991). The scheme shown suggests that apoAI, or degraded apoAI, may directly or indirectly, through interaction with receptors, initiate biochemical events within cells.
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novel cofactor of PKC. The addition of HDL, which inhibits apoAI phosphorylation, indicates that the presence of lipid masks the phosphorylation site on the apolipoprotein. Presumably, under physiological conditions, the reaction only occurs following dissociation of apoAI from HDL, possibly after receptor occupancy. Taken together with emerging evidence for signal transduction following HDL binding to cells and an expectation that the catabolism of apoAI either in the free or lipid associated form would be mediated by a specific process, the likelihood for the existence of an apoAI receptor is strengthened considerably. How convincing is the evidence that biochemical signalling is activated by HDL? As argued above, the fact that HDL circulates in plasma at concentrations above those needed for receptor saturation, does not negate a role for “active” HDL subclasses or moieties that reside at very low concentrations in the blood or extracellular fluids and which may have different functions. At least two biochemical processes have been identified that respond to HDL, or more probably apoAI, the first being mobilisation of intracellular cholesterol as discussed above and the other related to its capacity to stimulate several mitogenic effects (Biran et al., 1983; Favre et al., 1989, Jozan et al., 1985). ApoAI induced cholesterol efflux from cells is associated with activation of protein kinase C (Mendez et al., 1991). Recently, Garver et al. (1997) identified at least three phosphoproteins that were phosphorylated following incubation of skin fibroblasts with HDL. Two of these were phosphorylated only by HDL, not free apoAI, whereas one termed pp18 appeared to be responsive to HDL apolipoproteins. HDL was shown in a recent study to stimulate mitogen-activated protein kinases although the signalling pathways involved appear not to respond to HDL apolipoproteins and probably unrelated to apoprotein-mediated cholesterol translocation and efflux. Wu and Handwerger (1992) found that HDL and apoAI each caused a time and dose dependent increase in phosphorylation of a PMAinducible 80kDa protein in human trophoblasts, concomitant with an increase in human placental lactogen release. That PKC is involved in apolipoprotein-mediated cellular cholesterol efflux was strengthened by a recent report by Li et al. (1997) who used PKC inhibitors to selectively down-regulate PKC and found a substantially reduced cholesterol efflux to extracellular apoAI. Whereas the signalling events related to cholesterol efflux probably respond to low concentrations of active HDL moieties (e.g., apoAI), the strong mitogenic effect that has been observed with HDL in endothelial cells or smooth muscle cells increases up to 300–500 ug/ml HDL. An explanation for this phenomenon follows from recent observations by Walter et al. (1995) that HDL stimulates multiple signalling pathways in fibroblasts that appears to involve phosphatidylcholine (PC) turnover
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as well as phosphatidylinositol(PI) hydrolysis. The breakdown of PC followed HDL activation of phospholipases C and D, the former producing diacylglycerol and the latter phosphatidic acid, has important biochemical consequences since both products are known to activate second-messenger responses. The authors point out that whereas mitogen-induced hydrolysis of PI is rapid and transient, PC hydrolysis is sustained and that both are induced by growth factor-like activity of the HDL particle; thus mitogenic effects of HDL could be observed over a wide concentration range of HDL. In addition to the proposed down regulation of the membrane adhesion molecule HB2/ALCAM by HDL (discussed above), cytokineinduced expression of the soluble adhesion molecules VCAM and ICAM also appears to be regulated by HDL (Cockerill et al., 1995). The involvement of an HDL or apoAI receptor in this process is being investigated based on the hypothesis that HDL binding, via signal transduction, can confer gene regulation by altering the DNA binding of calcium-dependent transcription factors. In fact, endothelial adhesion molecule expression is coupled to events involving sphingosine kinase and NF-κB activation (Xia et al., 1998). Perhaps HDL inhibits these factors, thereby reducing VCAM and ICAM. Antiatherogenic effects of apoA-I include protection against cell death induced by oxidised LDL; since it appears to involve cell signalling (Suc et al., 1997) an apoA-I receptor, not yet identified may be responsible. Numerous biochemical pathways are affected by HDL. Some, though not all of them appear to be directly related to lipid metabolism, but all impinge in some way on atherogenesis, which will no doubt stimulate further attempts at unravelling the active receptor sites behind the pathways. These will probably involve detailed probing of sites present in receptors we already know about, and a continuing search for other membrane proteins which undoubtedly exist and play active roles in processing HDL. References Acton, S., Rigotti, A., Landschulz, K., Xu, S., Hobbs, H.H. and Krieger, M. (1996). Identification of scavenger receptor SR-BI as a high density lipoprotein receptor. Science , 271 , 518–520. Allan, C., Fidge, N., Morrison, J.R. and Kanellos, J. (1993). Monoclonal antibodies to human apolipoprotein AI: structural probes for the cellular binding domain of apoAI. Biochem J. , 290 , 449– 455. Babitt, J., Trigatti, B., Rigotti, A., Smart, E.J., Anderson, R.G.W., Xu, S. 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 , 13242–13249.
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Bachorik, P.S., Franklin, F.A., Virgil, D.G. and Kwiterovich, P.O. (1982). High affinity uptake and degradation of apolipoprotein Efree high density lipoprotein and low density lipoprotein in cultured porcine hepatocytes. Biochemistry , 21, 5675–5684. Banka, C.L., Black, A.S. and Curtiss, L.K. (1994). Localization of an Apolipoprotein A-I Epitope Critical for Lipoprotein-mediated Cholesterol Efflux from Monocytic Cells. J.Biol. Chem. , 269 , 10288–10297. Barbaras, R., Grimaldi, P., Negrel, R. and Ailhaud, G. (1986). Characterization of high-density lipoprotein binding and cholesterol efflux in cultured mouse adipose cells. Biochim. Biophys. Acta. , 888 , 143–156. Barbaras, R., Collet, X., Chap, H. and Perret, B. (1994). Specific Binding of Free Apolipoprotein A-I to a High-Affinity Binding Site on HepG2 Cells: Characterization of Two High-Density Lipoprotein Sites. Biochemistry , 33 , 2335–2340. Barbaras, R., Puchois, P., Fruchart, J.C., Pradines-Figueres, A. and Ailhaud, G. (1990). Purification of an apolipoprotein A binding protein from mouse adipose cells. Biochem. J. , 269 , 767–773. Biesbroeck, R., Oram, J.F., Albers, J.J. and Bierman, E.L. (1983). Specific high affinity binding of high density lipoprotein to cultured human skin fibroblasts and arterial smooth muscle cells. J.Clin. Invest. , 71 , 525–539. Biran, S., Horowitz, A.T., Fuks, Z. and Vlodavsky, I. (1983). High density lipoprotein and extracellular matrix promotes growth and plating efficiency of normal human mammary epithelial cells in serumfree medium. Int. J.Cancer. , 31 , 557–566. Bond, H.M., Morrone, G., Venuta, S. and Howell, K.E. (1991). Characterization and purification of proteins which bind high density lipoprotein. Biochem. J. , 279 , 633–641. Bowen, M.A., Patel, D.D., Li, X., Modrell, B., Malacko, A.R., Wang, W.C. et al. (1995). Cloning, Mapping, and Characterization of Activated Leukocyte-Cell Adhesion Molecule (ALCAM), a CD6 Ligand. J.Exp. Med. , 181 , 2213–2220. Bowen, M.A., Bajorath, J., Siadak, A.W., Modrell, B., Malacko, A.R., Marquardt, H. et al. (1996). The Amino-terminal Immunoglobulinlike Domain of Activated Leukocyte Cell Adhesion Molecule Binds Specifically to the Membrane-proximal Scavenger Receptor Cysteine-rich Domain of CD6 with a 1:1 Stoichiometry. J.Biol. Chem ., 271 , 17390–17396. Brinton, E.A., Oram, J.F., Chen, C.H., Albers, J.J. and Bierman, E.L. (1986). Binding of High Density Lipoprotein to Cultured Fibroblasts after Chemical Alteration of Apoprotein Amino Acid Residues. J.Biol. Chem. , 261 , 495–503. Brissette, L. and Noel, S.P. (1986). The effects of human low and high density lipoproteins on the binding of rat intermediate density lipoproteins to rat liver membranes. J.Biol. Chem. , 261 , 6847–6852. Brissette, L., Charest, M.C. and Falstrault, L. (1996). Selective uptake of cholesteryl esters of low-density lipoproteins is mediated by the
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lipoprotein-binding site in HepG2 cells and is followed by the hydrolysis of cholesteryl esters. Biochem. J. , 318 , 841–847. Carr, B.R., Parker, Jr. C.R., Milewich, L., Porter, J.C., MacDonald, P.C. and Simpson E.R. (1980). The role of low density, high density, and very low density lipoproteins in steriodogenesis by the human fetal adrenal gland. Endocrinology , 106 , 1854–1861. Chen, Y.I., Kraemer, F.B. and Reaven, G.M. (1980). Identification of specific high density lipoproteinbinding sites in rat testis and regulation of binding by human chorionic gonadotropin. J.Biol. Chem. , 255 , 9162–9167. Chiu, D.S., Oram, J.F., LeBoeuf, R.C., Alpers, C.E. and O’Brien, K.D. (1997). High-density lipoprotein binding protein (HBP) vigilin is expressed in human atherosclerotic lesions and colocalizes with apolipoprotein E. Arterioscler. Thromb. Vasc. Biol.. , 17 , 2250– 2358. Cockerill, G.W., Rye, K., Gamble, R.R., Vadas, M.A. and Barter, P.J. (1995). High-density lipoproteins inhibit cytokine-induced expression of endothelial cell adhesion molecules. Arterioscler. Thromb. Vasc. Biol. , 15 , 1987–1984. Dalton, M.B. and Swaney, J.B. (1993). Structural and funcitonal domains of apolipoprotein AI within high density lipoproteins. J.Biol. Chem. , 268 , 19274–19283. Davidson, W.S., Lund-Katz, S., Johnson, W.J., Anantharamaiah, G.M., Palgunachari, M.N., Segrest, J.P. et al. (1994). The Influence of Apolipoprotein Structure on the Efflux of Cellular Free Cholesterol to High Density Lipoprotein. J.Biol. Chem. , 269 , 22975–22982. de Crom, R.P.G., Haperen, R. van., Visser, P., Willemsen, R., Kamp, A. van der, W.M. (1994). Structural Relation Between HDL-Binding Proteins in Porcine Liver. Arterioscler. Thromb. , 14 , 305–312. Endemann, G., Stanton, L.W., Madden, K.S., Bryant, C.M., White, R.T. and Protter A.A. (1993). CD36 is a receptor for oxidized low density lipoprotein. J.Biol. Chem. , 268 , 11811–11816. Favre, G., Blancy, E., Tournier, J.F., Soula, G. (1989). Proliferative effect of high density lipoprotein (HDL) and HDL fractions (HDL1.2, HDL3) on virus transformed lymphoblastoid cells. Biochim. Biophys. Acta. , 1013 , 118–124. Fidge, N., Kagami, A. and O’Connor, M. (1985). Identification of a high density lipoprotein binding protein from adrenocortical membranes. Biochem. Biophys. Res. Commun. , 129 , 759–765. Fidge, N.H. and Nestel, P.J. (1985). Identification of apolipoproteins involved in the interaction of human high density lipoprotein 3 with receptors on cultured cells. J.Biol. Chem. , 260 , 3570–3575. Fidge, N. (1986). Partial purification of a high density lipoprotein binding protein from rat liver and kidney membranes. FEBS Letts. , 199 , 265–268. Fielding, P.E., Kawano, M., Catapano, A.L., Zoppo, A., Marcovina, S. and Fielding, C.J. (1994). Unique epitope of apolipoprotein A-I expressed in prebeta-1 high density lipoprotein and its role in the catalzyed efflux of cellular cholesterol. Biochemistry , 33 , 6981–
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8 Regulation of the Low Density Lipoprotein (B/E) Receptor Petri T.Kovanen* and Wolfgang J.Schneider Wihuri Research Institute, Helsinki, Finland, and Department of Molecular Genetics, Vienna Biocenter, University of Vienna, Vienna, Austria
LDL receptors from a wide variety of species are highly conserved class I membrane proteins with five domains, for which the relationships between structure and function are now well understood. Their most significant features are also conserved at the genomic level. The key elements regulating the promoter of the human LDL receptor gene are the so-called sterol-responsive elements (SREs), in particular SRE-1. Recent studies on the regulation of sterol metabolism have revealed that active soluble transcription factors are produced by a two-step proteolytic cleavage from membrane-bound precursors, known as the sterol-regulatory element binding proteins (SREBPs). The highly regulated first step in this proteolytic maturation of a transcription factor is exquisitely sterol-sensitive. The proteolytic activity of the enzyme catalyzing SREBP cleavage (SREBP cleaving activity, SCA) is modified by a SCAP (SREBP cleavage-activating protein); interestingly, the membrane-bound domains of SCAP and of another sterol-sensing protein, the HMG CoA reductase, share structural features. LDL receptor-mediated supply of cholesterol to cells is feed-back regulated, the key component being the sterol-sensitive transcriptional activity of the LDL receptor gene. The receptor itself has an important regulatory function: it keeps the level of cholesterol constant both intra- and extracellularly. The overall benefit from this LDL receptor-mediated regulatory system, the LDL receptor pathway, is the coordinated utilisation of the intra- and extracellular
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sources of cholesterol and maintenance of cholesterol homeostasis in the body. KEYWORDS: Cholesterol, DNA-Binding Proteins, Gene Expression Regulation, Lipoproteins, Promoter Regions, Receptors, LDL.
INTRODUCTION The LDL receptor is the key component in the feed-back regulated maintenance of cholesterol homeostasis in the body (Brown and Goldstein, 1986). In fact, as an active interface between the extra- and intracellular cholesterol pools, it is itself subject to regulation. LDLderived cholesterol and its intracellularly generated *Corresponding Author: Petri T.Kovanen, Wihuri Research Institute, Kalliolinnantie 4, Helsinki 00140, Finland
oxidized derivatives mediate a complex series of feedback control mechanisms that protect the cell from overaccumulation of cholesterol. First, LDL-derived sterols suppress the activities of 3-hydroxy-3methylglutaryl-CoA (HMG-CoA) synthase and HMG-CoA reductase, the two key enzymes in cellular cholesterol biosynthesis. Second, cholesterol itself activates the cytoplasmic cholesterol-esterifying enzyme acyl-CoA:cholesterol acyltransferase (ACAT; E.G. 2.3.1.26), which allows the cells to store excess cholesterol in the form of cholesteryl esters. Third, the cholesterol contained in LDL particles suppresses the synthesis of new LDL receptors, thus preventing further cellular entry of LDL and cholesterol overloading. The overall benefit from, and consequence of, this LDL receptormediated regulatory system, the so-called LDL receptor pathway, is the coordinated utilisation of the intra- and extracellular sources of cholesterol. Human fibroblasts and other mammalian cells are able to subsist in the absence of lipoproteins because they can synthesize cholesterol from acetyl-CoA. When LDL is available, however, the cells primarily use the LDL receptor to import LDL cholesterol, and their own synthetic activity is suppressed. Thus, within the cell, the level of cholesterol is kept constant even when the external supply of cholesterol in the form of lipoproteins can undergo large fluctuations. In vivo, the LDL receptors have two important functions: first, to supply cells with cholesterol; and second, to remove cholesterol-rich lipoprotein particles from the bloodstream, so preventing their accumulation in the circulation. These concepts have been derived from studies on cultured fibroblasts from normal subjects and from patients with familial
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hypercholesterolemia (FH). Lack of the above-described regulatory features in FH fibroblasts led to the conclusion that the abnormal phenotype is caused by lack of LDL receptor function, i.e., by an interruption of the normal LDL receptor pathway. In particular, the balance between the extracellular and intracellular cholesterol pools is disturbed. Clinically, the most important effect of LDL receptor deficiency is hypercholesterolaemia, which leads to accelerated development of atherosclerosis and its complications. In the following two sections, the LDL receptor is analysed in greater detail, with emphasis on the impact of mutations on its structure and function. In the section to follow, physiological regulation of LDL receptors is outlined.
RELATIONSHIPS BETWEEN THE STRUCTURE AND FUNCTION OF THE LDL RECEPTOR Studies at the levels of protein chemistry, molecular biology, and cell biology have led to a detailed understanding of the structure and function of the LDL receptor. Molecular cloning demonstrated that the mature receptor is a highly conserved, integral membrane glycoprotein consisting of five domains. In order of appearance from the amino terminus, these domains are: (1) a ligand-binding domain; (2) a domain having a high degree of homology with the precursor of the epidermal growth factor (EGF); (3) a domain that contains a cluster of O-linked carbohydrate chains; (4) a transmembrane domain; and (5) a short cytoplasmic domain. Until the three-dimensional structure of the 839residue receptor has been fully worked out, an arrangement of these domains as presented in Figure 8.1 may serve as a useful model. This domain mediates the interaction between the receptor and lipoproteins containing apo B-100 and/or apo E (Esser et al., 1988). This function is localised to a stretch at the amino terminus of the receptor, a sequence of 292 amino acid residues that comprises seven repeats, each of approximately 40 residues. These seven repeats are arranged in head-to-tail fashion; their high content of cysteines presumably mediates folding of the domain into a rigid structure with clusters of negatively charged residues on its surface (N, SDE in Figure 8.1). The negative charges are found at the carboxy terminus of each of the seven repeat units, with repeats 2–7 thought to participate in the binding of lipoprotein(s) to positively charged residues on apo B-100 or apo E. The fifth repeat is required for binding of apo E, while repeats 2–7 cooperatively recognize apo B-100. The structure of repeat 5 of the human LDL receptor has been determined to 1.7 A resolution by x-ray crystallography (Fass et al., 1997). The data suggest that a calcium ion is coordinated by the acidic residues in the carboxy-terminal region of
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the repeat. Thus, interaction with the positively charged residues of apolipoproteins B-100 and E may not occur, as previously thought, via these acidic receptor residues, because Ca2+ caging renders them unavailable for this purpose. Rather, interaction may occur with other acidic residues, or binding also may be mediated by other than ionic interactions. In this context, it is noteworthy that the fifth LDL receptor ligand binding repeat contains the sequence SDEE instead of the SDE present in the other six repeats.
Figure 8.1 Schematic representation of LDL receptor. The five domains of the class I membrane protein are indicated (see text). SDE, the core of a negatively charged cluster of residues in each of the seven ligand-binding repeats; YWTD, the consensus signature sequence of each of five 50-amino acid repeats in the domain homologous to the EGF precursor; A, B, and C, three six-cysteine domains with homology to the EGF precursor repeat motifs; CHO, carbohydrate; FDNPVY, the internalisation sequence in the cytoplasmic tail of the receptor.
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The Ligand Binding Domain The EGF Precursor Homology Domain This domain of the LDL receptor lies adjacent to the ligand binding site and comprises approximately 400 amino acids; its outstanding feature is the sequence similarity to parts of the EGF precursor, itself a membrane-bound protein. Of particular interest is the high degree of homology in three regions termed “growth factor repeats”. Two of these repeats (A and B in Figure 8.1) are located in tandem at the amino terminus, and the third (C) is at the carboxy terminus of the precursor homology region of the LDL receptor. Repeat A may have a supportive role in the binding of apo B-100 to the 40-residue repeats in the NH2terminal domain (Esser et al., 1988). The remainder of this domain is made up of five ~50-residue stretches that contain tetrapeptide sequences with a consensus of Tyr-Trp-Thr-Asp (YWTD in Figure 8.1). By deletion of the entire EGF precursor domain via site-specific mutagenesis, evidence was obtained for the possible involvement of this region in the acid-dependent dissociation of the receptor from LDL and its subsequent recycling. The O-linked Sugar Domain This domain of the human LDL receptor is a 58-amino-acid stretch, highly enriched in serine and threonine residues, located just outside the plasma membrane. Most, if not all, of the 18 hydroxylated amino acid side chains are glycosylated. In the course of receptor maturation, the O-linked oligosaccharides undergo posttranslational elongation: when the receptor leaves the endoplasmic reticulum, N-acetylgalactosamine is the sole O-linked sugar present, and upon processing in the Golgi complex, galactosyl and sialyl residues are added. Despite the detailed knowledge of the structure of this region, its function remains elusive. It may simply serve as a “stalk” enabling the binding domain to protrude into the extracellular milieu, so facilitating steric access of lipoprotein particles. The Membrane Anchoring Domain This domain of the LDL receptor lies carboxyterminally to the Olinked carbohydrate cluster. It consists of 22–25 hydrophobic amino acids, the sequence of which is the least conserved of all the receptor domains in seven mammalian species. This argues against any specific function other than anchoring, (such as signal transduction across the plasma membrane elicited by ligand binding, or formation of an ion
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channel). This argument is further supported by the fact that, in the absence of LDL, recycling of the receptor occurs constitutively. As would be expected, deletion of this domain in certain naturally occurring mutations leads to secretion of truncated receptors, as found in the mutant cell lines. The Cytoplasmic Tail This region of the LDL receptor constitutes a short stretch of 50 amino acid residues involved in the targeting of LDL receptors into clathrincoated pits. Naturally occurring mutations (Davis et al., 1986) and sitespecific mutagenesis (Davis et al., 1987b) have led to identification of an “internalisation signal”. This sequence, PheAsp-Asn-Pro-Val-Tyr (FDNPVY, Figure 8.1) is present in the human LDL receptor. The tetrapeptide NPXY (where X denotes any amino acid) has now been found in all (over 20) structurally elucidated members of the LDL receptor family. In the following section, the relationships between the receptor’s protein structure and gene organisation are described.
THE HUMAN LDL RECEPTOR GENE Intron/Exon Organisation and Naturally Occurring Mutations The ~ 48 kb LDL receptor gene contains 18 exons and is located on the distal short arm of chromosome 19. There is a strong correlation between the functional domains of the protein and the exon organisation in the gene. For instance, the 7 cysteine-rich repeats of the ligand-binding domain are coded for by exons 2 (repeat 1), 3 (repeat 2), 4 (repeats 3, 4, and 5), 5 (repeat 6) and 6 (repeat 7). The EGF precursor homology domain is encoded by 8 exons, organized in a manner very similar to the gene for the EGF precursor itself. The third domain is translated from a single exon between introns 14 and 15. The last exon encodes the 12 carboxyterminal amino acids of the receptor and about 2.5kb of untranslated mRNA, including three tandem Alu repetitive elements (Südhof et al., 1985); it should be noted that the human LDL receptor gene contains in excess of 30 Alu repeats, twice as many as expected from the frequency of such elements in the human genome. Thus, the LDL receptor gene is a compound of shared coding sequences. In fact, many more molecules containing some or all of these elements have been discovered and still more are likely to be found. Molecular genetic studies in FH patients have identified over 200 different mutations in the LDL receptor gene. These have been grouped
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into five categories according to their phenotypic effects on the receptor protein (Hobbs et al., 1990). Class 1: no detectable precursor These so-called “null” alleles are, in contrast to early predictions, relatively rare. The mutant alleles fail to produce receptor proteins, as determined by immuno-precipitation with many monoclonal and polyclonal anti-receptor antibodies, and thus fail to bind any LDL. The spectrum of these mutations includes point mutations causing premature termination codons early in the protein coding region, mutations in the promoter region blocking transcription (Koivisto et al., 1994), mutations that lead to abnormal splicing and/or instability of the mRNA, and large deletions. In one case (Lehrman et al., 1986), a 5-kb deletion has resulted from recombination between a region in exon 13 and an Alu sequence in intron 15; thus, this case is an example of the deleterious consequences of the preponderance of Alu sequences in the LDL receptor gene. Class 2: slow or absent processing of the precursor These alleles, probably accounting for at least half of all mutant LDLR alleles, specify transport-deficient receptor precursors which fail to move at normal rates from the endoplasmic reticulum to and through the Golgi compartment(s) and on to the cell surface. As a consequence, the sudden increase in apparent Mr from 120,000 to 160,000 typically observed during biosynthesis of the normal receptor (Tolleshaug et al., 1982) is lacking. Most of these mutations are complete: there is total absence of transport from the endoplasmic reticulum, and no receptors ever reach the cell surface. In one study of class 2 mutations, the FH Lebanese allele was analyzed (Lehrman et al., 1987). The mutated receptor had an apparent molecular weight of 100,000, did not undergo processing, and remained in the endoplasmic reticulum. As a result of a nonsense mutation (Cys-660 to Stop), the defective receptor consisted only of the binding domain and the major part of the EGF precursor homology domain; the termination codon occurred in a cysteine-rich region of growth-factor repeat C (cf. Figure 8.1), producing unpaired cysteines. Presumably, the failure of the truncated polypeptide to leave its site of synthesis was due to abnormal folding (Lehrman et al., 1987). Of particular interest are the natural and artificial mutations affecting the first cysteine-rich repeat in the ligand-binding domain of the LDL receptor. Affected receptors fail to bind certain anti-receptor monoclonal antibodies, but still bind LDL with high affinity (Van Driel et al., 1987). In a South African Xhosa patient with FH, a deletion of 6
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bp in exon 2 has led to loss of Asp-26 and Gly-27 from the first cysteine-rich repeat. The abnormal receptor is processed very slowly and degraded rapidly, and the number of receptors reaching the cell surface is grossly diminished. In contrast, when the first repeat is lacking entirely, the protein is transported normally (Van Driel et al., 1987). These mutations have shed further light on the importance of certain structural features for normal intracellular transport of the LDL receptor. Class 3: defective ligand binding These receptors in general reach the cell surface at normal rates, but are unable to bind LDL efficiently. Most of the class 3 receptors have normal or close to normal apparent molecular weights in SDSpolyacrylamide gel electrophoresis, and by definition, undergo the normal maturation process. In two cases, possibly characterized by the same mutant allele (FH London-2 and FH Paris-1, (Hobbs et al., 1990), exon 5, coding for the sixth cysteine-rich repeat in the ligand binding domain (Figure 8.1), is deleted. The deletion arises from homologous recombination between Alu sequences such that the resulting transcript contains exon 4 directly joined to exon 6. Inasmuch as the ends of exons 4, 5, and 6 all occur in the same reading frame (see above), the translation product is exactly 41 amino acids (coded for by exon 5) shorter than normal. It is of interest that this mutant receptor has lost its ability to bind LDL, but has apparently retained its activity towards apo E, as demonstrated by its capacity to bind rabbit -VLDL, an apo E- and apo B-containing class of lipoprotein particles believed to bind to the LDL receptor via apo E. This finding supports the notion that the fifth repeat holds the key to the binding of apo E. Class 4: internalisation-defective Here, one of the prerequisites for effective ligand internalisation— clustering of the LDL receptors in the coated pits—is not met. The failure of these “internalization-defective” receptors migrate to coated pits results from mutations that directly or indirectly disrupt the carboxy-terminal domain of the receptor. The paternal allele in patient J.D., the first patient identified with this phenotype (Brown and Goldstein, 1976; Davis et al., 1986) contains a single base change resulting in substitution of cysteine for tyrosine at position 807, located 18 residues into the cytoplasm. Since the cytoplasmic domain is directly affected, this structural disruption is likely responsible for the failure of the receptor to migrate into a coated pit, a notion further strengthened by expression studies of mutant
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alleles constructed in vitro (Davis et al., 1987b). Variants of class 4 mutations have been identified in which the receptors produced are secreted from the cells. Large deletions are observed, resulting, once again, from recombination between two repetitive Alu sequences, which lead to lack of both cytoplasmic and transmembrane domains; the majority of the truncated mutant proteins are, as expected, secreted from the mutant cells. The delineation of class 4 mutations has proved that the cytoplasmic domain of the LDL receptor, and possibly of other receptors in clathrincoated pits, contains information crucial for targeting these membrane proteins to the pits. The consensus sequence, (FD)NPXY, is the signature of endocytotic capability for all members of the LDL receptor gene family. Class 5: recycling-defective The grouping of these mutations into a separate class (Hobbs et al., 1990) appears to be the least rationalized of all classes. The classification is based on the observation that, upon deletion of the entire EGF precursor domain of the human LDL receptor by in vitro mutagenesis, the truncated receptor binds and internalizes -VLDL, but, in the acidic environment of the endosome, fails to release the ligand (Davis et al., 1987a). Consequently, instead of returning to the surface in an unoccupied state, the mutant receptor is rapidly degraded. In all of the so-called class 5 mutants, the affected part is the EGF precursor homology domain or the YWTD region thereof. However, these mutations could, in fact, be categorized as class 2 mutations, for the receptors take longer to reach the cell surface. These mutations also superficially resemble class 3 alleles, in that they are binding-deficient because of reduced surface expression as a consequence of slow processing and/or accelerated degradation. In summary, the molecular details of LDL receptor-mediated removal of VLDL-derived remnant lipoproteins (IDL and LDL) from plasma are now well understood. The occurrence of natural mutations in the LDL receptor gene has provided powerful tools for delineating the structural as well as the regulatory features of the LDL receptor. The Regulatory Part of the LDL Receptor Gene Transcription of the LDL receptor gene is efficiently regulated by sterols. The key elements mediating this regulation are three imperfect repeats (numbered 1–3) of 16bp each, located within 177bp (positions 234 to -58) (Südhof et al., 1987b). Repeats 1 and 3 bind the constitutive transcription factor, Sp1. The activity of repeats 1 and 3, although necessary, is insufficient for high level transcription (Dawson et al.,
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1988; Sanchez et al., 1995; Südhof et al., 1987a). Repeat 2, which does not bind Sp1, does, however, harbor a special element that is directly responsible for regulation by sterols. Mutational analysis of this element identified a 10-bp stretch, ATCACCCCAC, termed sterolresponsive element-1 (SRE-1), as the target of the SRE-1 binding proteins (SREBP-1 and -2) (Briggs et al., 1993, Wang et al., 1993). SREBPs are active only under conditions of sterol deprivation; when sterols are added to cells, the contribution of repeat 2 is abolished, and the rate of transcription falls. SREBP sites are also present in the sterolresponsive regions of the promoters of the genes for HMG-CoA synthase, HMG-CoA reductase, fatty acid synthase (Shimano et al., 1996), farnesyl diphosphate synthase (Ericsson et al., 1996), acetylCoA carboxylase (Magaña et al., 1997), and stearoyl CoA desaturase-1 (Kim and Spiegelman, 1996). Figure 8.2 depicts our current understanding of the coordination of molecular events underlying transcriptional regulation via SRE-1. The key molecules in the cholesterol feedback system, SREBP-1 and -2, are membrane-bound proteins of the endoplasmic reticulum and the nuclear envelope, whose activity is regulated by a unique proteolytic cascade. The tripartite structure of the 120–125 kDa SREBPs defines (i) an amino-terminal segment of ca. 500 residues, which is a transcription factor of the basic-helix-loop-helix-leucine zipper family, and projects into the cytosol, (ii) a 75-residue hairpin anchor consisting of two membrane-spanning regions separated by a short hydrophilic loop, and (iii) a carboxy-terminal domain of ca. 500 amino acids exposed to the cytosol. In cultured cells, the two SREBPs appear to be redundant and act independently. In sterol-depleted cells, two sequential proteolytic steps release the amino-terminal transcription factor, which now can enter the nucleus and activate transcription of the LDL receptor gene and other SREBP site-containing genes (Sakai et al., 1996). The two-step proteolytic release process requires an initial clip at site 1 (Figure 8.2), near or at an arginine (R) in the luminal loop, which separates the two membranespanning segments. Subsequently, a second protease(s) cleaves the protein at site 2 in the middle of the first transmembrane domain, thereby releasing the amino-terminal transcription factor into the cytoplasm. It is the site 1 protease that is strictly regulated by sterols, i.e., it is active in sterol-depleted cells and is silent when sterols are abundant. The cleavage at site 2 is insensitive to sterol levels, but occurs only after site 1 has been clipped. Site 2 proteolysis requires the sequence DRSR, found at the amino-terminal side of the first membrane-spanning domain of SREBP-1 and -2.
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Figure 8.2 Transcriptional regulation of the LDL receptor via SRE-1. SREBP precursor (thick bold line, ~1150 amino acids) is an ER-anchored (doublelined oval) hairpin precursor molecule, which undergoes a series of proteolytic cleavages. In sterol-depleted cells, activation of LDL receptor transcription is triggered by binding to SRE-1 in the promoter region of the receptor of a basic helix-loop-helix leucine-zipper family member (bHLH-Zip) that is released from the N-terminus of SREBP, so that it can enter the nucleus. The two sequential cleavages required to complete this process are indicated by the circled numbers 1 and 2. When cellular sterol levels are low, SCAP (SREBP cleavage-activating [SCA] protein), a protein spanning the ER membrane several times, activates SCA (the enzyme having SREBP-cleaving activity) in sterol-suppressible fashion, and SCA then cleaves SREBP at site 1. Sterol-dependent cleavage at site 1 is a
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prerequisite for subsequent cleavage at site 2; when cells are overloaded with sterol, proteolysis at site 1 is abolished. The C-terminal domain of SCAP forms a complex with that of SREBP-2, an interaction required for efficient SREBP cleavage (Sakai et al., 1997). Mutant forms of SCAP (e.g., D433N) have helped to delineate this process. Sterol-independent processing of SREBP is also indicated (CPP32). For further details, see text.
As so often is the case in complex pathways, the delineation of this multistep mechanism has been greatly facilitated by the availability of mutant cell lines. Mutant Chinese hamster ovary (CHO) cells have been isolated that can be grouped into two types of sterol-regulatory deficiency, i.e., sterol auxotrophs and sterol-resistant cells. Sterol auxotrophs cannot synthesise cholesterol or LDL receptors in response to sterol depletion. These cells cannot cleave the SREBPs at site 2, and consequently the transcription factor cannot enter the cytoplasm. Sterol-resistant cells cannot turn off the synthesis of cholesterol or of the LDL receptor when sterols accumulate. The reason for this deficiency is one of several mutations in the SREBP-2 gene, most often resulting in truncated proteins that lack the membrane-spanning region altogether (Yang et al., 1995). This constitutively soluble protein cannot be suppressed by sterols, and goes directly to the nucleus. Interestingly, although SREBP-1 appears structurally normal in these cells, its proteolytic processing is reduced, possibly through a regulatory effect mediated by the truncated SREBP-2. Another reason for the sterol-resistant phenotype, against the background of normal SREBPs, is failure to suppress the proteolytic cascade in response to sterol overload. In one particular instance, the 25-RA cells (Chang and Limanek, 1980), the SREBP cleavage at site 1 was shown to have become resistant to suppression by sterols. The gain-of-function point mutation affects a novel membrane protein, termed SREBP cleavageactivating protein (SCAP; Figure 8.2), in such a way that it overrides the suppression by sterols of an unidentified protease(s) (SCA? in Figure 8.2) involved in cleavage of SREBPs at site 1 (Hua et al., 1996). Wild-type SCAP exerts the same effect when overexpressed in cells by transfection, but for some unknown reason the mutant form (with an Asn instead of an Asp residue in position 443; Figure 8.2) in 25-RA cells is much more potent at the endogenous level. SCAP is a protein that spans the ER membrane several times; some of the membranespanning stretches are closely homologous to those in HMG CoA reductase, one of the prime proteins whose degradation is regulated by sterols. The occurrence of the same Asp-to-Asn substitution in SCAPs of three different sterol-resistant cell lines points to the importance of residue 443 in determining the activity of SCAP (Nohturfft et al.,
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1996). Thus, the genes for SCAP and SREBP-2 are the only ones identified so far in which a mutation gives rise to a sterol-resistant phenotype.
PHYSIOLOGICAL REGULATION OR LDL RECEPTORS Regulation of LDL Receptors by Cholesterol Studies on cultured cells The original in vitro experiments that led to the discovery of the LDL receptor involved sterol-mediated regulation of cellular cholesterol synthesis in cultured human skin fibroblasts (Brown et al., 1974). Cholesterol synthesis was found to be suppressed by addition of LDL to the fibroblasts. As we now know, LDL receptor-mediated uptake of LDL particles leads to their hydrolysis in lysosomes, with ensuing inflow of liberated LDL cholesterol into the cytoplasmic compartment of the cells (Brown and Goldstein, 1986). This cholesterol not only serves as building blocks for membrane synthesis, but also acts as a precursor of regulatory derivatives in a sophisticated feedback system which protects the fibroblasts from cholesterol accumulation. Thus, cholesterol is thought to initiate genetic repression of cholesterol synthesis and of LDL receptor synthesis following its conversion to a hydroxylated metabolite, i.e., oxysterol (Taylor and Kandutsch, 1985; Südhof et al., 1987a; Takagi et al., 1989; Smith et al., 1990). The size of the putative regulatory sterol pool is likely to depend on the size of the pool of unesterified cholesterol within the cell. Thus, processes that tend to increase the content of unesterified cholesterol within the cell will increase the flow of cholesterol into the sterol pool; in contrast, processes that tend to lower the content of unesterified cholesterol within the cell will decrease the flow of cholesterol into the sterol pool. The size of the cytoplasmic pool of unesterified cholesterol is determined by the relative rates of the processes that supply and processes that remove unesterified cholesterol from the cytoplasm (Figure 8.3). In the simple system of cultured fibroblasts, inflow of unesterified cholesterol is governed by LDL receptor-mediated uptake of LDL, and outflow of unesterified cholesterol by the rate of cell growth. Accordingly, an increase in LDL concentration and cessation of cell growth will increase the flow of cholesterol into the regulatory pool, whereas lowering of LDL concentration or stimulation of cell growth will decrease the flow of cholesterol into the regulatory pool. Finally, inhibition of cholesterol synthesis by HMG CoA reductase inhibitors also reduces the content of unesterified cholesterol and
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increases LDL receptor activity of fibroblasts (Goldstein et al., 1979b).
Figure 8.3 Sterol-dependent regulation of LDL receptor activity in cells. Cholesterol input into (left) and output from (right) the cytoplasmic pool of unesterified cholesterol in mammalian cells (compilation of data). Cholesterol input into the pool: Lipoprotein cholesterol is taken up by cells via LDL receptors or via other receptors of the LDL receptor family, notably the LDL-receptor related protein (LRP). Cholesterol is also synthesized by the cells, 3-hydroxy-3methylglutaryl-CoA (HMG-CoA) reductase being a key regulatory enzyme in the pathway of cholesterol biosynthesis. Cholesterol output from the pool: In liver cells, cholesterol is incorporated into VLDL, and converted into bile acids, 7 hydroxylase being the enzyme regulating bile acid synthesis. In steroidogenic cells, cholesterol is converted into steroid hormones, an outstanding example being the ACTH-driven steroid hormone synthesis in the adrenal cortex. In all growing and dividing cells, cholesterol is used as building blocks of the plasma membrane. Any excess of cholesterol is esterified by the
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acyl-CoA: cholesterol acyltransferase (ACAT). Unesterified cholesterol is also converted into oxysterols, which inhibit LDL receptor synthesis by suppressing the transcriptional activity of the LDL receptor gene. The amount of oxysterols in this regulatory sterol pool depends on the size of the pool of unesterified cholesterol. Thus, processes that increase the size of the pool of unesterified cholesterol will decrease LDL receptor synthesis, and processes that decrease its size will increase LDL receptor synthesis.
Downregulation of LDL receptor activity is a rather slow process (the life span of a receptor is about 20 hours), and it results merely from suppression of transcription of the LDL receptor gene, i.e., from inhibition of synthesis of new receptor molecules, without acceleration of their degradation. This allows the receptors to return to the cell surface after their internalisation, and to continue their recycling (each round-trip takes ~10 min) for at least 6 hours after cessation of their synthesis (Goldstein et al., 1979a). Thus, after exposure of cultured fibroblasts to high concentrations of LDL, the cells continue to ingest LDL at a uniform rate for several hours. This leads to a transient oversupply of cholesterol to the cells, and to a transient stimulation of cholesterol esterification by ACAT, so directing cholesterol into the cholesteryl ester storage pool. However, the ultimately complete downregulation of LDL receptors in fibroblasts prevents them from becoming engorged with cholesteryl esters, such as is seen in phagocytes, whose unabated uptake of lipoproteins is due to lack of a sterolregulated feedback mechanism (Goldstein et al., 1979e). The above basic concepts of sterol-dependent regulation of LDL receptors also apply to other cells. The mechanisms that lead to increased inflow and outflow of unesterified cholesterol from the cytoplasmic compartment are many, and they vary with the cell type (Figure 8.3). In hepatocytes, uptake of lipoprotein cholesterol may take place via the LDL receptors, and also via other receptors of the LDL receptor family, such as the LDL receptor-related protein (LRP) (Krieger and Hertz, 1994). It has been observed that LDL receptormediated uptake of cholesterol does not lead to efficient downregulation of LDL receptors, whereas uptake of VLDL does efficiently suppress LDL receptor expression in these hepatocytes (Kamps and van Berkel, 1992). The authors suggested that the differential effect of lipoprotein cholesterol on LDL receptor regulation may have resulted from the presence of several pools of unesterified cholesterol in the HepG2 cells. In hepatocytes, cholesterol is also utilised for synthesis and secretion of very low density lipoproteins (VLDL), and for synthesis and secretion of bile acids. Moreover, the
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activity of ACAT appears to be high in hepatocytes (Havekes et al., 1987; Salter et al., 1989). All these processes divert cholesterol from the regulatory pool, and may partly explain the relative resistance of hepatocytes to suppression of LDL receptor expression in response to LDL-derived cholesterol. Thus, in contrast to nonhepatic cells, which show almost complete suppression of LDL receptors in the presence of high plasma concentrations of LDL, the liver cells show only partial downregulation of their LDL receptors. The molecular mechanisms responsible for this differential response to an increase in extracellular lipoprotein cholesterol concentration have began to be unravelled only recently. In an elegant experiment, Dueland et al. (1992) examined the effect of 7 -hydroxylase on the. expression of the LDL receptor in nonhepatic cells (Chinese hamster ovary (CHO) cells). The CHO cells were transfected with a plasmid encoding 7 -hydroxylase, and expressed both the mRNA and the functional activity. Importantly, in the presence of serum, expression of the LDL receptor by transfected cells was more than 20-fold higher than that of nontransfected cells, despite a 50% increase in the content of cellular cholesteryl esters. Thus, in regard to resistance to downregulation of the LDL receptor expression, CHO cells expressing 7 -hydroxylase exhibited the liver phenotype. The paradoxical induction of LDL receptor mRNA in transfected cells containing large amounts of cholesterol suggests that 7 -hydroxylase indirectly induces the LDL receptor gene by metabolising, i.e., inactivating, inhibitory oxysterols. This may also be the case in hepatocytes. Steroidogenic cells are another example of a specialised cell with a more complex system of cellular cholesterol pools (cholesterol balance). Thus, stimulation of steroid hormone synthesis in the adrenal cortex by ACTH ultimately leads to increased LDL receptor expression in this organ (Brown et al., 1979). When cultured adrenocortical cells are stimulated acutely with ACTH, the initial response is a rapid output of steroids. The substrate for this initial burst of synthesis comes from cholesterol that is drained from the small pool of metabolically active free cholesterol in the cells (Figure 8.3). This sterol is immediately replenished by hydrolysis of the stored cholesteryl esters. In addition, cholesterol synthesis is activated through enhancement of the activity of HMG CoA reductase. If the stimulus to steroid secretion is prolonged, net cholesteryl ester hydrolysis eventually ceases, cholesterol synthesis declines, and the enhanced cholesterol output is balanced primarily by acceleration of the receptor-mediated uptake of cholesterol from LDL. Similarly, chorionic gonadotropin increases LDL receptor expression in cultured human granulosa cells, the LDLderived cholesterol becoming the primary substrate for steroid hormone synthesis in luteinised granulosa cells (Takagi and Strauss, 1989).
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Finally, stimulation of cell growth by mitogens will increase their need for cholesterol, and also enhances their LDL receptor expression. Cuthbert et al. (1986) have utilised proliferating lymphocytes for detection of heterozygous FH by assaying the functional activity of LDL receptors in freshly isolated lymphocytes. In this elegant assay, cholesterol synthesis of lymphocytes is blocked by an HMG CoA reductase inhibitor, which renders cell proliferation fully dependent on LDL-derived cholesterol. Lymphocytes from patients heterozygous for functional LDL receptor abnormalities can be distinguished from normal lymphocytes, since the former require twice the concentration of LDL for a given proliferation rate. Studies in vivo While the above studies at the cellular level provided insights into the regulation of cholesterol concentration within cells, animal studies have led to definition of the role LDL receptor in the regulation of plasma LDL levels. Most tissues have detectable levels of LDL receptors (Brown et al., 1980, Rudling et al., 1990b). Malignant cells in some types of leukemia and of solid tumours show very high levels of LDL receptors (Rudling et al., 1990a, Vitols et al., 1990). Such elevated LDL receptor activity may even lead to hypocholesterolaemia in patients with leukemia (Vitols et al., 1985). In normal tissues, the levels of LDL receptors are highest in the adrenal gland and in the ovarian corpus luteum. But when the weight of the whole organ is taken into consideration, the liver is found to produce by far the largest number of LDL receptors. It has been estimated that approximately 70% of the total body uptake of plasma LDL occurs in the liver by LDL receptor-dependent pathways (Pittman et al., 1982, Spady et al., 1983). In addition to removing LDL from plasma, the hepatic LDL receptors also remove its precursor, the intermediate density lipoproteins (IDL) particles (Goldstein et al., 1983). Thus, absence of functional LDL receptors in the liver leads to simultaneous overproduction and reduced degradation of LDL, the result being great increases in the plasma LDL concentration, as found in patients with FH. The liver is the organ primarily responsible for the removal of LDL from the plasma by the LDL receptor pathway (Brown et al., 1981). The parenchymal cells of the liver are separated from the plasma by a fenestrated endothelial surface, in which the hepatocytes are surrounded by high concentrations of LDL, so allowing optimal interaction with LDL receptors. The findings on cultured hepatocytes and on intact animals have led to reappraisal of the therapeutic possibilities in the treatment of hypercholesterolaemia. If functional LDL receptor genes are missing, their replacement is necessary to
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allow efficient removal of LDL from the circulation. This was done in a child with the homozygous form of FH by transplanting a liver from a normal donor to provide LDL receptors (Bilheimer et al., 1984). After transplantation, the patient’s plasma LDL levels declined to near normal. This extreme mode of therapy also defined the importance of the liver in controlling the plasma LDL levels in humans. More recently, hypercholesterolaemia in LDL receptor knockout mice was rapidly reversed to normal by adenovirus-mediated gene delivery (Ishibashi et al., 1993). In this animal model, injection of a recombinant replication-defective adenovirus encoding the human LDL receptor driven by the cytomegalovirus promoter restored expression of the LDL receptor protein in the liver, possibly paving the way for future modes of therapy of the homozygous form of FH. In the above examples, total lack of LDL receptors was overcome by providing LDL receptor genes. Interestingly, gene manipulation has produced significant effects on LDL receptor expression and on plasma cholesterol levels in normal animals also. Adenovirus-mediated LDL receptor gene transfer, with ensuing hepatic overexpression of LDL receptors, accelerates cholesterol clearance (Herz and Gerard, 1993), and prevents diet-induced hypercholesterolaemia in normal mice (Yokode et al., 1990). Thus, stimulation of hepatic LDL receptors can counteract the environmental challenge leading to hypercholesterolaemia, namely, a diet high in cholesterol and saturated fat. Experiments in a variety of animals, including primates, have shown that ingestion of a diet high in cholesterol and saturated fat leads to a build-up of cholesterol in the liver, and this represses transcription of the LDL receptor gene (Brown and Goldstein, 1986; Goldstein and Brown, 1990). In the human liver, the LDL receptor protein content correlates with the plasma LDL cholesterol concentration even within the normal range of plasma cholesterol levels (Soutar et al., 1986). Finally, in patients with heterozygous familial hypercholesterolaemia, LDL receptor activity can be upregulated with drugs that inhibit HMG-CoA reductase in the liver. In this way, one can exploit the normal regulation of receptor synthesis, stimulating the single normal gene in these patients to produce increased numbers of LDL receptors. This stimulation can be accomplished by maneuvers which deplete bile acids, and thus induce hepatocytes to convert more cholesterol into bile acids. The two mechanisms for stimulation of LDL receptor activity were originally delineated by treatment of normal dogs with mevinolin and colestipol (Kovanen et al., 1981). Subsequent studies in humans suffering from the heterozygous form of FH have indicated that HMG-CoA reductase, either alone or in combination with bile acid depletion with a bile acid-binding resin, can decrease plasma levels of LDL primarily by stimulating receptor-mediated catabolism of LDL (Bilheimer et al., 1983). Moreover, treatment of
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normolipidaemic subjects with a bile acid-binding resin increased the number of LDL receptors in liver membranes (Rudling et al., 1990b). The successful lowering of plasma LDL levels in FH heterozygotes with a cholesterol synthesis inhibitor, alone or in combination with a bile acid-binding resin, confirmed that the most important target for regulation of LDL receptors is the liver, and that this regulation appears to depend on a small pool of metabolically active cholesterol (oxygenated sterols). Thus, treatment with a bile acid-binding resin stimulates the synthesis and output of bile acids from the liver; the liver cell compensates for the elevated output of bile acids by converting more cholesterol into bile acids and thus diverts cholesterol from entering the regulatory sterol pool. This, again, results in stimulation of LDL receptor synthesis and in acceleration of LDL receptor-mediated uptake of plasma LDL. If an inhibitor of cholesterol synthesis is added to the regimen, even less cholesterol reaches the regulatory pool, and a more profound increase in LDL receptor activity ensues, leading to more efficient clearance of plasma LDL. Regulation of LDL Receptors by Non-steroidal Compounds The observation that, in cultured cells, addition of LDL to the incubation medium decreases transcription of LDL receptors by the cells has raised an important question: can the sterol-mediated regulatory system be bypassed by nonsteroidal compounds? Such agents may have effects on other cellular processes, rather than acting directly on cellular sterol metabolism. However, they may also exert their effect on LDL receptors indirectly by influencing cellular cholesterol metabolism, and thereby altering the size of the active sterol pool. Examples of such nonsteroidal compounds are hormones, such as ATCH, which stimulate steroid hormone synthesis in steroidogenic cells. Studies in cultured cells Studies with the human hepatoma cell line HepG2 have been helpful in the delineation of liver-specific factors responsible for the regulation of LDL receptor activity. Regarding human applications, it appears that the HepG2 cells are suitable for studies of sterol-mediated regulation of the LDL receptor, but that this may not be the case for studies on regulation by nonsteroidal compounds. Thus, although treatment of HepG2 cells with a phorbol ester (PMA) results in a dramatic increase in the number of LDL receptors on the cell surface (Kamps and van Berkel, 1993), treatment of cultured rat or human liver parenchymal cells with PMA did not lead to increased binding of LDL to these cells.
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These results suggest that protein kinase C-mediated regulation of LDL receptor activity is specific for HepG2 cells, and that, with regard to LDL receptor regulation, HepG2 cells do not reflect human hepatocytes. Despite the above limitation, the HepG2 system has been instrumental in the elucidation of the mechanism by which a novel nonsteroidal compound, oncostatin M, regulates LDL receptors. Oncostatin M is a growth-regulatory peptide secreted by macrophages and activated T lymphocytes. Grove et al. (1991) reported that oncostatin M upregulates LDL receptors in HepG2 cells by a novel mechanism that involves stimulation of tyrosine kinase activity. This activity, when stimulated by oncostatin M, appears to be relatively specific, since other peptides (epidermal growth factor and insulin), although they also increase tyrosine phosphorylation of proteins in HepG2 cells, do not significantly increase the number of LDL receptors. Thus, upregulation of the LDL receptor occurs regardless of the state of sterolmediated LDL receptor repression, and it is not the peptide that stimulates HepG2 cells to proliferate, but the effect depends on rapid stimulation of protein tyrosine phosphorylation. In fact, induction of the transcription factor Erg-1 by oncostatin M precedes the upregulation of LDL receptors in the HepG2 cells. The correlations among tyrosine kinase phosphorylation, Erg-1 induction, and LDL receptor regulation suggest that Erg-1 may be a nuclear signal transducer utilised by oncostatin M to induce transcription of the LDL receptor gene. This possibility is supported by the discovery of an Erg1 consensus sequence (GAGGGGGCG) approximately 330 base pairs upstream from the transcription initiation site of the LDL receptor promoter region (Südhof et al., 1985). Recently, activation of LDL receptor transcription by oncostatin M was found to depend on repeat 3 of the LDL receptor promoter (Liu et al., 1997). Thus, regulation of LDL receptors by oncostatin M is the first, and thus far the only, wellcharacterised mechanism by which a compound regulates LDL receptors directly, i.e., not via the regulatory sterol pool. Studies in vivo In the rat, hepatic LDL receptor expression and plasma LDL levels exhibit diurnal periodicity (Balasubramaniam et al., 1994). Interestingly, the rhythms showed an inverse correlation, plasma LDL levels being lowest at the onset of darkness when LDL receptor activity was at its peak. Maximal expression of LDL receptors occurred several hours before the peak activity of HMG CoA reductase, and appeared not to be influenced by cellular cholesterol levels during the 24-hour cycle. The activity of hepatic LDL receptors is under hormonal control. For
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example, oestrogen (Angelin et al., 1992, Kovanen et al., 1979), thyroid hormone (Brindley and Salter, 1991), insulin (Wade et al., 1989), and glucagon (Rudling and Angelin, 1993) have all been shown to stimulate LDL receptor expression and to lower plasma LDL cholesterol. Of particular interest is the demonstration that pituitary growth hormone (GH) stimulates hepatic LDL receptors in humans (Angelin and Rudling, 1994). The effect of growth hormone on hepatic LDL receptors was assessed by injecting growth hormone into patients with gallstones prior to operation, and hepatic LDL receptor binding activity was determined in liver biopsy specimens. Growth hormone administration induced a 2-fold upregulation of hepatic LDL receptors, and this was accompanied by a 25% decrease in the serum cholesterol level. Interestingly, the LDL receptor stimulation caused by the growth hormone treatment was of similar magnitude to that observed after 3 weeks of treatment with an HMG CoA reductase inhibitor. It is of interest that, at least in the rat, GH is important in maintaining the resistance of hepatic LDL receptors to suppression by dietary cholesterol. Thus, it is possible that GH directly stimulates hepatic LDL receptors. Elucidation of the mechanisms by which GH specifically stimulates LDL receptors will add to our understanding of the nonsteroidal regulation of LDL receptors in the liver, the most important organ for LDL receptor regulation. Finally, what of the role of fatty acids in regulating hepatic LDL receptors? It is well known that, in the steady state, plasma LDLcholesterol concentration is largely determined by lipids of two different types in the diet: (1) cholesterol itself, and (2) the fatty acids contained chiefly in triacylglycerols of the food. In general terms, it has been shown that when the diet contains saturated fatty acids, the plasma LDL-cholesterol level increases, whereas substituting unsaturated fatty acids for the more saturated ones leads to a decrease in the circulating LDL level. Recent animal studies, allowing measurement of absolute rates of cholesterol synthesis and transport in the liver under welldefined in vivo conditions, have led to a better understanding of the mechanisms underlying regulation of LDL metabolism by fatty acids. These studies suggest that fatty acids regulate hepatic LDL receptor activity by influencing the size of the regulatory sterol pool (Daumerie et al., 1992). Thus, the type of fatty acid determines the distribution of any excess cholesterol between the biologically inactive storage pool of cholesteryl esters and the putative small regulatory pool. This regulatory effect of the various fatty acids can be explained in terms of the known sensitivity of ACAT activity to the type of the long-chain fatty acid: the monounsaturated fatty acid 18:1 drives hepatic cholesteryl ester formation at a much higher rate that do the saturated long-chain fatty acids (Goodman et al., 1964). Could fatty acids control hepatic LDL receptors directly by
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interacting with the regulatory elements of the LDL receptor gene, such as the SREBPs? If the expression of SREBPs were inhibited more efficiently by saturated fatty acids (together with cholesterol) than by unsaturated fatty acids, the saturated fatty acids would lead to a more efficient downregulation of the LDL receptor and cause a larger increase in the plasma LDL concentrations (Chang, 1997). Clearly, future experimentation will permit molecular analysis of the regulatory effects of the Western diet on hepatic LDL receptors and on the level plasma LDL cholesterol, the main risk factor for coronary heart disease. References Angelin, B., Olivecrona, H., Reihnér, E., Rudling, M., Stålberg, D., Eriksson, M. et al. (1992). Hepatic cholesterol metabolism in estrogen-treated men. Gastroenterology , 103 , 1657–1663. Angelin, B. and Rudling, M. (1994). Growth hormone and hepatic lipoprotein metabolism. Current Opinion of Lipidology , 5 , 160– 165. Balasubramaniam, S., Szanto, A. and Roach, P.D. (1994). Circadian rhythm in hepatic low-density-lipoprotein (LDL)-receptor expression and plasma LDL levels. Biochemical Journal , 298 , 39–43. Bilheimer, D.W., Goldstein, J.L., Grundy, S.M., Starzl, T.E. and Brown, M.S. (1984). Liver transplantation to provide low density lipoprotein receptors and lower plasma cholesterol in a child with homozygous familial hypercholesterolemia. New England Journal of Medicine , 311 , 1658–1664. Bilheimer, D.W., Grundy, S.M., Brown, M.S. and Goldstein, J.L. (1983). Mevinolin and cholestipol stimulate receptor-mediated clearance of low density lipoprotein from plasma in familial hypercholesterolemia heterozygotes. Proceedings of the National Academy of Sciences of the United States of America , 80 , 4124– 4128. Briggs, M.R., Yokoyama, C., Wang, X., Brown, M.S. and Goldstein, J.L. (1993). Nuclear protein that binds sterol regulatory element of low density lipoprotein receptor promoter. I. Identification of the protein and delineation of its target nucleotide sequence. Journal of Biological Chemistry , 268 , 14490–14496. Brindley, D.N. and Salter, A.M. (1991). Hormonal regulation of the hepatic low density lipoprotein receptor and the catabolism of low density lipoproteins: relationship with the secretion of very low density lipoproteins. Progress in Lipid Research , 30 , 349–360. Brown, M.S. and Goldstein, J.L. (1976). Analysis of a mutant strain of human fibroblasts with a defect in the internalization of receptorbound low density lipoprotein. Cell , 9 , 663–674. Brown, M.S. and Goldstein, J.L. (1986). A receptor-mediated pathway for cholesterol homeostasis. Science , 232 , 34–47. Brown, M.S., Dana, S.E. and Goldstein, J.L. (1974). Regulation of 3
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hydroxy-3-methylglutaryl coenzyme A reductase activity in cultured human fibroblasts. Comparison of cells from a normal subject and from a patient with homozygous familial hypercholesterolemia. Journal of Biological Chemistry , 249 , 789–796. Brown, M.S., Kovanen, P.T. and Goldstein, J.L. (1979). Receptormediated uptake of lipoprotein-cholesterol and its utilization for steroid synthesis in the adrenal cortex. Recent Progress in Hormone Research , 35 , 215–257. Brown, M.S., Kovanen, P.T. and Goldstein, J.L. (1980). Evolution of the LDL receptor concept—from cultured cells to intact animals. Annals of the New York Academy of Sciences , 348 , 48–68. Brown, M.S., Kovanen, P.T. and Goldstein, J.L. (1981). Regulation of plasma cholesterol by lipoprotein receptors. Science , 212 , 628–635. Chang, T.Y. (1997). SREBPs, membrane lipid biosynthesis, and fatty acids. Journal of Clinical Investigation , 100 , 1905–1906. Chang, T-Y. and Limanek, J.S. (1980). Regulation of cytosolic acetoacetyl coenzyme A thiolase, 3-hydroxy-3-methylglutaryl coenzyme A synthase, 3-hydroxy-3-methylglutaryl coenzyme A reductase, and mevalonate kinase by low density lipoprotein and by 25-hydroxy-cholesterol in Chinese hamster ovary cells. Journal of Biological Chemistry , 255 , 7787–7795. Cuthbert, J.A., East, C.A., Bilheimer, D.W. and Lipsky, P.E. (1986). Detection of familial hypercholesterolemia by assaying functional low-density lipoprotein receptors on lymphocytes. New England Journal of Medicine , 314 , 879–883. Daumerie, C.M., Woollett, L.A. and Dietschy, J.M. (1992). Fatty acids regulate hepatic low density lipoprotein receptor activity through redistribution of intracellular cholesterol pools. Proceedings of the National Academy of Sciences of the United States of America , 89 , 10797–10801. Davis, C.G., Goldstein, J.L., Südhof, T.C., Anderson, R.G.W., Russell, D.W. and Brown, M.S. (1987a). Acid-dependent ligand dissociation and recycling of LDL receptor mediated by growth factor hemology region. Nature , 326 , 760–765. Davis, C.G., Lehrman, M.A., Russell, D.W., Anderson, R.G.W., Brown, M.S. and Goldstein, J.L. (1986). The J.D. mutation in familial hypercholesterolemia: Amino acid substitution in cytoplasmic domain impedes internalization of LDL receptors. Cell , 45 , 15–24. Davis, C.G., van Driel, I.R., Russell, D.W., Brown, M.S. and Goldstein, J.L. (1987b). The low density lipoprotein receptor. Identification of amino acids in cytoplasmic domain required for rapid endocytosis. Journal of Biological Chemistry , 262 , 4075– 4082. Dawson, P.A., Hofmann, S.L., van der Westhuyzen, D.R., Brown, M.S. and Goldstein, J.L. (1988). Sterol-dependent repression of low density lipoprotein receptor promoter mediated by 16-base pair sequence adjacent to binding site for transcription factor Sp1. Journal of Biological Chemistry, 263, 3372–3379.
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Dueland, S., Trawick, J.D., Nenseter, M.S., MacPhee, A.A. and Davis, R.A. (1992). Expression of 7 -hydroxylase in non-hepatic cells results in liver phenotypic resistance of the low density lipoprotein receptor to cholesterol repression. Journal of Biological Chemistry , 267 , 22695–22698. Ericsson, J., Jackson, S.M., Lee, B.C. and Edwards, P.A. (1996). Sterol regulatory element binding protein binds to a cis element in the promoter of the farnesyl diphosphate synthase gene. Proceedings of the National Academy of Sciences of the United States of America , 93 , 945–950. Esser, V., Limbird, L.E., Brown, M.S., Goldstein, J.L. and Russell, D.W. (1988). Mutational analysis of the ligand binding domain of the low density lipoprotein receptor. Journal of Biological Chemistry , 263 , 13282–13290. Fass, D., Blacklow, S., Kim, P.S. and Berger, J.M. (1997). Molecular basis of familial hypercholesterolaemia from structure of LDL receptor module. Nature , 388 , 691–693. Goldstein, J.L. and Brown, M.S. (1990). Regulation of the mevalonate pathway. Nature , 343 , 425–430. Goldstein, J.L., Anderson, R.G.W. and Brown, M.S. (1979a). Coated pits, coated vesicles, and receptor-mediated endocytosis. Nature , 279 , 679–685. Goldstein, J.L., Helgeson, J.A. and Brown, M.S. (1979b). Inhibition of cholesterol synthesis with com-pactin renders growth of cultured cells dependent on the low density lipoprotein receptor. Journal of Biological Chemistry , 254 , 5403–5409. Goldstein, J.L., Ho, Y.K., Basu, S.K. and Brown, M.S. (1979c). Binding site on macrophages that mediates uptake and degradation of acetylated low density lipoprotein, producing massive cholesterol deposition. Proceedings of the National Academy of Sciences of the United States of America , 76 , 333–337. Goldstein, J.L., Kita, T. and Brown, M.S. (1983). Defective lipoprotein receptors and atherosclerosis. Lessons from an animal counterpart of familial hypercholesterolemia. New England Journal of Medicine . 309 , 288–296. Goodman, D.S., Deykin, D. and Shiratori, T. (1964). The formation of cholesterol esters with rat liver enzymes. Journal of Biological Chemistry , 239 , 1335–1345. Grove, R.I., Mazzucco, C.E., Radka, S., Shoyab, M. and Kiener, P.A. (1991). Oncostatin M up-regulates low density lipoprotein receptors in HepG2 cells by a novel mechanism. Journal of Biologial Chemistry , 266 , 18194–18199. Havekes, L.M., De Wit, E.C.M. and Princen, H.M.G. (1987). Cellular free cholesterol in HepG2 cells is only partially available for downregulation of low-density-lipoprotein receptor activity. Biochemical Journal , 247 , 739–746. Herz, J. and Gerard, R.D. (1993). Adenovirus-mediated low density lipoprotein receptor gene transfer accelerates cholesterol clearance in normal mice. Proceedings of the National Academy of Sciences of
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the United States of America , 90 , 2812–2816. Hobbs, H.H., Russell, D.W., Brown, M.S. and Goldstein, J.L. (1990). The LDL receptor locus in familial hypercholesterolemia: mutational analysis of a membrane protein. Annual Review of Genetics , 24 , 133–170. Hua, X., Nohturfft, A., Goldstein, J.L. and Brown, M.S. (1996). Sterol resistance in CHO cells traced to point mutation in SREBP cleavageactivating protein. Cell , 87 , 415–426. Ishibashi, S., Brown, M.S., Goldstein, J.L., Gerard, R.D., Hammer, R.E. and Herz, J. (1993). Hypercholesterolemia in low density lipoprotein receptor knockout mice and its reversal by adenovirusmediated gene delivery. Journal of Clinical Investigation , 92 , 883– 893. Kamps, J.A.A.M. and van Berkel, T.J.C. (1992). Complete downregulation of low-density-lipoprotein-receptor activity in the human hepatoma cell line HepG2 by -migrating very-low-density lipoprotein and non-lipoprotein cholesterol. Different cellular regulatory pools of cholesterol. European Journal of Biochemistry , 206 , 973–978. Kamps, J.A. and van Berkel, T.J. (1993). Regulation of low-densitylipoprotein receptors in the human hepatoma cell line HepG2. Effect of phorbol 12-myristate 13-acetate and low-density lipoprotein. European Journal of Biochemistry , 213 , 989–994. Kim, J.B. and Spiegelman, B.M. (1996). ADD1/SREBP1 promotes adipocyte differentation and gene expression linked to fatty acid metabolism. Genes and Development , 10 , 1096–1107. Koivisto, U-M., Palvimo, J.J., Jänne, O.A. and Kontula, K. (1994). A single-base substitution in the proximal Sp1 site of the human low density lipoprotein receptor promoter as a cause of heterozygous familial hypercholesterolemia. Proceedings of the National Academy of Sciences of the United States of America , 91 , 10526–10530. Kovanen, P.T., Bilheimer, D.W., Goldstein, J.L., Jaramillo, J.J. and Brown M.S. (1981). Regulatory role for hepatic low density lipoprotein receptors in vivo in the dog. Proceedings of the National Academy of Sciences of the United States of America , 78 , 1194– 1198. Kovanen, P.T., Brown, M.S. and Goldstein, J.L. (1979). Increased binding of low density lipoprotein to liver membranes from rats treated with 17alpha-ethinyl estradiol. Journal of Biological Chemistry , 254 , 11367–11373. Krieger, M. and Hertz, J. (1994). Structures and functions of multiligand lipoprotein receptors: macrophage scavenger receptor and LDL receptor-related protein (LRP). Annual review of biochemistry , 63 , 601–637. Lehrman, M.A., Russell, D.W., Goldstein, J.L. and Brown, M.S. (1986). Exon-Alu recombination deletes 5 kilobases from the low density lipoprotein receptor gene, producing a null phenotype in familial hypercholesterolemia. Proceedings of the National Academy of Sciences of the United States of America , 83 , 3679–3683.
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Lehrman, M.A., Schneider, W.J., Brown, M.S., Davis, C.G., Elhammer, A., Russell, D.W. et al. (1987). The Lebanese allele at the LDL receptor locus: nonsense mutation produces truncated receptor that is retained in endoplasmic reticulum. Journal of Biological Chemistry , 262 , 401–410. Liu, J., Streiff, R., Zhang, Y.L., Vestal, R.E., Spence, M.J. and Briggs, M.R. (1997). Novel mechanism of transcriptional activation of hepatic LDL receptor by oncostatin M. Journal of Lipid Research , 98 , 2035–2048. Magaña, M.M., Lin, S.S., Dooley, K.A. and Osborne, T.F. (1997). Sterol regulation of acetyl coenzyme A carboxylase promoter requires two interdependent binding sites for sterol regulatory element binding proteins. Journal of Lipid Research , 38 , 1630– 1638. Nohturfft, A., Hua, X., Brown, M.S. and Goldstein, J.L. (1996). Recurrent G-to-A substitution in a single codon of SREBP cleavageactivating protein causes sterol resistance in three mutant Chinese hamster ovary cell lines. Proceedings of the National Academy of Sciences of the United States of America , 93 , 13709–13714. Pittman, R.C., Carew, T.E., Attie, A.D., Witztum, J.L., Watanabe, Y. and Steinberg, D. (1982). Receptor-dependent and receptorindependent degradation of low density lipoprotein in normal and in receptor-deficient mutant rabbits. Journal of Biological Chemistry , 257 , 7994–8000. Rudling, M.J. and Angelin, B. (1993). Stimulation of rat hepatic low density lipoprotein receptors by glucagon. Evidence of a novel regulatory mechanism in vivo. Journal of Clinical Investigation , 91 , 2796–2805. Rudling, M.J., Angelin, B., Peterson, C.O. and Collins, V.P. (1990a). Low density lipoprotein receptor activity in human intracranial tumors and its relation to the cholesterol requirement. Cancer Research , 50 , 483–487. Rudling, M.J., Reihnér, E., Einarsson, K., Ewerth, S. and Angelin, B. (1990b). Low density lipoprotein receptor-binding activity in human tissues: Quantitative importance of hepatic receptors and evidence for regulation of their expression in vivo. Proceedings of the National Academy of Sciences of the United States of America , 87 , 3469–3473. Sakai, J., Duncan, E.A., Rawson, R.B., Hua, X., Brown, M.S. and Goldstein, J.L. (1996). Sterol-regulated release of SREBP-2 from cell membranes requires two sequential cleavages, one within a transmembrane segment. Cell , 85 , 1037–1046. Sakai, J., Nohturfft, A., Cheng, D., Ho, Y.K., Brown, M.S. and Goldstein, J.L. (1997). Identification of complexes between the COOH-terminal domains of sterol regulatory element-binding proteins (SREBPs) and SREBP cleavage-activating protein. Journal of Biological Chemistry , 272 , 20213–20221. Salter, A.M., Ekins, N., Al-Seeni, M., Brindley, D.N. and Middleton, B. (1989). Cholesterol esterification plays a major role in
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determining low-density-lipoprotein receptor activity in primary monolayer cultures of rat hepatocytes. Biochemical Journal , 263 , 255–260. Sanchez, H.B., Yieh, L. and Osborne, T.F. (1995). Cooperation by sterol regulatory element-binding protein and Sp1 in sterol regulation of low density lipoprotein receptor gene. Journal of Biological Chemistry , 270 , 1161–1169. Shimano, H., Horton, J.D., Hammer, R.E., Shimomura, I., Brown, M.S. and Goldstein, J.L. (1996). Overproduction of cholesterol and fatty acids causes massive liver enlargement in transgenic mice expressing truncated SREBP-1a. Journal of Clinical Investigation , 98 , 1575– 1584. Smith, J.R., Osborne, T.F., Goldstein, J.L. and Brown, M.S. (1990). Identification of nucleotides responsible for enhancer activity of sterol regulatory element in low density lipoprotein receptor gene. Journal of Biological Chemistry , 265 , 2306–2310. Soutar, A.K., Harders-Spengel, K., Wade, D.P. and Knight, B.L. (1986). Detection and quantitation of low density lipoprotein (LDL) receptors in human liver by ligand blotting, immunoblotting, and radioimmunoassay. LDL receptor protein content is correlated with plasma LDL cholesterol concentration. Journal of Biological Chemistry , 261 , 17127–17133. Spady, D.K., Bilheimer, D.W. and Dietschy, J.M. (1983). Rates of receptor-dependent and -independent low density lipoprotein uptake in the hamster. Proceedings of the National Academy of Sciences of the United States of America , 80 , 3499–3503. Südhof, T.C., Goldstein, J.L., Brown, M.S. and Russell, D.W. (1985). The LDL receptor gene: a mosaic of exons shared with different proteins. Science , 228 , 815–822. Südhof, T.C., Russell, D.W., Brown, M.S. and Goldstein, J.L. (1987a). 42bp element from LDL receptor gene confers end-product repression by sterols when inserted into viral TK promotor. Cell , 48 , 1061–1069. Südhof, T.C., Van der Westhuyzen D.R., Goldstein, J.L., Brown, M.S. and Russell, D.W. (1987b). Three direct repeats and a TATA-like sequence are required for regulated expression of the human low density lipoprotein receptor gene. Journal of Biological Chemistry , 262 , 10773–10779. Takagi, K., Alvarez, J.G., Favata, M.F., Trzaskos, J.M. and Strauss, J.F. III (1989). Control of low density lipoprotein receptor gene promoter activity. Ketoconazole inhibits serum lipoprotein but not oxysterol suppression of gene transcription. Journal of Biological Chemistry , 264 , 12352–12357. Takagi, K. and Strauss, J.F. 3d. (1989). Control of low density lipoprotein receptor gene expression in steroidogenic cells. Canadian Journal of Physiology & Pharmacology , 67 , 968–973. Taylor, F.R. and Kandutsch, A.A. (1985). Use of oxygenated sterols to probe the regulation of 3-hydroxy-3-methylglutaryl-CoA reductase and sterologenesis Methods in Enzymology , 110 , 9–19.
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Tolleshaug, H., Goldstein J.L., Schneider, W.J. and Brown M.S. (1982). Posttranslational processing of the LDL receptor and its genetic disruption in familial hypercholesterolemia. Cell , 30 , 715– 724. Van Driel, I.R., Goldstein, J.L., Südhof, T.C. and Brown, M.S. (1987). First cysteine-rich repeat in ligand-binding domain of low density lipoprotein receptor binds Ca2+ and monoclonal antibodies, but not lipoproteins. Journal of Biological Chemistry , 262 , 17443–17449. Vitols, S., Angelin, B., Ericsson, S., Gahrton, G., Juliusson, G., Masquelier, M. et al. (1990). Uptake of low density lipoproteins by human leukemic cells in vivo: Relation to plasma lipoprotein levels and possible relevance for selective chemotherapy. Proceedings of the National Academy of Sciences of the United States of America, 87 , 2598–2602. Vitols, S., Gahrton, G., Björkholm, M. and Peterson, C. (1985). Hypocholesterolaemia in malignancy due to elevated low-densitylipoprotein-receptor activity in tumor cells: Evidence from studies in patients with leukaemia. Lancet , ii , 1150–1154. Wade, D.P., Knight, B.L. and Soutar, A.K. (1989). Regulation of lowdensity-lipoprotein-receptor mRNA by insulin in human hepatoma HepG2 cells. European Journal of Biochemistry , 181 , 727–731. Wang, X., Briggs, M.R., Hua, X., Yokoyama, C., Goldstein, J.L. and Brown, M.S. (1993). Nuclear protein that binds sterol regulatory element of low density lipoprotein receptor promoter. II. Purification and characterization. Journal of Biological Chemistry , 268 , 14497– 14504. Yang, J., Brown, M.S., Ho, Y.K. and Goldstein, J.L. (1995). Three different rearrangements in a single intron truncate sterol regulatory element binding protein-2 and produce sterol-resistant phenotype in three cell lines. Role of introns in protein evolution. Journal of Biological Chemistry , 270 , 12152–12161. Yokode, M., Hammer, R.E., Ishibashi, S., Brown, M.S. and Goldstein, J.L. (1990). Diet-induced hypercholesterolemia in mice: Prevention by overexpression of LDL receptors. Science , 250 , 1273–1275.
9 The Role of the Microsomal Triglyceride Transfer Protein in the Assembly and Secretion of Plasma Lipoproteins John R.Wetterau and David A.Gordon Bristol-Myers Squibb, P.O. Box 4000, Princeton, N.J. 08543, USA
The microsomal triglyceride transfer protein (MTP) is a heterodimeric lipid transfer protein composed of a unique large subunit and the multifunctional protein, protein disulfide isomerase (PDI). In vitro assays show that MTP transports triglyceride, cholesteryl ester, and phospholipid molecules between membranes. In vivo, MTP is found in the lumen of the endoplasmic reticulum (ER) of hepatocytes and enterocytes and is required for the production of apoB containing lipoproteins; very low density lipoproteins (VLDL) in the liver, and chylomicrons in the intestine. An absence of functional MTP is the cause of the rare human genetic disease, abetalipoproteinaemia. Subjects with this disease have an absence of all apoB containing lipoproteins and have plasma cholesterol levels around 40mg/dl. Current evidence suggests that MTP plays a critical role early in the assembly of a lipoprotein particle. Although there are experimental barriers which make it difficult to define the precise steps leading to a mature VLDL or chylomicron particle, breakthroughs in our understanding of the assembly process and the discovery of selective MTP inhibitors have aided efforts to elucidate the role of MTP in the assembly pathway. Recent studies suggest that MTP mediated lipid transfer is vital for the folding and stability of nascent apoB as it is translated and translocated into the lumen of the ER. KEYWORDS: Very low density lipoprotein,
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B, microsomal protein disulfide
VERY LOW DENSITY LIPOPROTEIN AND CHYLOMICRON ASSEMBLY Newly synthesised chylomicrons and very low density lipoproteins (VLDL) have lipid cores composed of triglyceride with some cholesteryl ester which is surrounded by a monolayer of phospholipid, free cholesterol, and protein (reviewed by Havel and Kane, 1995). The primary protein component of hepatic VLDL is apolipoprotein B-100. This is a single polypeptide with a molecular weight of 512,000. Computer modeling has been used by Segrest et al. (1994) to help further elucidate the nature of apoB. The amino terminal 20% of apoB contains an alpha helix rich domain typical of globular proteins which is stabilized by seven disulfide bonds (Yang et al., 1990). The remainder of the protein contains two amphipathic beta sheet domains and two amphipathic alpha helix domains which impart the lipid binding properties to the protein. Newly made VLDL also contain small amounts of apoE and apoC proteins. The primary structural protein of intestinal chylomicrons is the 241,000 molecular weight protein, apoB48. ApoB-48 is a product of the same gene which encodes apoB-100 (For a recent review see Chan et al., 1997). In the intestine, the gene product is deaminated posttranscriptionally at cytidine 6666 of the mRNA to produce a uridine. This converts codon 2153 of the message to a stop codon. Some species, including mice and rats, are also capable of editing apoB mRNA in the liver. These species produce apoB-48 particles in the liver as well as the intestine. Newly synthesized chylomicrons also contain apoAI and apoAIV. Lipoproteins are assembled in a complex, multistep process in which lipid is sequentially added to apoB. Although many of the details of the process are yet to be elucidated, the main steps in the assembly process have been established. ApoB is synthesized on the rough endoplasmic reticulum (ER) and translocated into the lumen of the ER. The initial lipid component of the lipoprotein is added cotranslationally to apoB (Borén et al., 1992). The apoB may then form a mature lipoprotein particle in the lumen of the ER which is eventually secreted from the cell. Liver derived cells or cell lines used to study the assembly process have shown that newly synthesized apoB may have additional fates (Figure 9.1). Depending upon the form of apoB, the cell or cell line studied, and their culture conditions, apoB may enter one of several pathways which may either lead to its degradation or assembly into a lipoprotein which is subsequently secreted. For example, apoB may be
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degraded
Figure 9.1 Newly synthesized apoB may have one of many different fates. ApoB may follow a pathway leading to its degradation, secretion as a small dense lipoprotein particle, or as a mature, lipid rich, lipoprotein particle.
cotranslationally as it emerges into the lumen of the endoplasmic reticulum or it may associate with the ER membrane. In most cases, the membrane associated apoB appears to be destined for degradation. Alternatively, apoB may associate with lipid to form a small lipoprotein particle with the density of high density lipoproteins (HDL) or low density lipoproteins (LDL). Some of these small lipoprotein particles are degraded intracellularly, while others are secreted from the cell or converted to more lipid rich, mature lipoprotein particles which are then secreted. This latter pathway has become known as the two step model for the assembly of plasma lipoproteins. Following their assembly, the lipoproteins are transported to the Golgi apparatus where they are packaged into secretory vesicles for transport out of the cell by exocytosis. In some cases, the Golgi may also play a role in the maturation of a lipoprotein particle or its degradation. A two step model for lipoprotein assembly was initially suggested by the studies of Alexander et al. (1976) who, based upon their electron microscopy studies, suggested that nascent apoB particles associate with lipid at the interface between the rough and smooth endoplasmic reticulum. Later, Spring et al. (1992) found that expression of various truncated versions of apoB-100 in HepG2 cells allowed the production
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of lipoprotein particles. However, they were unable to produce VLDL density particles leading them to propose that they were only able to observe the first step in the assembly of VLDL in this cell line. A clear example of the two-step assembly of VLDL is found in the assembly of apoB-48 VLDL in a rat liver derived cell line, McArdle RH-7777. Borén et al. (1994) found that when these cells were cultured in the absence of oleic acid, supplements the apoB-48 was incorporated into HDL density particles which accumulated within the cells because they were very slowly secreted. However, if the cells were grown in media supplemented with oleic acid, apoB-48 was secreted in VLDL density particles. In an experiment to demonstrate the two step nature of lipoprotein assembly, they pulse labelled the apoB and then chased the cells in oleic acid free media. After the labelled apoB-HDL particles were formed within the cells, they supplemented the growth media with oleic acid. Labelled apoB-48 VLDL were secreted, clearly demonstrating that the small HDL density particles were converted to large VLDL density particles. While the two steps of apoB-48 VLDL assembly can be resolved in McArdle RH-7777 cells, the two steps have yet to be clearly separated and defined for apoB-100 lipoproteins or apoB-48 particles in enterocytes or in vivo. However, pulse-chase experiments in both rats (Swift, 1995) and rabbit hepatocytes (Cartwright et al., 1997) provide strong support for the proposal that large VLDL particles are made from small precursor particles. In cell culture models, apoB secretion is highly regulated co- and posttran-slationally by lipid availability. There appears to be little transcriptional regulation of apoB (reviewed by Sniderman and Cianflone, 1993 and Dixon and Ginsberg, 1993). A portion of newly synthesized apoB is incorporated into a lipoprotein particle which is secreted, while the remainder is degraded. For example, in HepG2 cells grown in lipid deficient media, less than 25% of the apoB synthesized is secreted (Dixon et al., 1991). Oleic acid supplementation of the growth media dramatically increases the amount of apoB secreted from the cells, and in addition, decreases the density of the particles produced (Ellsworth et al., 1986). Cholesteryl ester availability has also been proposed to control the amount of apoB containing lipoproteins produced. It is not clear how the regulation of apoB-containing lipoprotein production by lipid availability observed in cell culture relates to the in vivo regulation of apoB production. Although apoB expression does not appear to be limiting for lipoprotein production in cell culture models, it may be in vivo. For instance, mouse models which have decreased apoB mRNA expression due to disruption of one apoB allele have decreased plasma apoB levels (Farese et al., 1995). Elucidation of the details of lipoprotein assembly has been hampered by difficulties in approaching the problem experimentally. The assembly is a complex, multistep process which occurs within the
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endoplasmic reticulum and possibly the Golgi apparatus. Pulse chase approaches to study assembly are complicated by the time it takes to synthesize a protein as large as apoB-100, estimated to be 14min in HepG2 cells (Boström et al., 1986). This time is significant relative to the time it takes to secrete newly synthesized apoB, approximately 30min. Thus, one is not looking at a well synchronised process in a pulse-chase study. Interpretation of the results has also been complicated by the large number of different cell culture systems studied and the various culture conditions used to maintain the cells. In addition, it is well established that the cell lines used to study lipoprotein assembly have altered lipid metabolism and physiology compared to an hepatocyte or enterocyte. Further complications are introduced when cells are transfected with apoB constructs which result in inappropriate levels of apoB expression. This may create new regulatory processes, or the introduction of assembly or degradation pathways which normally do not occur.
MICROSOMAL TRIGLYCERIDE TRANSFER PROTEIN The microsomal triglyceride protein (MTP) is a soluble protein originally isolated and characterized from bovine liver. The protein is a heterodimer consisting of the multifunctional protein, protein disulfide isomerase (PDI) and a unique 97kD subunit (Wetterau et al., 1990). In addition to its role in MTP, PDI has disulfide isomerase and chaperone (Cai et al., 1994) activities which play a role in folding newly synthesized proteins within the lumen of the ER. PDI is also a subunit of the tetrameric enzyme, prolyl-4-hydroxylase. cDNAs encoding the large subunit of human, bovine (Sharp et al., 1993), hamster (Lin et al., 1994), and mouse (Nakamuta et al., 1996) MTP have been cloned. The protein is highly conserved between species (greater than 85% amino acid identity between the human protein and that of the other species), however it is not highly homologous to any previously described protein. Using a low level of homology, analysis of structural predictions, and a comparison of gene structures, Shoulders et al. (1994) proposed that the MTP large subunit is a member of the vitellogenin gene family. Vitellogenin is cleaved to produce three polypeptides which comprise lipovitellin, a lipoprotein complex that is a component of yolk in egg-laying animals. PDI is absolutely required for MTP activity. Dissociation of the two subunits of MTP using a variety of nondenaturing approaches results in a loss of MTP activity and the aggregation of the MTP large subunit. Although little disulfide isomerase activity is expressed by intact MTP (Wetterau et al., 1990), PDI activity is readily detected following
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treatments of MTP that dissociate the large subunit from PDI (Wetterau et al., 1991). Expression studies in insect Sf9 cells support the biochemical studies; the expression of active MTP requires the coexpression of PDI and the MTP large subunit (Ricci et al., 1995). In the absence of PDI expression, the MTP large subunit forms an insoluble aggregate. The molecular role of PDI in the transfer protein is not completely clear. Mutational analysis has demonstrated that the disulfide isomerase activity of PDI is not required for MTP to express its transfer activity (Lamberg et al., 1996). PDI contains a KDEL endoplasmic reticulum retention sequence which may play a role in keeping MTP in the ER. The large subunit of MTP which does not contain an ER retention sequence, may utilize its association with PDI to maintain its ER localization. Although this targeting function of PDI in MTP has not been demonstrated directly, there is experimental evidence for a similar role for PDI in prolyl-4-hydroxylase (Vuori et al., 1992). MTP activity is typically characterized by measuring the rate of MTP mediated transport of radiolabelled lipid molecules between synthetic small unilamellar, phosphatidylcholine vesicles. Triglyceride (TG) and cholesteryl ester (CE) are typically incorporated into the vesicle membranes at a concentration around 0.5mol%. In these assays, bovine MTP has an almost two fold preference for TG transport compared to CE, and a 25 fold preference for TG transfer compared to phosphatidylcholine when the percents of donor lipid transferred per time are compared (Wetterau and Zilversmit, 1985). However, because phosphatidylcholine is present in excess over neutral lipid (200:1 mole ratio, phospholipid:neutral lipid), MTP actually transports many more phospholipid than TG or CE molecules in this assay. Although the kinetics of MTP mediated lipid transfer with the rat, mouse, hamster, and human proteins have not been characterized in detail, they all have been found to have relative lipid transfer activities similar to that of the bovine protein. In vitro assays show that MTP binds and shuttles lipid molecules between membranes (Atzel and Wetterau, 1993; Atzel and Wetterau, 1994). MTP with bound lipid, an intermediate in the transfer reaction, has been isolated and characterized. There appears to be three or more lipid molecule binding sites on MTP with one site for TG, one site for CE, and up to three sites for phosphatidylcholine. It is not clear if the TG, CE, and one of the PC sites represent one, two or even three separate sites. In a one site model, the TG, CE, and phospholipid molecules would compete for binding to the same site. The triglyceride and cholesteryl ester and some of phospholipid binding sites on MTP appear to play a role in the lipid transfer reaction because MTP rapidly transports TG, CE, and phospholipid from these sites on MTP to a membrane, or from a membrane to these sites on MTP. One or two of
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the phospholipid binding sites does not rapidly transport phospholipid molecules to a membrane, suggesting that this lipid binding site or sites is not actively participating in shuttling lipid molecules between membranes. Lipid transfer proteins typically promote the exchange of lipid molecules between membranes in in vitro assays. The ability of MTP to promote the net transport of lipid was investigated by measuring the ability of MTP to transport TG and CE into membranes which do not contain TG or CE (Wetterau and Zilversmit, 1985). MTP was readily able to transport TG and CE from membranes rich in these lipid to membranes deficient in them, indicating that MTP could promote the net movement of TG and CE. In contrast, the only other mammalian protein known to catalyze neutral lipid transfer, the cholesteryl ester transfer protein, was not able to promote the net movement of TG and CE in these studies. Thus the apparent mass transfer mediated by MTP appears to be an intrinsic property of the protein and not a function of the assay used to characterize it. MTP activity and protein are found within the lumen of microsomes isolated from liver and intestine. In rat liver, equal levels of MTP activity are isolated from rough and smooth microsomal fractions (Wetterau and Zilversmit, 1986). MTP is very prevalent within the lumen of the ER, representing up to 1 % of the soluble protein. In hamster, the duodenum contains 10 fold more MTP large subunit mRNA and fourfold more lipid transfer activity than the liver (Lin et al., 1994). Along the length of the intestine, MTP message levels decrease, with the colon containing about 5% the message levels of the duodenum. Northern blot analysis of various human tissues has revealed that in addition to liver and intestine, lower message levels can be found in ovary, testis, and kidneys (Shoulders et al., 1993). The possible role, if any, of MTP in these tissues is not known.
DEFECTS IN MTP CAUSE ABETALIPOPROTEINAEMIA Abetalipoproteinaemia is a recessive defect in the production of chylomicrons and VLDL (reviewed by Kane and Havel, 1989). Subjects produce virtually no apoB containing lipoproteins resulting in plasma cholesterol levels of around 40mg/dl. The neurodegenerative disorders associated with the defect are secondary to fat soluble vitamin deficiencies, and in particular, a vitamin E deficiency. Development of these disorders can be prevented by dietary vitamin supplements. Due to the defect in lipoprotein production, fat accumulates in the hepatocytes and enterocytes of abetalipoproteinaemic subjects. With time, individuals with abetalipoproteinaemia learn to avoid high fat
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foods that tend to produce gastrointestinal discomfort due to fat malabsorption. The first direct evidence that MTP is involved in lipoprotein assembly came from the demonstration that an absence of functional MTP causes abetalipoproteinaemia. In an initial set of experiments, MTP activity levels in intestinal biopsies obtained from normal and abetalipoproteinaemic subjects were compared (Wetterau et al., 1992). MTP activity could be readily detected in five normal subjects (Figure 9.2), as well as one subject with Anderson’s disease and another subject with homozygous hypobetalipoproteinaemia. These latter two subjects have genetic diseases independent of abetalipoproteinaemia which also result in an absence of intestinal lipoprotein production. In contrast, MTP activity was not detectable in four unrelated abetalipoproteinaemic subjects (Figure 9.2). Western blot analysis of the intestinal proteins revealed that the large subunit of MTP was undetectable in the abetalipoproteinaemic subjects. To determine the molecular basis for the absence of MTP activity and protein, the gene encoding the large subunit of MTP was sequenced in the four abetalipopro
Figure 9.2 MTP activity in intestinal biopsies obtained from five normal subjects (left panel) or four unrelated abetalipoproteinemic subjects (right panel). The results are expressed as the percent donor membrane triglyceride transferred to acceptor membranes per hour per g intestinal protein. Reprinted with permission from: Wetterau, J.R., Aggerbeck, L.P., Bouma, M.-E., Eisenberg, C., Munck, A., Hermier, M., Schmitz, J., Gay, G., Rader, D.J. and Gregg, R.E. (1992). Science, 258, 999–1001. Copyright, 1992 American Association for the Advancement of Science.
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teinaemic subjects (Sharp et al., 1993; Ricci et al., 1995; Rehberg et al., 1996). Mutations which explained the absence of the large subunit were identified in all four subjects (see below for additional details). In addition to these four subjects, the sequence of the genes encoding MTP in an additional eight abetalipoproteinaemic individuals also reveal mutations which render MTP inactive (Narcisi et al., 1995). These studies led to two conclusions. First, abetalipoproteinaemia is caused by mutations in the gene encoding the large subunit of MTP which render MTP inactive. Second, MTP is required for the efficient production of chylomicrons and VLDL. Most mutant alleles identified to date readily predict defects in MTP. These consist of various frameshift, nonsense, and splice site mutations, all of which predict severely truncated MTP large subunits. However, two MTP mutations identified were rather subtle and required further characterization to fully understand why they resulted in an absence of detectable MTP activity and protein. One was a nonsense mutation causing the MTP large subunit to be truncated at the carboxyl terminus by only 30 amino acids (Ricci et al., 1995). A second was a missense mutation resulting in arginine 540 being changed to a histidine residue (Rehberg et al., 1996). The basis for the disruption of MTP in these two instances was further revealed through expression studies performed in insect Sf9 cells. In both cases, the mutations produced similar findings. When wild type MTP large subunit was co-expressed with PDI, active MTP was produced. However, when the mutated large subunits were co-expressed with PDI, MTP activity was not detectable, although the mutant protein could be readily detected by Western blot analysis. Further characterization of the MTP large subunit demonstrated that it had formed an insoluble aggregate within the cell. The production of aggregated large subunits suggested that both the nonsense and missense mutations produced a protein which was unable to associate with PDI in a normal fashion. Proper association with PDI is required for the MTP large subunit to form a soluble, active transfer protein (see above). In the abetalipoproteinaemic subjects, the aberrantly folded MTP large subunit is probably rapidly degraded. However, the high levels of expression obtained using the baculovirus expression system allowed the MTP large subunit to be detected in the Sf9 cells.
RECONSTITUTION OF LIPOPROTEIN ASSEMBLY Early attempts to reproduce lipoprotein assembly in a nonhepatic, nonintestinal cell line revealed that Chinese hamster ovary cells (CHO) did not produce a lipoprotein particle when they were transfected with a plasmid expressing the amino terminal 53% of apoB (apoB-53), even
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though apoB protein was produced (Thrift et al., 1992). In contrast, when apoB-41 was expressed in a mouse mammary derived cell line, a lipoprotein particle was produced in which over 50% of the total lipid was TG (Herscovitz et al., 1995). To further characterize the role of MTP in lipoprotein assembly, various investigators have co-expressed MTP with apoB or truncated forms of apoB in three different nonhepatic, nonintestinal cell lines; HeLa (Gordon et al., 1994), COS (Leiper et al., 1994; Patel and Grundy, 1996; Wang et al., 1996), and Sf21 (Gretch et al., 1996). Except for the very short forms of apoB (less than 20 to 30% the size of apoB-100), MTP co-expression resulted in increased apoB secretion. The level of increase was dependent upon the cell line used, culture conditions, and the nature of the apoB fragment expressed. The production of lipoproteins by HeLa cells was characterized in detail by Gordon et al. (1994). When cells were transiently transfected with a plasmid expressing apoB-53, the protein was detected in cell homogenates, indicating it was produced. However, only trace quantities of protein were found in the media. When similar transient transfections were performed in HeLa cells stably transfected with MTP, apoB was found both in the cells and the media. A greater than 30 fold increase in the efficiency of apoB secretion was found in the cells expressing MTP compared to the cells which did not. The lipoproteins produced had a density of HDL. This density was not unexpected because similar density particles were produced when apoB-53 was expressed in a liver derived cell line, McArdle RH-7777. In liver derived cell lines, the number of apoB particles produced and their density can be regulated by the growth conditions. For example, in HepG2 cells, the presence of oleic acid in the growth medium increases the number of particles secreted and the density of the particles is decreased, indicating that they are more lipid rich. The regulation of lipoprotein production by lipid supplements in the growth media of HeLa cells expressing MTP and apoB was tested by Gordon et al. (1994). In the absence of lipid supplements, about 55% of the apoB was secreted on particles with a density of HDL (d=1.063–1.21 g/ml), while the remainder (45%) of the apoB was observed in the lipid poor bottom fraction (d>1.21 g/ml). When cultured in the presence of oleic acid and cholesterol, the number of particles secreted was doubled and the density of the particles decreased (Figure 9.3). A greater fraction (80%) of the lipoproteins had a density similar to HDL, while 15% had a lower density (<1.063 g/ml). Only a small percentage of the apoB was recovered in the bottom
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Figure 9.3 ApoB secretion from HeLa cells expressing MTP is regulated by the availablity of lipid in the growth media. HeLa cells stably expressing MTP were transiently transfected with apoB-53 and then incubated in lipid-poor or lipid rich media for 24 hours. The total apoB and apoB with the density of VLDL and LDL, HDL, or d>1.21 g/ml were quantitated by Western blot analysis followed by densi-tometry. Reprinted with permission from: Gordon, D.A., Jamil, H., Sharp, D., Mullaney, D., Yao, Z., Gregg, R.E. and Wetterau, J. (1994). Proc. Natl. Acad. Sci. USA, 91, 7628–7632. Copyright, 1994 National Academy of Sciences. U.S.A.
fraction. Thus in HeLa cells expressing MTP, the nature of the apoB lipoproteins produced and the regulation of their production is similar to liver derived cell lines. When truncated versions of apoB are expressed in liver derived cell lines, approximately 25% of the protein must be expressed before it associates with lipid to form a lipoprotein particle (Spring et al., 1992; Yao et al., 1991). Smaller fragments are secreted in a lipid depleted form. Wang et al. (1996) expressed a variety of apoB fragments in COS-7 cells. Secretion of apoB fragments equal or larger than apoB-23 was increased by co-expression with MTP. Regardless of the size of the apoB fragment expressed (apoB-23 through apoB-94), the majority of the lipoproteins had an HDL density. Gretch et al. (1996) found that the amino terminus of apoB was necessary for MTP dependent reconstitution of lipoprotein production in insect Sf21 cells. Furthermore, Ingram and Shelness (1997) showed that the amino
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terminus of apoB needs to fold correctly in order to initiate MTP dependent lipid transfer to the nascent apoB polypeptide.
THE STEPS IN LIPOPROTEIN ASSEMBLY MEDIATED BY MTP Recently, considerable attention has been focused on identifying the specific MTP mediated steps in the multistep pathway leading to a mature lipoprotein particle. The above mentioned two-step model for lipoprotein assembly will be used to discuss the specific role or roles of MTP in lipoprotein assembly. In this model, apoB initially forms a small dense lipoprotein particle. This is subsequently converted to a lipid rich, mature lipoprotein by the addition of lipid. The studies of abetalipoproteinaemia as well as the reconstitution experiments have provided evidence that MTP is required for an early step in lipoprotein assembly. For instance, it can be predicted that if MTP mediated only the second step of assembly, then abetalipoproteinaemic individuals should produce small dense apoB lipoproteins which have been shown to be secretion competent in cell culture. Abetalipoproteinaemic subjects do not produce significant quantities of these small dense apoB lipoproteins (Kane and Havel, 1989), suggesting MTP is required for an early step. Similarly, expression of apoB in non-hepatic, non-intestinal cell lines should also lead to the secretion of small dense lipoprotein particles if MTP mediated only the second step. In general, most if not all of the apoB expressed in the absence of MTP is degraded before lipoproteins can form (Thrift et al., 1992). Expression of MTP along with apoB in nonhepatic, non-intestinal cell lines results in the formation of a small dense particle reminiscent of the primordial lipoprotein precursor formed in the first step of lipoprotein assembly (Gordon et al., 1994, Leiper et al., 1994 and Wang et al., 1996). Lipid laden VLDL particles are not produced. Thus MTP coexpression with apoB only reconstitutes a process reminiscent of the early phase of lipoprotein assembly, further supporting the suggestion that MTP is required for an early step in the assembly pathway. The development of highly specific inhibitors of MTP has greatly facilitated the elucidation of the role and step(s) in lipoprotein assembly mediated by MTP. A number of such inhibitors have been described (Jamil et al., 1996,; Haghpassand et al., 1996; Benoist et al., 1996). MTP inhibitors can be divided into two classes; reversible inhibitors and nonreversible photoaffinity inhibitors which permanently inactivate MTP by forming a covalent adduct between the inhibitor molecule and MTP. All of the inhibitors reported to date have similar levels of potency when measured in an in vitro assay for MTP activity and all
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show a highly selective, dose-dependent inhibition of apoB secretion when administered to HepG2 cells. Interestingly, one inhibitor which inhibits apoB secretion from HepG2 cells, BMS-200150, was shown to selectively block MTP mediated triglyceride and cholesteryl ester transfer compared to phospholipid transfer (Jamil et al., 1996). Therefore, inhibition of MTP mediated neutral lipid transfer is sufficient to block lipoprotein secretion. Inhibitor studies carried out by Benoist et al. (1996) and Benoist and Grand-Perret (1997) in HepG2 cells were particularly informative with regard to which steps in lipoprotein assembly are mediated by MTP. Using a pulse-chase protocol, they showed that addition of inhibitor in close temporal proximity to a short 10 min pulse (in other words, at the initiation of translation), effectively blocked apoB secretion. However, the ability of the inhibitor to block apoB secretion disappeared as early as 10 min into the chase, consistent with MTP being required for an early, but not a late step, in the assembly pathway. Although apoB secretion was blocked, analysis of the cell extracts showed that there was no accumulation of apoB intracellularly. In fact, the incorporation of radiolabelled amino acids into apoB was reduced 2–3 fold by the inhibitor, even though total cellular protein did not appear to be affected. This could be explained if inhibition of MTP causes apoB to be degraded cotranslationally or immediately post-translationally. When an experimental protocol was used to synchronize apoB-100 translation prior to the radiolabelled amino acid pulse, they found that in the presence of the MTP inhibitor, there was no effect on the shorter forms of apoB, however, the amount of apoB greater than 65% the size of apoB-100 was decreased significantly. This suggests apoB-100 is being degraded cotranslationally in the absence of MTP activity. Thus inhibition of MTP mediated lipid transfer in HepG2 cells causes a rapid increase in the degradation of apoB which occurs in close temporal proximity to translation. This conclusion is further supported by their experiments showing that co-treatment of the cells with various inhibitors of protein degradation blocked the ability of the MTP inhibitor to stimulate apoB decay (Benoist and Grand-Perret, 1997). Under these conditions, the apoB simply accumulated within the cell and was not secreted, suggesting it was improperly folded and retained within the endoplasmic reticulum. From these studies, it appears that MTP-mediated lipid transfer either stabilizes the apoB polypeptide as it is synthesized and enters the lumen of the ER or it assists the translocation of apoB into the lumen of the ER where it is protected from degradative processes that occur in the cytoplasm. Beyond protecting apoB from degradation, MTP is also required to ensure proper initiation of lipoprotein assembly and the formation of a secretion competent particle. These findings were further extended by Rusiñol et al. (1997). Using
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purified microsomes, a reconstituted in vitro system for lipoprotein assembly was established. It was shown that if microsomes from liver were used, apoB was capable of translocating into the microsomal lumen cotranslationally. Interestingly, if they used microsomes derived from the pancreas which does not contain MTP, apoB was still capable of translocating into the lumen. Therefore, MTP was not required for the translocation of newly synthesized apoB into the lumen of the microsomes. This evidence along with the previously described studies, strongly supports the hypothesis that the main role for MTP in the very early steps of the lipoprotein assembly pathway is related to the proper folding and stabilization of the apoB polypeptide as it enters the lumen of the endoplasmic reticulum. This is believed to occur via transfer of lipid to the amphipathic lipid binding domains of apoB. Inhibitors have also been utilized to investigate whether MTP also mediates the second step of lipoprotein assembly for apoB-48. In this case, a photoaffinity MTP inhibitor was utilized in the McArdle RH7777 cell line (Gordon et al., 1996). As mentioned previously, this class of inhibitor permanently and selectively inactivates MTP when the inhibitor is activated by irradiation with ultraviolet light. Covalent inactivation of MTP in McArdle RH-7777 cells under culture conditions which only promote the first step in apoB-48 lipoprotein assembly, blocked apoB secretion. However, inactivation of MTP after the first step was complete, but before the second step was activated, did not affect the loading of lipid onto the nascent particles and the production of apoB-48 VLDL once the second step was initiated by addition of oleic acid. Thus, in this cell culture model, MTP only mediates the first step in apoB-48 assembly and not the loading of core neutral lipid in the second step. While these results are clear, a subsequent report has shown MTP inhibition does affect the production of apoB-48 VLDL in McArdle RH-7777 cells using a similar but not identical approach (Wang et al., 1997). The specific experimental differences that might explain the different outcomes from the two studies are not clear. From the available data, one could conclude that normal MTP activity is not required for the second step in lipoprotein assembly, but under some experimental conditions, a loss of MTP activity may affect its efficiency. Additional data indicating that MTP mediates an early step in the lipoprotein assembly pathway comes from studies of the physical association between MTP and apoB. Using co-immunoprecipitation techniques, two independent studies have shown a stable interaction between MTP and apoB (Wu et al., 1996 and Patel and Grundy, 1996). This interaction occurs within the amino terminal 13% of the apoB polypeptide and is stable to high salt and low pH indicating the association is hydrophobic in nature. The binding of MTP to apoB is transient, reaching a peak 10min after initiation of translation, and
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diminishing rapidly thereafter. That MTP appears to associate with only the amino terminus of apoB and that the peak extent of the interaction occurs 10min after initiation of translation, indirectly supports the hypothesis that MTP mediates an early step in the lipoprotein assembly pathway. The association between apoB and MTP has recently been confirmed in a cell free system by Hussain et al. (1997). Additional investigation is needed to confirm the biological importance of the MTP-apoB physical association. Studies to date show MTP prefers to associate with a domain on apoB which is believed to not bind lipid. Since the only known activity of MTP is lipid transfer, it is unclear how a physical interaction with this non-lipid binding segment would promote lipidation of apoB. In addition, a stable physical interaction between MTP and apoB is not compatible with a shuttle mechanism of MTP mediated lipid transfer (Atzel and Wetterau, 1993). However, the kinetics of the lipid transfer reaction were studied in an in vitro system and it is unclear how well this in vitro system recapitulates what occurs in vivo. It is a formal possibility that lipid transfer has a different kinetic mechanism in vivo. A model for the role of MTP in lipoprotein assembly, based upon the data described above, is shown schematically in Figure 9.4. Translation and processing of apoB begins much like that for any other secretory protein. The signal peptide is cleaved and the amino terminus is inserted into the lumen of the endoplasmic reticulum. This domain is folded into a globular structure with multiple disulfide linkages (Figure 9.4, panel A). The correct folding of this domain is critical to MTP dependent lipoprotein assembly and is perhaps promoted by the physical interaction of MTP with this site. MTP binds lipid molecules from the inner leaflet of the endoplasmic reticulum membrane and then associates with the amino terminus of the nascent apoB polypeptide (Figure 9.4, panel B). The lipid molecule is then transferred to a newly formed amphipathic lipid binding domain (Figure 9.4, panel C). This allows the lipid binding domain to fold correctly so that the hydrophilic surfaces orient outward towards the aqueous milieu of the ER lumen while the hydrophobic surfaces orient towards the inside of the developing spherical particle containing neutral lipid in the core (Figure 9.4, panel D). This stabilizes the higher order structure of the apoB polypeptide. If MTP is absent or the lipid supply is insufficient, the amphipathic domains cannot orient properly, exposing the hydrophobic lipid binding surfaces to the unfavourable aqueous environment. This can
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Figure 9.4 Model for the role of MTP in the assembly of apoB containing lipoproteins. In this model, apoB is translated and translocated into the lumen of the endoplasmic reticulum. Within the ER, MTP mediated addition of lipid to the lipid binding domains of apoB allows it to form a nascent apoB lipoprotein particle. Bulk core lipid is then added to make a mature lipoprotein particle which is then secreted from the cell. Reprinted with permission from: Gordon, D.A. (1997). Recent advances in elucidating the role of the microsomal triglyceride transfer protein in apolipoprotein B lipoprotein assembly. Curr. Opin. Lipid., 8, 131–137. Copyright, 1997 Rapid Sciences Publishers.
have a number of negative outcomes including misfolding, aggregation, or binding to the inner leaflet of the ER membrane. The end result is the degradation of the newly synthesized protein. However, if MTP is present and the lipid supply is sufficient, lipidation of the newly forming lipid binding domains continues until translation of the protein is complete and a small primordial precursor particle is released (Figure 9.4, panel E). Finally, the mature particle is formed by addition of bulk neutral lipid to the core. It appears this process may not require MTP for apoB-48, however, this remains a subject of controversy requiring further study. Also, the case of apoB-100 has yet to be addressed experimentally. The resultant mature lipoprotein particle is transported to the Golgi apparatus and subsequently secreted from the cell.
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with two amphipathic beta-strand domains. Detection by the computer program LOCATE. Arterioscler. Thromb ., 14 , 1674–1685. Sharp, D., Blinderman, L., Combs, K.A., Kienzle, B., Ricci, B., WagerSmith, K. et al. (1993). Cloning and gene defects in microsomal triglyceride transfer protein associated with abetalipoproteinemia. Nature , 365 , 65–69. Shoulders, C.C., Brett, D.J., Bayliss, J.D., Narcisi, T.M.E., Jarmuz, A., Grantham, T.T. et al. (1993). Abetalipoproteinemia is caused by defects of the gene encoding the 97 kDa subunit of a microsomal triglyceride transfer protein. Hum. Mol. Genetics , 2 , 2109–2116. Shoulders, C.C., Narcisi, T.M.E., Read, J., Chester, S.A., Brett, D.J., Scott, J. et al. (1994). The abetalipoproteinemia gene is a member of the vitellogenin family and encodes an alpha-helical domain. Nat. Structural Biol ., 1 , 285–286. Sniderman, A.D. and Cianflone K. (1993). Substrate delivery as a determinant of hepatic apoB secretion. Arterioscler. Thromb ., 13 , 629–636. Spring, D.J., Chen-Lui, L.W., Chatterton, J.E., Elovson, J., and Schumaker, V.N. (1992). Lipoprotein assembly. Apolipoprotein B size determines lipoprotein core circumference J.Biol. Chem ., 267 , 14839–14845. Swift, L.L. (1995). Assembly of very low density lipoproteins in rat liver: a study of nascent particles recovered from the rough endoplasmic reticulum. J.Lipid Res ., 36 , 395–406. Thrift, R.N., Drisko, J., Dueland, S., Trawick, J.D. and Davis, R.A. (1992). Translocation of apolipoprotein B across the endoplasmic reticulum is blocked in a nonhepatic cell line. Proc. Natl. Acad. Sci. USA , 89 , 9161–9165. Vuori, K., Pihlajaniemi, T., Myllylñ, R. and Kivirikko, K.I. (1992). Site-directed mutagenesis of human protein disulphide isomerase: effect on the assembly, activity and endoplasmic reticulum retention of human prolyl 4-hydroxylase in Spodoptera frugiperda insect cells. EMBO J . 11 , 4213–4217. Wang, S., McLeod, R.S., Gordon, D.A. and Yao, Z. (1996). The microsomal triglyceride transfer protein facilitates assembly and secretion of apolipoprotein B-containing lipoproteins and decreases co-translational degradation of apolipoprotein B in transfected COS7 cells. J.Biol. Chem ., 271 , 14124–14133. Wang, Y., McLeod, R.S. and Yao, Z. (1997). Normal activity of microsomal triglyceride transfer protein is required for the oleateinduced secretion of very low density lipoproteins containing apolipoprotein B from McA-RH7777 cells. J.Biol. Chem ., 272 , 12272–12278. Wetterau, J.R. and Zilversmit, D.B. (1985). Purification and characterization of microsomal triglyceride and cholesteryl ester transfer protein from bovine liver microsomes. Chem. Phys. Lipids , 38 , 205–222. Wetterau, J.R. and Zilversmit, D.B. (1986). Localization of intracellular triacylgylcerol and cholesteryl ester transfer activity in rat tissues.
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Biochim. Biophys. Acta ., 875 , 610–617. Wetterau, J.R., Combs, K.A., Spinner, S.N. and Joiner, B.J. (1990). Protein disulfide isomerase is a component of the microsomal triglyceride transfer protein complex. J.Biol. Chem ., 265 , 9800– 9807. Wetterau, J.R., Combs, K.A., McLean, L.R., Spinner, S.N. and Aggerbeck, L.P. (1991). Protein disulfide isomerase appears necessary to maintain the catalytically active structure of the microsomal triglyceride transfer protein. Biochemistry , 30 , 9728– 9735. Wetterau, J.R., Aggerbeck, L.P., Bouma, M.-E., Eisenberg, C., Munck, A., Hermier, M. et al. (1992). Absence of microsomal triglyceride transfer protein in individuals with abetalipoproteinemia Science , 258 , 999–1001. Wu, X., Zhou, M., Huang, L.-S., Wetterau, J. and Ginsberg, H.N. (1996). Demonstration of a physical interaction between microsomal triglyceride transfer protein and apolipoprotein B during the assembly of apoB-containing lipoproteins. J.Biol. Chem ., 271 , 10277–10281. Yang, C.-Y., Kim, T.W., Weng, S.-A., Lee, B., Yang, M. and Gotto, A.M. (1990). Isolation and characterization of sulfhydryls and disulfide peptides of human apolipoprotein B-100. Proc. Natl. Acad. Sci. USA , 87 , 5523–5527. Yao, Z., Blackhart, B.D., Linton, M.F., Taylor, S.M., Young, S.G. and McCarthy, B.J. (1991). Expression of carboxyl-terminally truncated forms of human apolipoprotein B in rat hepatoma cells. Evidence that the length of apolipoprotein B has a major effect on the buoyant density of the secreted lipoproteins. J.Biol. Chem ., 266 , 3300–3308.
10 Role of Endothelial Lipases in Disease Ira J.Goldberg and Catherine H.Tuck Department of Medicine, Columbia University College of Physicians & Surgeons, 630 West 168th Street, New York NY 10032, USA
INTRODUCTION In 1943 while studying postprandial lipaemia in dogs, Hahn (1943) first noted that injection of heparin led to the rapid clearance of chylomicrons from plasma. This was due to the release of two enzymes, lipoprotein lipase (LpL) and hepatic triglyceride lipase (HL) into the bloodstream. The post-heparin lipases regulate the plasma concentrations of the major classes of circulating lipoproteins and modulate the uptake of lipids into tissues. Moreover, a number of human lipid disorders are due to abnormalities of these enzymes. The regulation of these activities by pharmacological, hormonal, and genetic differences in humans and animals has been intensively studied in the last several decades.
STRUCTURE OF POST-HEPARIN LIPASES LpL, HL and pancreatic lipase belong to a gene family. They have a similar active serine catalytic site. Hydrolysis of substrates in different lipoproteins is determined by variations in the lipid binding regions of the lipase protein. The lid-structure (Figure 10.1) is critical in this respect since substituting the HL lid onto LpL allows the mutated LpL to act on smaller lipoproteins (Kobayashi et al., 1996). Other important domains are required for LpL to interact with apoCII, its activator, and with heparan sulfate proteoglycans. This latter region has been studied by assessing LpL binding to heparin. Although there are a number of basic amino acid rich regions of LpL, the carboxyl-terminal domains of the protein modulate its interaction with heparin (Nielsen et al., 1997). LpL is widely believed to be most active as a dimeric molecule that is composed of two identical subunits that are arranged in a head to tail configuration (Wong et al., 1997). Although LpL will hydrolyze most
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triacylglycerols, its preferred fatty acid is oleate. Saturated fatty acids appear to be a less preferred substrate than unsaturated fatty acids. Fatty acids in the 1 position of triglyceride and phospholipids are hydrolysed in preference to those in the 2 position.
Figure 10.1 Schematic of lipoprotein lipase structure. LpL is thought to function as a dimeric protein containing two identical subunits arranged in a head to tail configuration. Like most other lipases, including hepatic lipase, it has a lipidbinding lid that controls its ability to use substrate lipid particles. A catalytically active serine resides in a “pocket” within the amino terminal half of the molecule.
SYNTHESIS AND PROCESSING OF LIPASES LpL is synthesised by a number of cells and tissues. The major sites of
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LpL synthesis are skeletal and cardiac muscle and adipose tissue; lesser amounts of LpL are made in the kidney, adrenal, brain and macrophages (Zechner, 1997). In addition, LpL is synthesised in the liver in the neonatal period and perhaps during cachexia. The events that control LpL transcription are an area of active research. LpL production may also be modulated by changes in mRNA translation due to proteins that bind to the 3′ untranslated region of the message (Ranganathan and Kern, 1997). However, major changes in LpL activity and actions occur without changes in LpL gene expression or translation, so post-translational processes are critical regulatory steps. Once translated, the LpL protein undergoes a series of intracellular processing events that control its activity; unglycosylated LpL is inactive. In addition, LpL is most active as a dimer. This dimerization process does not require the presence of heparan sulfate proteoglycans (Berryman and Bensadoun, 1995). LpL must transfer from its cells of origin to the luminal surface of capillary endothelial cells that are exposed to the large triglyceride-rich lipoproteins contained in the blood. This process is depicted in Figure 10.2. From tissue culture studies it appears that the newly synthesised protein is first associated with the surface of the cells. Some of this LpL is re-internalised and then degraded (Berryman and Bensadoun, 1995). The remainder is dissociated from the cell surface, perhaps via the actions of an endothelial cell heparanase (Pillarisetti et al., 1997). The newly released LpL, either alone or in tandem with a fragment of digested glycosaminoglycan, transfers to the endothelial cell. As suggested in the figure, LpL not associated with glycosaminoglycans is unstable and may rapidly be inactivated, shown as dissociation into monomers. Although there has been an extensive search for an LpLspecific transporter for transcytosis, none has been found. Therefore it is possible that LpL transfers from the abluminal to the luminal side of endothelial cells by moving around or non-specifically through the endothelial cells. LpL associates with the luminal endothelial surface and its binding to and release from these cells could affect its activity in vivo. Both LpL and HL associate with highly negatively charged molecules, heparan sulfate proteoglycans, on the cell surface; a specific ten-unit high affinity LpL-binding oligosaccharide has been isolated (Parthasarathy et al., 1994). LpL associates with a number of other proteins including members of the LRP receptor family and regions of apolipoprotein B (Goldberg, 1996). Perturbations of endothelium with tumour necrosis factor and perhaps other cytokines and lipolysis products will release the bound LpL into the bloodstream. Some active LpL is found
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Figure 10.2 Extracellular transport of lipoprotein lipase. LpL is a somewhat unique molecule in that it must transfer from its site of synthesis, shown in myocytes and adipocytes, to the luminal surface of the endothelial cell where it interacts with circulating triglyceride-containing lipoproteins. LpL may be released from the adipocyte surface alone, or in association with a piece of glycosaminoglycan (GAG). The heparin-like GAG may prevent the LpL from being inactivated (shown as a monomer). Alterations of LpL activity that occur with feeding-fasting are not due to changes in the amount of LpL protein and might reflect alterations in this LpL transport system.
in the bloodstream associated with lipoproteins and even more inactive LpL is present on lipoproteins in pre-heparin blood (Vilella et al., 1993). Most of this LpL is rapidly removed by the liver either via uptake by LRP or proteoglycans (Wallinder et al., 1979). It is possible that by acting as a ligand for LRP, LpL will increase removal of associated lipoproteins. Although less thoroughly investigated, HL appears to have a similar synthetic route. It is made in hepatocytes and requires carbohydrate addition and processing. Like LpL, some HL is found on the surface of its cell of origin, the hepatocyte. HL is thought to transfer to adjacent endothelial cells lining the space of Disse.
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REGULATION OF LIPOLYTIC ACTIONS OF ENDOTHELIAL-BOUND LPL Delivery of fatty acids derived from triglyceride and other lipophilic substances such as cholesterol and fat-soluble vitamins requires uptake of whole lipoproteins or dissociation and assimilation of selected lipids. LpL might participate in both types of lipid uptake. Large triglyceriderich lipoproteins probably do not cross the endothelial barrier except when it is disrupted, e.g., in advanced atherosclerotic lesions. Most fatty acids delivered to cells are thought to be produced by luminal endothelial surface LpL hydrolysis of lipoprotein triglyceride to free fatty acids. Studies of chylomicron uptake by tissues indicate that muscle takes up some chylomicron core lipid (Hultin et al., 1996). Thus, intact lipoproteins or portions of them that have been dissociated during lipolysis must cross the endothelial barrier. In sinusoidal spaces, like the space of Disse in the liver, triglyceride-rich lipoproteins can directly interact with parenchymal cells. Several processes regulate LpL actions at the surface of the endothelial cell. Aside from the amount of LpL itself, apoCII is required to fully activate the enzyme. Both triglyceride-rich lipoproteins and HDL contain apoCII. Since newly formed chylomicrons that are isolated from the lymph before their entry into the circulation contain very little apoCII, chylomicron CII in the bloodstream must have transferred from other lipoproteins, most likely from HDL. Possibly for this reason, a characteristic of many human hypoalphalipoproteinaemias is moderate fasting and post-prandial hypertriglyceridaemia. Excess apoCIII inhibits LpL-mediated lipolysis either by displacing apoCII or by direct actions on the enzyme. This was first observed in vitro and subsequently has been confirmed by studies in transgenic mice overexpressing apoCIII. Humans with a genetic apoCIII deficiency have very low circulating concentrations of triglyceride and rapid removal of VLDL (Ginsberg et al., 1986). Since apoCIII also decreases uptake of remnant lipoproteins by blocking their interaction with the LDL receptor related protein (LRP), this action might also affect lipoprotein catabolism in these patients. The lipoproteins must physically come into contact with the lipases for lipolysis to begin. The diameter of the vessel, its tortuosity, the flow rate of the blood and any margination of the particles that allows them to contact the vessel wall must modulate this. Larger diameter particles are more likely to come into contact with the vessel wall and are also more likely to be able to interact with multiple LpL molecules at once. In part for this reason and in part because larger triglyceride-rich lipoproteins are more likely to be removed from the bloodstream by the
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liver, chylomicron clearance is much more rapid than VLDL, 5–15 minutes versus 4–6 hours. Another reason for the rapid removal of chylomicrons might relate to a greater exposure of substrate lipids. This could occur because less of the surface is occupied by apoB; chylomicrons contain the smaller B48 rather than larger apoB100 molecules. Both chylomicrons and large, nascent VLDL require some triglyceride hydrolysis for their efficient removal from the circulation. In vitro, an emulsion concentration of approximately 5 mols/mL is required for optimal LpL activity; i.e., this amount of triglyceride leads to saturation of the enzyme. These data are in good agreement with the in vivo observation suggesting that LpL is saturated at a plasma concentration of approximately 500mg/dL (Brunzell et al., 1973); 5 mols/mL equals 440mg/dL. When VLDL triglyceride concentration exceeds this amount, there is also an impairment of chylomicron removal as the two lipoproteins compete for the limited amount of enzyme.
NON-ENZYMATIC ACTIONS OF LIPASES Because they interact with both lipoproteins and cell and matrix proteoglycans, lipases can form a molecular bridge between these molecules. This must occur when circulating triglyceride-rich lipoproteins are hydrolysed by these enzymes. LDL and oxidised LDL will also be anchored to proteoglycans by LpL. This bridging action increases LDL cellular uptake and association with matrix proteoglycans (Goldberg, 1996) and does not require the LpL to be enzymatically active. Moreover, the interaction appears to involve an association between LpL and a region in the amino-terminal 20% of apoB. The physiologic and pathophysiologic importance of the nonenzymatic effects of the lipases is currently an area of active investigation. Similarly, HL might increase uptake of chylomicron remnants into the liver via non-enzymatic processes. Presumably the HL binds to these particles and, like LpL, either serves as the ligand for their uptake or concentrates them along the hepatocyte surface such that they are more likely to be internalised by the usual lipoprotein receptors.
REGULATION OF PLASMA LIPOPROTEINS BY POST-HEPARIN LIPASES LpL is the essential enzyme required for clearance of chylomicrons. In addition, catabolism of larger VLDL and initiation of the conversion of VLDL to LDL requires LpL. LpL will also hydrolyse triglyceride and
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phospholipid in LDL and will convert triglyceride-rich LDL into smaller denser LDL. Triglyceride in HDL is also a substrate for LPL actions. Surface lipid from chylomicrons transfers to HDL, increases circulating HDL lipids, and decreases the catabolic rate of the HDL. For this reason, LpL activity is positively correlated with HDL. Hepatic lipase has a different substrate specificity than LpL. HL preferentially hydrolyses triglyceride and phospholipids in small VLDL, LDL, and HDL. Thus, HL appears to be the primary enzyme responsible for conversion of larger HDL and LDL into smaller lipoproteins in the same class. In many studies, post-heparin plasma HL activity inversely correlates with HDL levels. HL also mediates one, but not the only, pathway required for removal of chylomicron remnants.
PHYSIOLOGICAL ACTIONS Delivery of Lipids to Tissues Free fatty acids produced by LpL are used for energy by skeletal and cardiac muscle and are re-esterified and stored within adipose tissue. In addition, by reducing the size of the lipoproteins and exposing receptor binding regions of apoB100 and perhaps by increasing association of apoE with lipoproteins, LpL will increase lipoprotein uptake by tissues. In addition to fatty acids, LpL is thought to increase uptake of fatsoluble vitamins by tissues. LpL increases both vitamin A and E uptake by cultured cells. Limited in vivo data suggest that this is also true in vivo. But since there are other routes for the uptake of these vitamins, this might be a secondary and redundant pathway. Caloric Distribution by LpL Exclusive of its actions to alter the circulating levels of lipoproteins, LpL affects the distribution of calories between tissues. As a marker for adipocyte differentiation and fat stores, LpL is correlated with obesity. After weight loss, LpL activity increases. A number of adipose genes that are stimulated by greater insulin sensitivity are also increased after weight loss. Although it has been postulated that genetic regulation of adipose LpL might modulate the propensity to weight gain, LpL is likely to be but one of many factors important in this respect. Humans and genetically manipulated mice that have no adipose LpL do not have a defect in fat development. Although less plasma lipoprotein free fatty acids are internalised in LpL-deficient fat, more de novo fatty acids are produced from carbohydrates (Weinstock et al., 1997). Partitioning of more fatty acids into muscle occurs with exercise. It is, however,
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unclear whether more fatty acid oxidation in muscle will modulate the development of obesity. What is clear is that lack of LpL in muscles of humans and mice does not lead to an obvious defect in muscle actions. Among the tissues that do not make LpL in the adult is the liver. In the neonatal period, however, the liver is one of the most active LpLproducing organs. Why is that so and why is it necessary for the LpL gene to be suppressed in adulthood? Although it has been postulated that liver LpL will cause the re-uptake of newly synthesised lipoproteins, studies in genetically modified mice do not support this. Rather, it appears that prenatal and perhaps neonatal animals require fatty acid delivery to the liver. Peripheral use of fatty acids for muscle actions and adipose development is not required until after birth. Unless the liver LpL is extinguished, the conversion to peripheral LpL use may not occur. Moreover, besides being futile, excessive hepatic uptake of fatty acids may lead to fatty livers.
PHYSIOLOGICAL REGULATION OF LPL LpL is regulated by both exercise and food intake. One of the most remarkable regulatory events is the reduction in muscle LpL and increase in adipose tissue LpL that occurs with feeding. This then allows the redistribution of calories towards storage in fat and away from oxidation in the muscle. Carbohydrates, and not just fat alone, are required for increased LpL production in postprandial adipose tissue. This along with recent data showing that overexpression of GLUT-4 in adipose tissue leads to an abnormal LpL regulation (Jensen et al., 1996), suggests that the LpL production is modulated by a metabolic effect of cellular glucose. Similarly, one of the metabolic changes accompanying chronic exercise is an increase in production of LpL activity within the muscle bed and a reduction in adipose LpL. Presumably this allows for more efficient uptake of fatty acids into exercising muscle.
HUMAN DISEASES Genetic LpL Deficiency or Type 1 Hyperlipoproteinaemia The function of LpL in lipoprotein metabolism was confirmed when Havel and Gordon (1960) showed that patients with severe fasting chylomicronaemia had a defect such that their post-heparin plasma was unable to hydrolyse chylomicrons in vitro. Multiple molecular reasons for defects in LpL have been established. Most commonly there is a defect in either the catalytic or lipid-binding region of the molecule due
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to an amino acid mutation (Lalouel et al., 1992). Deletion mutations and LpL that has defective heparin binding have also been reported. Genetic LpL deficiency causing type 1 hyperlipoproteinaemia occurs in approximately 1 per million persons. Type 1 hyperlipoproteinaemia also occurs without defects in the LpL molecule. Autoimmune hyperchylomicronaemia with antibodies to LpL or heparin has been reported in association with multiple myeloma, collagen-vascular disease, or lymphomas (Pruneta et al., 1997). Molecular defects in the activator apoCII also cause severe fasting chylomicronaemia; in this situation post-heparin LpL activity is normal but serum from the patient is unable to activate the patients LpL or a purified source of LpL (Breckenridge et al., 1978). A strain of mice called cld, combined lipase deficiency, has a defect in the intracellular molecular processing of LpL and HL molecules. A similar defect has not been described in humans. Type 1 hyperlipoproteinaemia usually presents in childhood. The children are sometimes fussy eaters, have episodes of vomiting and often are empirically switched to lower-fat diets. During childhood these children will occasionally present with severe abdominal pain due to pancreatitis and have severely lipaemic plasma. Other physical findings include hepatosplenomegaly, lipaemia retinalis, and eruptive xanthomas. These findings are usually seen when the triglyceride concentration exceeds 2,000 mg/dL; triglyceride levels as high as 20,000 mg/dL can occur. Heterozygous LpL deficiency is a relatively common genetic mutation in some locally inbred populations, for example in areas of French Canada. Most carriers are normal, although if stressed with a fat load a defect in chylomicron clearance will be evident (Harlan et al., 1967). Low HDL is common, and mild hypertriglyceridemia occurs especially in older carriers. As might be expected, heterozygous genetic LpL deficiency can lead to more severe hypertriglyceridaema when additional reasons for this condition exist. For example, severe hypertriglyceridaemia and pancreatitis have been seen in pregnant women and persons with diabetes who have one abnormal LpL allele (Ma et al., 1993). Treatment Treatment of Type 1 hyperlipoproteinaemia due to LpL or apoCII genetic mutations is aimed at prevention of the severe hyperchylomicronaemia that can cause pancreatitis. The prescription is to avoid intake of fats that will be absorbed as chylomicrons. Fat intake is limited to 5–10% of calories; cooking fat is often substituted with medium-chain triglyceride oils that do not form chylomicrons. Healthy lifestyle habits such as regular physical activity are encouraged.
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Triglyceride-raising medications such as the oestrogen in birth control pills must be avoided. Pregnancies should be monitored closely. In some cases medications that reduce triglyceride production such as fish oils or niacin might have some benefit. Acute episodes of pancreatitis due to hypertriglyceridaemia should be managed similarly to other causes of this potentially life-threatening disease. This includes giving intravenous fluids, observation, and elimination of all food. In cases due to heterozygous LpL defects, an infusion of low dose insulin along with sufficient amounts of glucose to prevent hyperglycaemia may stimulate the limited endogenous LpL. Patients with especially severe hypertriglyceridaemia have been treated with plasmapheresis to remove the chylomicrons from the bloodstream. Genetic apoCII defects may be corrected by infusion of normal plasma that will supply the missing apoCII. Why pancreatitis occurs is not completely understood. Perhaps, lipolysis of chylomicron triglyceride occurs within the pancreas because of the small amount of pancreatic lipase that escapes from the acinar cell. In the presence of a high local concentration of substrate, considerable amounts of free fatty acids and lysolecithin may be generated. High concentrations of these lipids are toxic to cell membranes. This could cause damage to adjacent acinar cells, release more pancreatic lipase, and initiate a cycle of lipolysis and further cell damage. An occasional manifestation of even greater degrees of hypertriglyceridaemia is the hyperchylomicronaemia syndrome (Chait and Brunzell, 1992). In this syndrome patients have decreased mental status and dyspnoea. The symptoms are thought to relate to a reduction in oxygen delivery due to either blood hyperviscosity or a defect in oxygen diffusion through the lipid-filled blood. This condition resolves with reduction of triglyceride levels. LpL and Atherosclerosis A number of other variations in the LpL gene have been reported. Some of these are probably polymorphisms with no clinical significance. Others are found more commonly in subjects with hypertriglyceridaemia and/or low HDL (Blades et al., 1993) and probably represent true defects in LpL activity. In addition, a variation in the LpL promoter has been found. Although not found in most patients, there may be some individuals with familial combined hyperlipoproteinaemia (elevated VLDL and LDL) who have heterozygous defects in LpL. A truncation variant of LpL has been described and is associated with higher HDL concentrations. LpL genetic variations have been studied as a cause of lipoprotein abnormalities and coronary artery disease. Since subjects with LpL deficiency have low plasma HDL levels, LpL defects are seen more
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frequently in subjects with coronary artery disease. In contrast, subjects with familial hypercholesterolaemia may have less disease if LpL activity is reduced (Zambon et al., 1993), presumably because they produce less LDL from its precursor triglyceride-rich lipoproteins. Type 1 hyperlipoproteinaemic patients were thought to be immune from coronary heart disease because the circulating lipoproteins are too large to cross the endothelial cell barrier. This dogma has recently been challenged by a report of several patients with LpL deficiency that had well documented coronary artery disease (Benlian et al., 1996).
DISORDERS OF HEPATIC LIPASE HL deficiency is even rarer than LpL deficiency. Homozygous carriers have hypertriglyceridaemia and hypercholesterolaemia, a pattern similar to that seen in type 3 hyperlipoproteinaemia. In contrast to those subjects, HDL cholesterol is normal despite the hypertriglyceridaemia. The initial reported cases with this disorder had premature coronary artery disease, presumably due to the large number of remnant lipoproteins that accumulated in the circulation (Breckenridge et al., 1982).
DIAGNOSIS Lipase defects are currently diagnosed by measurement of the activity of LpL and HL in post-heparin blood. A patient with a compatible clinical presentation should be tested while fasting. An intravenous injection of 60 units/kg of intravenous heparin is given and the blood sample collected 15 minutes later in a non-anticoagulant containing tube. The post-heparin blood should not clot; if it does the injection was faulty or the patient might have an antibody against heparin. The tube should be placed on ice and the plasma separated and frozen, preferably at −70°C. Lipase hydrolysis of triglyceride can be determined employing radioactive emulsion systems (Nilsson-Ehle and Schotz, 1976) or non-radioactive measurements of liberated free fatty acids. Since post-heparin plasma contains both LpL and HL, the two enzyme activities must be differentiated. This can be done in several ways. Specific antibodies against each enzyme are available. An additional method is to assess the increase in activity when serum is added; only LpL activity is stimulated by serum addition. HL can be measured by performing the assay in a buffer solution that contains 1 M NaCl which inactivates only LpL. In some situations, a genetic diagnosis might be useful; PCR-based restriction length polymorphisms or lipase gene sequencing can be done.
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LIPASE MODULATION BY HORMONES Insulin LpL is regulated by insulin. The most common lipoprotein abnormality found in diabetic patients is an elevated level of triglyceride (Ginsberg, 1991). In part this is due to increased triglyceride synthesis due to release of free fatty acids from adipose tissue and their re-assembly into VLDL triglyceride. A second abnormality is a relative deficiency of LpL. LpL is normal or slightly reduced in untreated type 2 diabetes. In adipose tissue, LpL activity and actions are increased by insulin infusions into normal and diabetic humans. Similarly, insulin treatment increases postheparin LpL activity. Therefore control of diabetes improves LpL and reduces plasma triglyceride. Several groups have studied the effects of insulin on HL activity, but the results are not completely consistent. Insulin deficiency has been reported to experimentally increase or decrease activity. Data on regulation of HL in humans are also not totally consistent. Post-heparin plasma HL activity decreased when normal and diabetic patients were studied after a hyperinsulinaemic infusion. Whether these changes were directly attributable to the insulin or the concomitant alterations in plasma lipoproteins or other metabolites could not be determined. Intraperitoneal insulin increases HL and leads to reduced triglyceride apoB ratios in VLDL and IDL. Sex Steroids Regional LPL activity and deposition of fat seem to be influenced by sex steroids. LpL activity in the femoral region is higher in females than in males (Howard et al., 1987). Pre-menopausal females have higher femoral than abdominal LpL activity but after menopause, these regional differences in LpL activity disappear. Although short-term replacement of oestrogen and progesterone given to post-menopausal women does not change overall post-heparin LPL activity, when postmenopausal women without hormone replacement were compared to women who had undergone hormone replacement for three years, those on hormone replacement had a reappearance of increased LpL in the femoral regions (Rebuffè-Scrive et al., 1987), implying that sex steroids are causally related to the increase in femoral adipose tissue deposition and LpL activity. Hypogonadal males tend to have higher LpL activity in abdominal fat versus femoral fat; short-term testosterone replacement equalises the two activities by lowering abdominal LpL but does not change femoral LPL (Rebuffè-Scrive et al., 1987). It appears then that sex steroids can
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change LpL distribution and fat cell distribution. The mechanism whereby sex steroids accomplish this change in body fat distribution is a very important unanswered question. Oestrogen profoundly reduces post-heparin HL activity. This effect likely depends on the first-pass metabolism of oestrogen by the liver, as transdermal estradiol does not effect HL activity. Although oestrogen also increases HDL levels and the changes in HDL and HL are correlated, kinetic studies have shown that HDL levels are increased due to increased A1 production, not decreased catabolism (Brinton, 1996). Therefore the decrease in HL activity appears to have no obvious effect on HDL clearance, implying that other routes exist for HDL clearance besides HL activity. Androgens and anabolic steroids (which have androgenic activity but cannot be aromatised to oestrogen and are usually given orally so that they have a large first-pass effect on the liver) increase HL activity, by about 25% and 130%, respectively (Thompson et al., 1989). Interestingly, in a kindred of patients with HL deficiency and high HDL2 levels, anabolic steroids did not increase HL yet decreased HDL2 levels, implying that anabolic steroids can decrease HDL by mechanisms other than HL action (Bausserman et al., 1997). Growth Hormone Post-heparin LpL and HL activities are low in acromegalic subjects with growth hormone-secreting tumours (Tan et al., 1997). Conversely, growth hormone-deficient adults have high LpL and HL activities compared to controls. Administration of growth hormone to growth hormone-deficient subjects decreases LpL and variably increases or decreases HL (Oscarsson et al., 1996; Asayama et al., 1984). Thyroid Hormone Hypothyroid subjects have decreased HL and LpL activities, associated with increased LDL and moderately increased HDL and triglyceride levels. After therapy with T3, HL and LpL increase, LDL levels are normalised, and triglycerides and HDL decrease (Valdemarsson et al., 1982). When T3 is given to normal subjects, HL increases while LpL is not changed; similarly, hyperthryoid subjects have increased HL but normal LpL levels when compared to controls (Valdemarsson et al., 1984). T3-mediated increases in HL production may be via posttranslational mechanisms.
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CHRONIC HUMAN DISEASES Chronic diseases, especially those associated with major changes in body weight or composition, are associated with alterations in LpL activity. Patients with chronic cachexia, e.g., due to cancer or AIDS, have reduced post-heparin plasma LpL. In part this may be due to decreased adipose tissue LpL production secondary to tumour necrosis factor; actions on the adipocytes (Roubenoff, 1997). Patients with renal failure also have a reduction in LpL; this may in part relate to circulating renal toxins (Goldberg, 1992).
PHARMACOLOGIC APPROACHES TO ALTERING LIPOPROTEIN LIPASE One approach to reducing plasma triglyceride levels is to increase the activity of LpL. Fibric acid drugs have long been known to do this, perhaps because they stimulate the PPAR class of transcription factors. A new class of drugs, called NO-1886, was discovered in Japan (Tsutsumi et al., 1993). This agent stimulated LpL activity in postheparin plasma, adipose tissue, and hearts of experimental animals. Triglyceride levels were reduced and HDL increased and in at least one model atherosclerosis was reduced. Therefore, modulation of LpL appears to be a target that has the possibility of improving lipoprotein profiles and reducing vascular disease. References Asayama, K., Amemiya, S., Kusano, S. and Kato, K. (1984). Growthhormone-induced changes in postheparin plasma lipoprotien lipase and hepatic triglyceride lipase activities. Metabolism , 33 , 129–31. Bausserman, L.L., Saritelli, A.L. and Herbert, P.N. (1997). Effects of short-term stanozolol administration on serum lipoproteins in hepatic lipase deficiency. Metabolism , 46 , 992–996. Benlian, P., De Gennes, J.L., Foubert, L., Zhang, H., Gagne, S.E. and Hayden, M. (1996). Premature atherosclerosis in patients with familial chylomicronemia caused by mutations in the lipoprotein lipase gene. New England Journal of Medicine , 335 , 848–854. Berryman, D.E. and Bensadoun, A. (1995). Heparan sulfate proteoglycans are primarily responsible for the maintenance of enzyme activity, binding, and degradation of lipoprotein lipase in Chinese hamster ovary cells. Journal of Biological Chemistry , 270 , 24525–24531. Blades, B., Vega, G.L. and Grundy, S.M. (1993). Activities of lipoprotein lipase and hepatic triglyceride lipase in post-heparin
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plasma of patients with low concentrations of HDL cholesterol. Arteriosclerosis and Thrombosis , 13 , 1227–1235. Breckenridge, W.C., Little, J.A., Steiner, G., Chow, A. and Poapst, M. (1978). Hypertriglyceridemia associated with deficiency of apolipoprotein C-II. New England Journal of Medicine , 298 , 1265– 1273. Breckenridge, W., Little, J., Allaupovic, P., Wang, C.S., Kuksis, A., Kaksis, G. et al. (1982). Lipoprotein abnormalities associated with a familial deficiency of hepatic triglyceride lipase. Atherosclerosis , 45 , 161–179. Brinton, E.A. (1996). Oral estrogen replacement therapy in postmenopausal women selectively raises levels and production rates of lipoprotein A-I and lowers hepatic lipase activity without lowering the fractional catabolic rate. Arteriosclerosis, Thrombosis and Vascular Biology , 16 , 431–440. Brunzell, J.D., Hazzard, W.R., Porte, D.J. and Bierman, E.L. (1973). Evidence for a common, saturable triglyceride removal mechanism for chylomicrons and very low density lipoproteins in man. Journal of Clinical Investigation , 52 , 1578–1585. Chait, A. and Brunzell, J.D. (1992). Chylomicronemia syndrome. Advances in Internal Medicine , 37 , 249–273. Ginsberg, H.N., Le, N.A., Goldberg, I.J., Gibson, J.C., Rubinstein, A., Wang, I.P. et al. (1986). Apolipoprotein B metabolism in subjects with deficiency of apolipoproteins CIII and AI: evidence that apolipoprotein CIII inhibits catabolism of triglyceride-rich lipoproteins by lipoprotein lipase in vivo. Journal of Clinical Investigation , 78 , 1287–1295. Ginsberg, H.N. (1991). Lipoprotein physiology in nondiabetic and diabetic states: relationship to atherogenesis. Diabetes Care , 14 , 839–855. Goldberg, I.J. (1992). Lipoprotein metabolism in normal and uremic patients. American Journal of Kidney Disease , 21 , 87–90. Goldberg, I.J. (1996). Lipoprotein lipase and lipolysis: central roles in lipoprotein metabolism and atherosclerosis. Journal of Lipid Research , 37 , 693–707. Hahn, P.F. (1943). Abolishment of alimentary lipemia following injection of heparin. Science , 98 , 19–20. Harlan, W.R., Winesett, P.S. and Wasserman, A.J. (1967). Tissue lipoprotein lipase in normal individuals and in individuals with exogenous hypertriglyceridemia and the relationship of this enzyme to assimilation of fat . Journal of Clinical Investigation , 46 , 239– 247. Havel, R.J. and Gordon, R.S., Jr. (1960). Idiopathic hyperlipemia: metabolic studies in an affected family. Journal of Clinical Investigation , 39 , 1777–1790. Howard, B.V., Xiaoren, P., Harper, I, Kuusi, T. and Taskinen, M.R. (1987). Lack of sex differences in high density lipoproteins in Pima Indians: studies of obesity, lipase activities and steroid hormones. Arteriosclerosis , 7 , 292–300.
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Hultin, M., Savonen, R. and Olivecrona, T. (1996). Chylomicron metabolism in rats: lipolysis, re-circulation of triglyceride-derived fatty acids in plasma FFA, and fate of core lipids as analyzed by compartmental modelling. Journal of Lipid Research , 37 , 1022– 1036. Jensen, D.R., Tozzo, E., Eckel, R.H. and Kahn, B.B. (1996). Adiposespecific overexpression of GLUT-4 in transgenic mice alters lipoprotein lipase activity. American Journal of Physiology , 270 , R785–792. Kobayashi, J., Applebaum-Bowden, D., Dugi, K.A., Brown, D.R., Kashyap, V.S., Parrott, C. et al. (1996). Analysis of protein structure-function in vivo: adenovirus-mediated transfer of lipase lid mutants in hepatic lipase-deficient mice. Journal of Biological Chemistry , 271 , 26296–26301. Lalouel, J., Wilson D. and Iverius, P. (1992). Lipoprotein lipase and hepatic triglyceride lipase: molecular and genetic aspects. Current Opinion in Lipidology , 3 , 86–95. Ma, Y., Liu, M.S., Ginzinger, D., Frohlich, J., Brunzell, J.D. and Hayden, M.R. (1993). Gene-environment interaction in the conversion of a mild-to-severe phenotype in a patient homozygous for a Ser172 Cys mutation in the lipoprotein lipase gene. Journal of Clinical Investigation , 91 , 1953–1958. Nielsen, M.S., Brejning, J., Garcia, R., Zhang, H., Hayden, M.R., Vilaro, S. et al. (1997). Segments in the C-terminal folding domain of lipoprotein lipase important for binding to the low density lipoprotein receptor-related protein and to heparan sulfate proteoglycans. Journal of Biological Chemistry , 272 , 5821–5827. Nilsson-Ehle, P. and Schotz, M.C. (1976). A stable, radioactive substrate emulsion for assay of lipoprotein lipase. Journal of Lipid Research , 17 , 536–541. Oscarsson, J., Ottosson, M., Johansson, J.D., Wiklund, O., Marin, P., Bjorntorp, P. et al. (1996). Two weeks of daily injections and continuous infusion of recombinant growth hormone (GH) in GHdeficient adults. II. Effects on serum lipoproteins and lipoprotien and hepatic lipase activity . Metabolism , 45 , 370–377. Parthasarathy, N., Goldberg, I.J., Sivaram, P., Mulloy, B., Flory D.M. and Wagner, W.D. (1994). Oligosaccharide sequences of endothelial cell surface heparan sulfate proteoglycan with affinity for lipoprotein lipase. Journal of Biological Chemistry , 269 , 22391–22396. Pillarisetti, S., Paka, S., Sasaki, A., Vanni-Reyes, T., Yin, B., Parthasarathy, N. et al. (1997). Endothelial cell heparanase modulation of lipoprotein lipase activity: evidence that heparan sulfate oligosaccharide is an extracellular chaperone. Journal of Biological Chemistry , 272 , 15753–15759. Pruneta, V., Moulin, P., Labrousse, F., Bondon, P.J., Ponsin, G. and Berthezene, F. (1997). Characterization of a new case of autoimmune type I hyperlipidemia: long-term remission under immunosuppressive therapy. Journal of Clinical Endocrinology and Metabolism , 82 , 791–796.
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Ranganathan, G., Vu, D. and Kern, P.A. (1997). Translational regulation of lipoprotein lipase by epinephrine involves a transacting binding protein interacting with the 3′ untranslated region. Journal of Biological Chemistry , 272 , 2515–2519. Rebuffè-Scrive, M., Lonnroth, P., Marin, P., Wesslau, C., Bjorntorp, P. and Smith, U. (1987). Regional adipose tissue metabolism in men and postmenopausal women. International Journal of Obesity , 11 , 347–355. Roubenoff, R. (1997). Inflammatory and hormonal mediators of cachexia. Journal of Nutrition , 127, (5 Suppl): 1014S–1016S. Tan, K.C., Shiu, S.W., Janus, E.D. and Lam, K.S. (1997). LDL subfractions in acromegaly: relation to growth hormone and insulinlike growth factor-I. Atherosclerosis , 129 , 59–65. Thompson, P.D., Cullinane, E.M., Sady, S.P., Chenevert, C., Saritelli, A.L., Sady, M.A. et al. (1989). Contrasting effects of testosterone and stanozolol on serum lipoprotein levels. JAMA, 261 , 1165–1168. Tsutsumi, K., Inoue, Y., Shima, A., Iwasaki, K., Kawamura, M. and Murase, T. (1993). The novel compound NO-1886 increases lipoprotein lipase activity with resulting elevation of high density lipoprotein cholesterol, and long-term administration inhibits atherogenesis in the coronary arteries of rats with experimental atherosclerosis. Journal of Clinical Investigation , 92 , 411–417. Valdemarsson, S., Hedner, P. and Nilsson-Ehle, P. (1982). Reversal of decreased hepatic lipase and lipoprotien lipase activities after treatment of hypothyroidism. European Journal of Clinical Investigation , 12 , 423–428. Valdemarsson, S., Hedner, P. and Nilsson-Ehle, P. (1984). Treatment of hyperthyroidism: effects on hepatic lipase, lipoprotein lipase, LCAT and plasma lipoproteins Scandinavian Journal of Clinical and Laboratory Investigations , 44 , 183–189. Vilella, E., Joven, J., Fernandez, M., Vilaro, S., Brunzell, J.D., Olivecrona, T. et al. (1993). Lipoprotein lipase in human plasma is mainly inactive and associated with cholesterol-rich lipoproteins. Journal of Lipid Research , 34 , 1555–1564. Wallinder, L., Peterson, J., Olivecrona, T. and Bengtsson-Olivecrona, G. (1979). Rapid removal to the liver of intravenously injected lipoprotein lipase. Biochimica Biophysica Acta , 575 , 166–173. Weinstock, P.H., Levak-Frank, S., Hudgins, L.C., Radner, H., Friedmann, J.M., Zechner, R. et al. (1997). Lipoprotein lipase controls fatty acid entry into adipose tissue, but fat mass is preserved by endogenous synthesis in mice deficient in adipose tissue lipoprotein lipase. Journal of Clinical Investigation , 94 , 10261– 10266. Wong, H., Yang, D., Hill, J.S., Davis, R.C., Nikazy, J. and Schotz, M.C. (1997). A molecular biology-based approach to resolve the subunit orientation of lipoprotein lipase. Proceedings of the National Academy of Sciences USA , 94 , 5594–5598. Zambon, A., Torres, A., Bijvoet, S., Gagne, C., Moorjani, S., Lupien, P.J. et al. (1993). Prevention of raised low-density lipoprotein
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cholesterol in a patient with familial hypercholesterolaemia and lipoprotein lipase deficiency. Lancet , 341 , 1119–1121. Zechner, R. (1997). The tissue-specific expression of lipoprotein lipase: implications for energy and lipoprotein metabolism. Current Opinion in Lipidology , 8 , 77–88.
11 Relationship of the Cholesteryl Ester Transfer Protein (CETP) to Atherosclerosis Laurent Lagrost Laboratoire de Biochimie des Lipoprotéines—INSERM U498, Hôpital du Bocage—BP 1542, 21034 Dijon Cedex, France
Cholesteryl ester transfer protein (CETP) is an hydrophobic glycoprotein that facilitates the transfer of neutral lipids between lipoproteins, as well as between lipoproteins and cells. On the one hand, CETP appears as a proatherogenic factor that promotes the transfer of cholesteryl esters from the antiatherogenic high density lipoproteins (HDL) toward the potentially atherogenic apoB-containing lipoproteins. This leads to the formation of smallsized HDL and low density lipoprotein (LDL) subspecies that are known to constitute reliable markers of the atherosclerotic risk. On the other hand, CETP exhibits some antiatherogenic properties, in particular through its ability to generate pre -HDL that are highly efficient in promoting both the cellular cholesterol efflux and the esterification of cell-derived cholesterol in reverse cholesterol transport. In fact, CETP can be regarded as a janusfaced protein, with pro- or antiatherogenic properties depending on the metabolic context, as well as on plasma CETP levels. Ideally, plasma CETP levels should be high enough to generate optimal lipoproteins for reverse cholesterol transport, but it should not exceed an optimal value, approximating 2 mg/1, in order to restrain cholesterol load of atherogenic lipoproteins. Since hypercholesterolaemia is known to be associated with an abnormal rise in CETP production, CETP inhibition might be of potential relevance in atherosclerosis prevention in dyslipidaemic patients
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with high CETP levels, an hypothesis that deserves further attention. KEYWORDS: cholesteryl ester transfer protein; high density lipoproteins; low density lipoproteins; atherosclerosis.
INTRODUCTION In contrast to surface lipoprotein components that can spontaneously skip from one lipoprotein particle to another, the highly hydrophobic core neutral lipids, i.e., cholesteryl esters and triglycerides, cannot exchange through a simple aqueous diffusion process. Nevertheless, the transfer of neutral lipids between lipoprotein particles does occur in plasma of many species, and this is due to the activity of the plasma cholesteryl ester transfer protein (CETP). Human CETP is now well identified and characterised, and studies of the last twenty years demonstrated that it can markedly influence the quantity and quality of plasma lipoproteins. However, the consequences of the CETP-mediated lipid transfer reactions on the atherosclerotic process paradoxically remain a matter of controversy, probably reflecting the complexity of CETP activity in vivo. After a brief overview of CETP structure and function, that have been extensively reviewed in recent articles (Barter and Rye, 1994; Lagrost, 1994 and 1997; Melchior and Marotti, 1995; Tall, 1995; Yamashita et al., 1997), this chapter will mainly focus on the current state of knowledge concerning the role of CETP in lipoprotein metabolism and the consequences of CETP activity in terms of atherogenesis.
MOLECULAR STRUCTURE OF CETP The human CETP gene contains 16 exons. It exists as a single copy per haploid genome and is localised in the 16q12–16q21 region of chromosome 16 (Lusis et al., 1987; Agellon et al., 1990). Human CETP is an hydrophobic, acidic glycoprotein that is synthesised with a prepeptide of 17 aminoacid residues (Drayna et al., 1987). The mature protein found in human plasma contains 476 aminoacid residues; it has 95% homology with cynomolgus monkey CETP (Pape et al., 1991) and 80% homology with rabbit (Nagashima et al., 1988) and hamster (Jiang et al., 1991) CETP. Whereas the liver is the main source of CETP in monkey and rabbit, a widespread tissue distribution has been reported in humans, with substantial levels of CETP mRNA being detectable in the spleen, liver, small intestine, adrenal glands and adipose tissue (Drayna et al., 1987; Quinet et al., 1993). Sequence analysis reveals
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that CETP is a member of the lipid transfer/lipopolysaccharide binding protein (LT/LBP) family that contains three other related proteins: the phospholipid transfer protein (PLTP) (Day et al., 1994), the lipopolysaccharide binding protein (LBP) (Schuman et al., 1990), and the bactericidal permeability increasing protein (BPI) (Gray et al., 1989).
MECHANISM OF ACTION AND FUNCTION OF CETP CETP was first identified as a neutral lipid transfer protein that is mainly involved in the net mass transfer of cholesteryl esters from high density lipoproteins (HDL) to very low density lipoproteins (VLDL) as well as in the reciprocal net mass transfer of triglycerides from VLDL to HDL. In fact, all plasma lipoproteins can serve as substrates in CETP-mediated lipid transfer reaction. The direction of net lipid flux is determined mainly by the size and the disequilibrium state of cholesteryl ester and triglyceride pools between donor and acceptor lipoproteins (Lagrost, 1994). The binding of CETP to the lipoprotein surface is the initial event of the neutral lipid transfer reaction (For a review, Lagrost, 1994). This binding might involve the electrostatic interaction of two positively-charged basic aminoacid residues of CETP, i.e., Lys233 and Arg259, with negatively-charged groups localised at the lipoprotein surface (Jiang et al., 1995). As a result, cholesteryl ester transfer activity of CETP is markedly influenced by the overall electrostatic charge of lipoproteins (Nishida et al., 1993; Masson et al., 1996). A similar effect of lipoprotein electronegativity on the phospholipid transfer activity of PLTP has recently been reported (Desrumaux et al., 1998). In addition to the lipoproteinbinding sites, CETP contains a highly hydrophobic domain in the Cterminal region of the protein that carries its functional specificity, i.e., the binding and transfer of hydrophobic core neutral lipids in the second step of the CETP-mediated lipid transfer reaction (Albers et al., 1996). Whereas the C-terminal domain of CETP differs significantly from those of other members of the LT/LBP family, the N-terminal regions of CETP, PLTP, LBP, and BPI show significant homologies (Albers et al., 1996). Over the last decade, it has become more and more evident that CETP activity cannot be restricted to the simple molecular exchange of cholesteryl esters and triglycerides between lipoprotein substrates and that only an integrated analysis of the metabolic properties of CETP can accurately reflect its implication in lipoprotein metabolism in vivo. From a metabolic point of view, CETP provides a bridge between plasma apoB-containing lipoproteins and apoAI-containing lipoproteins through which they can exchange neutral lipid species.
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From a biochemical point of view, the CETP-mediated lipid transfer reaction in plasma leads to the replacement of the nonhydrolysable cholesteryl esters in the core of cholesteryl ester-rich lipoproteins, i.e., HDL and low density lipoproteins (LDL), by triglycerides that are mainly derived from VLDL. Subsequently, hepatic lipase (HL) acts on triglycerideenriched lipoproteins producing core lipid-depleted, smallsized LDL and HDL particles (Lagrost, 1997). In the case of HDL, HL leads to the formation of unstable intermediates with redundant surface components that can secondarily split up into pre- HDL and small HDL particles (for a review, Lagrost, 1997). It is noteworthy that CETP can also promote the size redistribution of HDL through a selfsufficient process involving the CETP-mediated fusion of HDL particles in the absence of apoB-containing lipoproteins (Rye et al., 1997). In fact, CETP activity is not restricted to the lipoprotein intravascular remodelling, with evidence in support of for role of CETP in the movement of cholesterol between lipoproteins and cells. In earlier studies, Stein et al. (1995; 1996) demonstrated that CETP may be involved in the efflux of cholesteryl ester from the vascular interstitium, providing a putative mechanism for the removal of cholesterol excess. In turn, CETP has been reported to facilitate the transfer of cholesteryl esters from HDL into several cell types, including HepG2 cells, smooth muscle cells, fibroblasts and adipocytes (Granot et al., 1987; Benoist et al., 1997). However, the direct role of CETP in cholesteryl ester movements between lipoproteins and cells remains puzzling; Rinninger and Pittman (1989) reported that the CETP-facilitated uptake of HDL cholesteryl esters by cultured HepG2 cells might indirectly involve the transfer of cholesteryl esters from HDL into more buoyant particles that are secreted by the cells and which are subsequently taken up by the apoB/E receptor pathway. Not only the cholesteryl ester movements, but also the efflux and esterification of free cholesterol in the first steps of the reverse cholesterol transport pathway can be enhanced by CETP (Atger et al., 1995; Francone et al., 1996). The stimulation by plasma CETP of both the efflux and the esterification of cell-derived cholesterol have been interpreted as direct consequences of the generation of more efficient lipoprotein particles, i.e., nascent pre- HDL, during the CETPmediated HDL remodelling (Atger et al., 1995; Francone et al., 1996). Interestingly, CETP is not only present in plasma but has also been identified as a membrane-bound protein that might locally exert a direct physiological role in the transfer of unesterified cholesterol between lipoproteins and cells (Ruiz-Noriega et al., 1994; Radeau et al., 1995).
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CETP AND ATHEROSCLEROSIS Predictions From in Vitro Studies Although functional studies with isolated lipoproteins and cultured cells do not provide direct insights into the pro- or antiatherogenic properties of CETP, the integration of various experimental data may help to predict the pathophysiological consequences of alterations in CETP activity in vivo. A dichotomous classification of “potentially beneficial” and “potentially deleterious” effects of CETP in terms of atherogenicity is proposed in Table 11.1. Epidemiological studies have established that the incidence of atherosclerosis correlates positively with the cholesterol content of the plasma apoB-containing lipoprotein fraction but negatively with the cholesterol content of the apoAIcontaining lipoprotein fraction. In this sense CETP activity may be regarded as a proatherogenic factor supplying the atherogenic lipoproteins with cholesteryl esters at the expense of the antiatherogenic HDL. In addition, CETP activity, in combination with HL activity, favours the emergence of small HDL and LDL subspecies that have been shown to be reliable markers of atherosclerotic risk (Austin and Krauss, 1986; Cheung et al., 1991; Johansson et al., 1991). Moreover, the facilitated uptake of cholesteryl esters by adipocytes (Benoist et al., 1997) and smooth muscle cells (Granot et al., 1987) might constitute an additional unfavourable effect of CETP, potentially leading to the accumulation of cholesterol in peripheral tissues. Conversely, the CETP-mediated movements of cholesteryl esters between lipoproteins and cells represent one step in the pathway of reverse cholesterol transport, i.e., the metabolic pathway leading to the transport of cholesterol from peripheral tissues back to the liver. Indeed, CETP has been reported to facilitate both the efflux of cholesteryl esters from the vascular interstitium (Stein et al., 1985, 1986) and the uptake of cholesteryl esters by liver cells (Granot et al., 1987). The positive role of CETP in reverse cholesterol transport is further supported by its ability to produce pre- HDL that are known to constitute the preferential acceptors of cellular cholesterol (Castro and Fielding, 1988). Finally, recent studies by Christison et al. (1995) suggested that CETP, by transferring potentially atherogenic cholesteryl ester hydroperoxides from lesion LDL to the plasma HDL might facilitate their hepatic detoxification. Taken together, results from in vitro studies do not lead to a definitive conclusion regarding the pathophysiological role of CETP activity. There is a clear need for further evaluation of the relevance of CETP to the development of atherosclerosis in humans and in animal models.
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Table 11.1 Metabolic properties of cholesteryl ester transfer protein
Potentially deleterious effects
Potentially beneficial effects
– increase in cholesterol content of apoBcontaining lipoproteins
– formation of nascent, pre HDL
– decrease in cholesterol content of high density lipoproteins
– facilitation of cholesteryl ester uptake by liver cells
– formation of small-sized
HDL and LDL – efflux of cholesteryl esters from the vascular interstitium
– facilitation of cholesteryl ester uptake in peripheral cells (smooth muscle cells, adipocytes,…)
– detoxification of oxidized cholesteryl esters
Corresponding references are quoted in the main text.
Animal Studies Plasma CETP and diet-induced atherosclerosis A significant correlation between coronary artery intimal plaque development and plasma CETP levels was reported in cynomolgus monkeys fed an atherogenic diet (Quinet et al., 1991). However, the role of CETP in promoting coronary atherosclerosis in cholesterol-fed animals could be indirect, simply reflecting a positive correlation of CETP with LDL cholesterol levels (Quinet et al., 1991). Indeed, only LDL cholesterol level, but not plasma CETP levels, appeared as an independent, significant predictor of monkey coronary atherosclerosis in a multiple regression analysis (Quinet et al., 1991). Interspecies comparisons In earlier studies, Ha and Barter (1982) reported a wide variability of CETP activity levels among various vertebrate species. They proposed a three-group subdivision, distinguishing between species with either low, intermediate, or high CETP activity. Interestingly, species with low CETP activity, among them rat and mouse, are resistant to atherosclerosis, while species with substantial CETP activity levels, among them rabbit and to a lesser extent man, are more susceptible to atherosclerotic lesion development. Thus, when analysing plasma CETP activity levels in the light of the atherosclerosis susceptibility of vertebrate species, CETP arises as one putative participant in the atherosclerosis process in vivo. Overall, it is noteworthy that the
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lipoprotein metabolism of various vertebrate species differs in many other respects, and conclusions drawn from interspecies comparisons are rather hazardous. Transient inhibition of CETP activity in vivo In a first attempt to explore the effect of CETP inhibition on lipoprotein metabolism in vivo, rabbits were injected with anti-CETP antibodies (Abbey and Calvert, 1989). When compared with control animals, antiCETP-injected rabbits with at least 50% inhibition of plasma cholesteryl ester transfer activity displayed significant increases in the size and the cholesteryl ester content of HDL (Abbey and Calvert, 1989). More recently, an alternative strategy, based on the intravenous injection of antisense oligodeoxynucleotides against CETP, was used to block endogenous CETP expression in rabbits (Sugano et al., 1996). When targeted to the liver by using asialoglycoprotein carrier molecules, antisense oligodeoxynucleotides produced a substantial inhibition of plasma CETP activity in cholesterol-fed rabbit and this was accompanied by a decrease in the plasma VLDL/LDL cholesterol levels and a concomitant increase in the plasma HDL cholesterol levels (Sugano and Makino, 1996). Most interestingly, injection of cholesterol-fed rabbits with antisense oligodeoxynucleotides resulted in less atherogenicity, indicating that CETP inhibition in high CETP/hypercholesterolaemic states constitutes a promising approach in atherosclerosis prevention (Sugano et al., 1998). Expression of functional CETP in CETP-deficient animals In earlier studies (Ha et al., 1985; Quig and Zilversmit 1986; Gavish et al., 1987; Groener et al., 1989), human CETP-containing fractions were injected intravenously in the rat, an animal that normally lacks plasma CETP activity. Overall, the CETP-mediated neutral lipid transfers were associated with significant decreases in the size and the cholesteryl ester content of HDL, with significant increases in the cholesteryl ester content of VLDL and LDL and in the triglyceride content of HDL, as well as with a significant reduction in the residence time of HDL-associated cholesteryl esters. Again, while these observations came in support of an important role of CETP in determining the metabolic behaviour of plasma lipids, they did not bring indications in term of atherogenesis, due to the short expression periods studied. The production of transgenic mice expressing either human (Agellon et al., 1991) or monkey (Marotti et al., 1992) CETP genes provided a unique approach to investigate the long term effect of CETP on both the lipoprotein metabolism and the atherosclerosis development. In
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agreement with data obtained in CETP-injected rats (Ha et al., 1985; Quig and Zilversmit, 1986; Gavish et al., 1987; Groener et al., 1989), expression of active CETP in transgenic mice is mainly characterised by significant drops in the size and the cholesterol content of HDL (Agellon et al., 1991; Marotti et al., 1992). Moreover, detailed studies in CETP-transgenic mice revealed that CETP expression is associated with a number of other metabolic alterations (see Table 11.2). Not only HDL cholesterol, but also apoAI levels are markedly reduced in CETPtransgenic mice, as a result of an increased fractional catabolic rate of apoAI (Hayek et al., 1993; Melchior et al., 1994). The latter point might reflect the
Table 11.2 Alterations in lipoprotein metabolism in CETP-transgenic mice HDL – decrease in HDL cholesterol and apoAI levels – increase in HDL cholesteryl ester and apoAI fractional catabolic rates – reduction in HDL size – increase in HDL triglyceride content – increase in pre -HDL-mediated cellular cholesterol efflux – increase in LCAT-mediated cholesterol esterification in HDL VLDL/LDL – increase in VLDL/LDL cholesterol content – increased -VLDL levels in transgenic mice expressing a human receptor binding-deficient apoE variant. – decrease in hepatic LDL receptor, HMG-CoA reductase, and 7 hydroxylase mRNA levels.
-
Corresponding references are quoted in the main text.
predominance of small HDL with high turnover that have been demonstrated to constitute highly efficient acceptors of cellular cholesterol in CETP transgenic mice (Atger et al., 1995; Francone et al., 1996). Expression of CETP in mice has also been associated with a significant decrease in the abundance of mRNAs encoding hepatic LDL receptor, 3-hydroxy-3-methylglutaryl-coenzyme A (HMGCoA) reductase and 7 -hydroxylase in mice, probably reflecting the cellular response to cholesterol accumulation in the liver of these mice (Jiang et al., 1993). The development of fatty liver is accelerated in CETP transgenic mice; this might be of pathological relevance in the process of hepatic steatosis (Blake et al., 1994). Accumulation of the highly
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atherogenic, cholesteryl ester-rich -VLDL has been observed in plasma from hyperlipidemic transgenic mice expressing simian CETP in addition to an apoE variant that is defective for receptor binding (Fazio et al., 1994). Overall, the lipoprotein phenotype associated with CETP expression in mouse (decreases in the size and cholesterol content of the HDL fraction and increases in the cholesterol content of proatherogenic apoB-containing lipoproteins) is known to be associated with heart disease susceptibility. Predictably, a significant increase in the severity and progression of atherosclerotic lesions was observed in CETP-transgenic mice fed a high fat/ high cholesterol diet (Marotti et al., 1993; Hayek et al., 1995), indicating that CETP-mediated alterations in plasma cholesterol distribution are potentially atherogenic. However, the proatherogenic effect of CETP has been limited to non-hypertrigly-ceridaemic CETP transgenic mice. Surprisingly, decreased early atherosclerotic lesions were observed in hypertriglyceridaemic mice expressing both human apo CIII and CETP transgenes; this was in spite of a marked reduction in HDL cholesterol levels in these animals (Hayek et al., 1995). These observations indicate that the role of CETP in atherogenesis may be influenced by its effects on the dynamics of HDL transport rather than on the level of circulating HDL. It is noteworthy that the coexpression of human apo CIII and CETP in transgenic mice is associated with an even more dramatic reduction in HDL size when compared with CETP-transgenic mice (Hayek et al., 1995). Since small HDL particles exhibit an enhanced ability to promote cholesterol efflux from the arterial wall, the CETP-mediated production of large amounts of optimal acceptors of cellular cholesterol in hypertriglyceridaemic apoCIII/CETPtransgenic mice might well have counteracted the deleterious effect of the CETP-mediated transfer of cholesteryl esters from HDL towards the potentially atherogenic apoB-containing fractions. Finally, it remains possible that the expression of the apolipoprotein transgene might have produced some uncontrolled metabolic variations in CETPtransgenic mice. Indeed, recent studies have demonstrated an unexpected disappearance of plasma lipid transfer inhibitory activity in transgenic mice overexpressing human apo AI (Masson et al., 1997). Human Studies Familial CETP deficiency The first inherited CETP deficiency found in the Japanese population was shown to relate to a G to A substitution at intron 14 of the CETP gene, resulting in abnormal splicing of CETP pre-messenger RNA (Brown et al., 1989). To date, eight additional mutations in the human CETP gene have been reported (for a review, Yamashita et al., 1997).
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All of them suppress the expression of normal, active CETP in plasma. Homozygotes exhibit no plasma cholesteryl ester transfer activity, while heterozygotes have plasma CETP activity levels that range between fifty and a few percents of normal, depending on the mutation site. Hyperalphalipoproteinaemia was first identified as the main characteristic of CETP-deficient subjects; it has been suggested that CETP gene defects might constitute one of the major cause of marked hyperalphalipoproteinaemia in the Japanese population (Hirano et al., 1993; Inazu et al., 1994). Several additional lipoprotein alterations that are associated with CETP deficiency are summarised in Table 11.3. Complete CETP deficiency is characterised by the emergence of large, apoE-containing HDL (Koizumi et al., 1985; Yamashita et al., 1988; Chiba et al., 1997) while partial CETP deficiency is only associated with a significant increase in the plasma HDL2 to HDL3 ratio as compared with unaffected subjects (Inazu et al., 1990). In fact, large, apoE-rich HDL do not appear in plasma of hyperalphalipoproteinemic subjects who have detectable plasma CETP activity, suggesting that they might constitute a better substrate for CETP than apoE-poor HDL (Chiba et al., 1997). Not only the structure, but also the kinetics of plasma HDL have been reported to be altered in CETP deficiency. Ikewaki et al., (1993) demonstrated that apoAI and apoAII catabolism is markedly delayed in CETP-deficient homozygotes, while the production rates were quite normal. Although the resulting elevation of plasma HDL and apoAI levels may prevent atherosclerosis development by detoxifying oxidised lipids (Parthasarathy et al., 1990; Bowry et al., 1992), the accumulation of plasma HDL due to an increased residence time might actually reflect a blockade rather than a facilitation of reverse cholesterol transport. In addition, both cellular cholesterol efflux and cholesterol esterification were reported to be impaired in HDL from CETP-deficient patients (Ohta et al., 1995). The presence of triglyceride-rich, polydisperse LDL constitute another characteristic of complete CETP deficiency in humans, indicating that CETP plays an important role in LDL maturation in vivo (Sakai et al., 1991, 1995; Bisgaier et al., 1991). Triglyceride-rich LDL from CETPdeficient patients were functionally abnormal; as expected from previous in vitro studies (Aviram et al., 1988; McKeone et al., 1993) they exhibited a decreased affinity for the LDL receptors in normal human fibroblasts (Sakai et al., 1995). Nevertheless, LDL levels were quite low in CETP-deficient patients compared with controls. This was shown to relate both to an increased fractional catabolic rate of LDL apo B and a decreased production rate of VLDL and IDL apo B (Ikewaki et al., 1995). Since CETP expression in transgenic mice was reported to reduce hepatic LDL receptor mRNA (Jiang et al., 1993), it would be predicted that LDL receptor expression would be upregulated in CETP deficiency.
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Table 11.3 Alterations in lipoprotein metabolism in CETP-deficient subjects HDL – increase in HDL cholesterol and apoAI levels – increase in large, apoE-rich HDL (homozygotes) – increase in HDL2/HDL3 ratio – increase in LpAI and LpAIAII levels – delayed catabolism of apoAI and apoAII – reduction in HDL-mediated cholesterol efflux from macrophage foam cells – impaired LCAT-mediated cholesterol esterification in HDL LDL – decrease in LDL cholesterol and apoB levels – abnormal polydisperse, triglyceride-rich LDL – decreased affinity of LDL particles for LDL receptors – increased catabolic rate of LDL Corresponding references are quoted in the main text.
With regard to the relationship between CETP and atherogenesis, no clear conclusions can be drawn from studies in CETP-deficient patients, with arguments in favour of both antiatherogenic and proatherogenic consequences of the CETP deficiency. An earlier, anecdotal study of a few CETP deficient subjects with no coronary artery disease and with a trend toward longevity, suggested that CETP deficiency might be associated with decreased atherosclerosis and increased life span (Inazu et al., 1990; Brown et al., 1989). However, the putative beneficial effect of CETP deficiency has been challenged by more recent studies. Indeed, Sakai et al. (1995) reported a CETPdeficient homozygote with corneal arcus and coronary artery disease in spite of marked hyperalphalipoproteinaemia. Hirano et al. (1995) also reported several CETP-deficient heterozygotes with atherosclerotic cardiovascular disease. A significantly higher prevalence of coronary heart disease was observed among aged CETP-deficient JapaneseAmerican men from Honolulu (Zhong et al., 1996), as well as among CETP-deficient Japanese patients from the Omagari City area (Hirano et al., 1997). Interestingly, in the Honolulu heart program (Zhong et aL, 1996), the effect of partial CETP deficiency was actually dependent on the plasma HDL cholesterol levels; CETP-deficient men with HDL
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cholesterol greater than 60 mg/dl showed a low prevalence of coronary heart disease (Zhong et al., 1996). In the Omagari study (Hirano et al., 1997), the relationship of HDL cholesterol level to atherosclerosis was more complex, with hyperalphalipoproteinemic CETP-deficient subjects still showing an increased incidence of ischaemic changes in electrocardiograms. It is noteworthy that, in more detailed studies (Sakai et al., 1995), CETP-deficient individuals with established atherosclerotic disease were shown to exhibit concomitant reduction in hepatic lipase activity. The latter point is of considerable interest, as no evidence of coronary artery disease was reported among a subgroup of hyperalphalipoproteinemic CETP-deficient heterozygotes with normal HL activity (Hirano et al., 1995). Since it is now well accepted that hepatic lipase deficiency by itself is a strong, independent risk factor for coronary artery disease (Busch et al., 1994), it would represent a confounding variable in a study of the relationship between CETP and atherogenesis. Therefore, CETP deficiency per se might not represent a risk factor in atherogenesis unless it is associated with low HL activity. The relationship of CETP to atherogenesis should be examined in the light of its effect on the structural and functional properties of plasma HDL. Recent studies in the Omagari City population, where CETP deficiency is extremely frequent, showed a striking, and significant Ushaped relationship between HDL cholesterol levels and the cardiovascular risk (Hirano et al., 1997). Variations of plasma HDL cholesterol levels on both sides of the 70 mg/dl value were associated with a greater incidence of ischaemic changes in electrocardiograms (Hirano et al., 1997). Given that CETP is one of the main determinants of plasma HDL cholesterol levels, appropriate CETP levels might then contribute to an optimal lipoprotein metabolism and to a low atherosclerotic risk. In an attempt to determine the effect of low plasma CETP activity on the cardiovascular risk in hypercholesterolaemia, Haraki et al. (1997) recently searched for alterations in cardiovascular risk in CETP-deficient heterozygotes with familial hypercholesterolaemia (FH), in whom a high LDL cholesterol is usually associated with a low level of HDL cholesterol. In spite of a significant increase in the levels of plasma HDL cholesterol, partial CETP deficiency did not prevent coronary heart disease in FH patients. However, it must be noted that LDL cholesterol levels were not significantly lower in CETP-deficient FH patients than in FH patients without deficiency. In addition, CETP mass, that was only moderately lowered in FH patients with partial CETP deficiency, was still quite high in this population due to the expected rise in CETP production rate in response to high plasma cholesterol levels (McPherson et al., 1997). Thus, future studies of FH patients with complete, rather than partial CETP deficiency will bring more insight into the putative protective effect of the suppression of plasma CETP activity in
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hypercholesterolaemic patients. Clinical observations Variations of plasma CETP have been investigated under a number of pathological conditions that are known to be associated with dyslipidaemia and cardiovascular disease (for a review, Lagrost, 1994). However, conflicting observations have been made in different laboratories, probably because of the variety of methods used. These have included i) net mass transfer assays of cholesteryl esters and triglycerides, ii) isotopic transfer assays of labelled cholesteryl esters and iii) CETP mass immunoassays (Lagrost, 1994). In terms of atherosclerosis susceptibility, the transfer of HDL cholesteryl esters to apoB-containing lipoproteins is markedly increased in patients with peripheral vascular disease (Rühling et al., 1989) and coronary artery disease (Bhatnagar et al., 1993) as compared to healthy controls with normal and comparable lipid levels, suggesting that CETP might constitute a risk factor in atherogenesis, independent of total plasma lipid levels. In addition, in an homogeneous population of healthy subjects with no conventional risk factors, CETP mass concentration was shown to directly relate to the carotid wall thickness (Föger et al., 1995). Conversely, the development of angina pectoris in normolipidaemic elderly patients has been found to be associated with significant decreases in plasma CETP activity (Miyashita et al., 1992). Recent studies by Kuivenhoven et al., (1998) brought new insights into the complex relationship of CETP to atherosclerosis. In a cohort of 807 men with angiographically documented coronary atherosclerosis, the pravastatin-mediated decrease in plasma CETP concentration was associated with a significant retardation of atherosclerosis progression in patients with B1B1 CETP gene polymorphism who originally displayed high CETP mass levels and high coronary atherosclerosis progression. In contrast, a decrease in plasma CETP concentration was associated with no changes or even a non-significant tendency toward an increased atherosclerosis progression in pravastatin-treated patients with B2B2 CETP gene polymorphism who originally displayed low CETP mass levels and slower coronary atherosclerosis progression when compared with B1B1 carriers (Kuivenhoven et al., 1998). These observations were explained in terms of a critical CETP concentration that would be required for both efficient reverse cholesterol transport and low atherosclerosis susceptibility. A reduction of CETP mass level would be beneficial only in patients with a high plasma CETP mass concentration, but not in patients with CETP mass concentrations in the low range (Kuivenhoven et al., 1998).
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CONCLUSION Although the metabolic action of CETP at the interface of the apoBcontaining lipoprotein and apoAI-containing lipoprotein pathways suggests that it should constitute a key determinant of atherosclerosis development in vivo, there has been no consensus over the last decade. CETP has been alternatively described as a pro- or an antiatherogenic factor. In fact, the more recent studies indicate that the apparently opposite effects of CETP on atherosclerosis may simply reflect the activity of CETP in different metabolic contexts. Currently, CETP appears as a janus-faced protein, with its relationship to atherosclerosis depending on whether its predominant effect is to increase the cholesterol content of proatherogenic particles or to facilitate the antiatherogenic, HDL-mediated reverse cholesterol transport. Ideally, plasma CETP level should be high enough to be able to generate optimal substrates for reverse cholesterol transport, but it should not exceed an optimal value, approximating 2mg/l, in order to limit the cholesterol load in atherogenic lipoproteins. It must be borne in mind that plasma cholesterol levels, in particular plasma LDL cholesterol levels, are known to increase CETP gene expression and plasma CETP levels. Thus, hypercholesterolaemic patients with high atherosclerotic risk are likely to have abnormally elevated CETP concentrations. In this case it could be postulated that a reduction of plasma CETP levels would potentially slow atherosclerosis progression. References Abbey, M., and Calvert, G.D. (1989). Effects of blocking plasma lipid transfer protein activity in the rabbit. Biochim. Biophys. Acta ., 1003 , 20–29. Agellon, L.B, Quinet, E.M., Gillette, T.G., Drayna, D.T., Brown, M.L., and Tall, A.R. (1990). Organization of the human cholesteryl ester transfer protein gene. Biochemistry , 29 , 1372–1373. Agellon, L.B., Walsh, A.M., Hayek, T., Moulin, P., Jiang, C.X., Shelanski, S.A., et al. (1991). Reduced high density lipoprotein cholesterol in human cholesteryl ester transfer protein transgenic mice. J.Biol. Chem ., 266 , 10796–10801. Albers, J.J., Tu, A.Y., Wolfbauer, G., Cheung, M.C. and Marcovina, S.M. (1996). Molecular biology of phospholipid transfer protein. Curr. Opin. Lipidol ., 7 , 88–93. Atger, V., de la Llera Moya, M., Bamberger, M., Francone, O., Cosgrove, P., Tall, A. et al. (1995). Cholesterol efflux potential of sera from mice expressing human cholesteryl ester transfer protein and/or human apolipoprotein AI. J.Clin. Invest ., 96 , 2613–2622. Austin, M.A. and Krauss, R.M. (1986). Genetic control of low density
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monkey plasma lipoproteins. J.Clin. Invest ., 87 , 1559–1566. Quinet, E.M., Huerta, P., Nancoo, D., Tall, A.R., Marcel, Y.L. and McPherson, R. (1993). Adipose tissue cholesteryl ester transfer protein mRNA in response to probucol treatment: cholesterol and species dependence. J.Lipid Res ., 34 , 845–852. Radeau, T., Lau, P., Robb, M., McDonnell, M., Aihauld, G. and McPherson, R. (1995). Cholesteryl ester transfer protein (CETP) mRNA abundance in human adipose tissue: relationship to cell size and membrane cholesterol content. J.Lipid Res ., 36 , 2552–2561. Rinninger, F. and Pittman, R.C. (1989). Mechanism of the cholesteryl ester transfer protein-mediated uptake of high density lipoprotein cholesteryl esters by HepG2 cells. J.Biol. Chem ., 264 , 6111–6118. Rühling, K., Zabel-Langhennig, R., Till, U. and Thielmann, K. (1989). Enhanced net mass transfer of HDL cholesteryl esters to apoBcontaining lipoproteins in patients with peripheral vascular disease. Clin. Chim. Acta , 184 , 289–296. Ruiz-Noriega, M., Silva-Cardenas, I., Delgado-Coello, B., ZentellaDehesa, A. and Mas-Oliva, J. (1994). Membrane-bound CETP mediates the transfer of free cholesterol between lipoproteins and membranes. Biochem. Biophys. Res. Commun ., 202 , 1322–1328. Rye, K.A., Hime, N.J. and Barter, P.J. (1997). Evidence that cholesteryl ester transfer protein-mediated reductions in reconstitued high density lipoprotein size involve particle fusion. J.Biol. Chem ., 272 , 3953–3960. Sakai, N., Matsuzawa, Y., Hirano, K.I., Yamashita, S., Nozaki, S., Ueyama, Y. et al. (1991). Detection of two species of low density lipoprotein particles in cholesteryl ester transfer protein deficiency. Arterioscler. Thromb ., 11 , 71–79. Sakai, N., Yamashita, S., Hirano, K.I., Ishigami, M., Arai, T., Kobayashi, K. et al. (1995). Decreased affinity of low density lipoprotein (LDL) particles for LDL receptors in patients with cholesteryl ester transfer protein deficiency. Eur. J.Clin. Invest ., 25 , 332–339. Schuman, R.R., Leong, S.R., Flaggs, G.W., Gray, P.W., Wright, S.D., Mathison, J.C. et al. (1990). Structure and function of lipopolysaccharide binding protein. Science , 264 , 1429–1431. Stein, Y., Stein, O., Olivecrona, T. and Halperin, G. (1985). Putative role of cholesteryl ester transfer protein in removal of cholesteryl ester from vascular interstitium, studied in a model system in cell culture. Biochim. Biophys. Acta , 834 , 336–345. Stein, Y., Halperin, G. and Stein, Y. (1986). Cholesteryl ester efflux from extracellular and cellular elements of the arterial wall. Model systems in culture with cholesteryl linoleyl ether. Arteriosclerosis , 6 , 70–78. Sugano, M. and Makino, N. (1996). Changes in plasma lipoprotein cholesterol levels by antisense oligodeoxynucleotides against cholesteryl ester transfer protein in cholesterol-fed rabbits. J.Biol. Chem ., 271 , 19080–19083. Sugano, M., Makino, N., Sawada, S., Otsuka, S., Watanabe, M.,
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Okamoto, H. et al. (1998). Effect of antisense oligonucleotides against cholesteryl ester transfer protein on the development of atherosclerosis in cholesterol-fed rabbits. J.Biol. Chem ., 273 , 5033– 5036. Tall, A.R. (1995). Plasma lipid transfer proteins. Annu. Rev. Biochem ., 64 , 235–257. Yamashita, S., Matsuzawa, Y., Okazaki, M., Kako, H., Yasuki, T., Akioka, H. et al. (1988). Small polydiperse low density lipoproteins in familial hyperalphalipoproteinemia with complete deficiency of cholesteryl ester transfer activity. Atherosclerosis , 70 , 7–12. Yamashita, S., Sakai, N., Hirano, K.I., Arai, T., Ishigami, M., Maruyama, Y. et al. (1997). Molecular genetics of plasma cholesteryl ester transfer protein. Curr. Opin. Lipidol , 8 , 101–110. Zhong, S., Sharp, D.S., Grove, J.S., Bruce, C, Yano, K., Curb, J.D. et al. (1996). Increased coronary heart disease in Japanese-American men with mutation in the cholesteryl ester transfer protein gene despite increased HDL levels. J.Clin. Invest ., 97 , 2917–2923.
12 Familial Lecithin: Cholesterol Acyltransferase Deficiency Syndromes P.Haydn Pritchard, Amir F.Ayyobi and John S.Hill Healthy Heart Program, St. Paul’s Hospital, 1081 Burrard Street, Vancouver, BC Canada, V6Z 1Y6
Lecithin: cholesterol acyltransferase (LCAT) is a plasma glycoprotein which plays an important role in the reverse cholesterol transport (RCT) pathway. The biochemistry of this protein is well understood and its naturally occurring defects result in LCAT deficiency syndromes, which present with unusual characteristics. Due to the important role of LCAT in the RCT pathway, risk of premature CAD in these patients has been carefully considered. However, the current data are inconclusive, despite the new insights from analyses of transgenic animal models of abnormal LCAT metabolism. Traditionally, LCAT deficiency syndromes have been divided into two disorders, Familial LCAT deficiency (FLD) and fish-eye disease (FED). This classification was based on the biochemical and clinical presentation of the disorders. There are several limitations with the traditional classification of LCAT mutants. We have proposed a new classification scheme based on the molecular basis of LCAT defects, to address these issues. • Class 1—Null mutations causing FLD. There is total loss of LCAT activity and LCAT mass is virtually absent. • Class 2—Missense mutation causing FLD. There is a total loss of LCAT activity and LCAT mass can vary from normal to absent. • Class 3—Missense mutations and minor deletions causing FED. There is a partial loss of activity against HDL or both LDL and HDL. LCAT mass
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is reduced. • Class 4—missense mutations causing FED. There is a partial loss of LCAT activity against HDL only and LCAT mass is reduced. A fifth class is designated for a small group of mutations that cannot be clearly defined in any of the above. KEYWORDS: LCAT, deficiency, familial, genes, LDL.
INTRODUCTION Historical Perspective The first case of familial LCAT deficiency was described over three decades ago by Kare Norum and collaborators (Norum and Gjone, 1968; Gjone and Norum, 1968b; Torsvik et al., 1968). The three probands from this family were characterised by complete absence of LCAT activity accompanied by reduced cholesteryl ester and plasma LCAT concentration. The major remarkable findings of these patients were lipoprotein abnormalities—high density lipoproteins (HDL) deficiency, lipid changes in low density lipoproteins (LDL) and very low density lipoproteins (VLDL) particles, corneal opacification, anaemia, proteinurea and renal disease (Glomset et al., 1989). These findings have prompted a great deal of attention from both clinical and basic scientists, which has led to a better understanding of pathophysiology of this disorder. Traditionally, the phenotypic and laboratory findings have provided the basis of classification for the variants of this disorder. These are familial LCAT deficiency and fisheye disease. It was not until 1992 that Skretting and co-workers described the genetic defect underlying the disorder in this first case of familial LCAT deficiency (Skretting et al., 1992). The DNA sequence analysis of the LCAT gene in these probands revealed a homozygous single point mutation (nucleotide substitution) in the LCAT gene resulting in the substitution of a methionine by a lysine residue at position 252 of the secreted mature protein. Further in vitro expression and study of this protein has revealed an inactive but normally secreted protein (Hill, 1994). The first case of fish eye disease, however, was described by Carlson and coworkers in two Swedish families, in 1979 (Carlson and Philipson, 1979; Carlson, 1982). Affected individuals presented with severe hypoalphalipoproteinaemia, and elevated triglyceride levels. Corneal opacity was also present in these patients and was the underlying reason
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for the unusual name of the disorder. These individuals had no other medical complications and appeared to be protected against premature coronary artery disease (CAD). However, recent findings provide some evidence to the contrary (Kuivenhoven et al., 1996a; Kuivenhoven et al., 1997). Lecithin: Cholesterol Acyltransferase The Lecithin: cholesterol acyltransferase gene has been localised to the q21–22 region of chromosome 16, encompassing 4.2 Kilobases (Teisberg et al., 1975). The coding region of the gene is contained within 6 exons separated by 5 introns (McLean et al., 1986a). Expression of this gene results in a 1550 nucleotide mRNA encoding a protein of 416 residues with a hydrophobic leader sequence of 24 amino acids and four N-linked glycosylation sites (McLean et al., 1986a; McLean et al., 1986b; Yang et al., 1987). The expressed product has a calculated molecular weight (MW) of 47,090 daltons with an apparent MW of 67,000. Thus, about 25% of the protein mass is due to its carbohydrate content (McLean et al., 1986a; McLean et al., 1986b; Yang et al., 1987; O et al., 1993a; Qu et al., 1993; Francone et al., 1993). LCAT is the enzyme responsible for the synthesis of the majority of cholesteryl esters in plasma. This plasma protein is expressed in the liver and is secreted into the plasma compartment where it is found to be associated with circulating HDL and LDL particles, and catalyses the esterification of cholesterol with a fatty acid obtained from the sn-2 position of phosphatidylcholine (McLean et al., 1986; Francone et al., 1989; Jonas, 1991). LCAT activity may be promoted by several apolipoproteins acting as co-factors. Although the most effective activator is apoA-I, other apolipoproteins such as apoA-II, A-IV, C-II, and C-III can also function as activators (Jonas, 1991). These methods employ either the natural substrate particles, plasma—primarily HDL-lipoproteins, or HDL analogues such as proteoliposome containing apoA-I. LCAT activity measured using plasma lipoproteins is defined as the plasma cholesterol esterification rate (CER), whereas the activity determined using synthetic substrates is regarded as LCAT activity reflecting the concentration of LCAT protein in plasma (Pritchard, 1997; Hill, 1994). A review of the methods used in measuring LCAT activity and their value in differential diagnosis of LCAT deficiency syndrome has recently been published (Pritchard, 1997). LCAT protein concentration in plasma has been successfully determined by a number of groups using various immunochemical techniques (Hill et al., 1993a; Klein et al., 1995; Qu et al., 1995; Hill et al., 1993b; Albers and Utermann, 1981; Albers et al., 1981). However,
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one must exercise caution in interpretation of the current data based on LCAT mass analyses, since there is a large inter-laboratory variation of the assays. Modulation of LCAT Activity in Transgenic Animals Over expression of human LCAT In recent years, human LCAT has been expressed in mice (Francone et al., 1995; Vaisman et al., 1995; Mehlum et al., 1995) and rabbits (Brousseau et al., 1996). Mehlum et al. (1995) generated a transgenic mouse strain expressing the human LCAT to assess the effect of an increase in LCAT activity in plasma. This mouse strain expressed 70 times more LCAT than normal human plasma resulting in a 50% decrease in plasma triglyceride. LDL and VLDL concentrations were also reduced to 50% of normal plasma. On the other hand, HDLcholesterol levels increased by 20% while accommodating the increase in apoA-I and apoA-II. Another study by Vaisman et al. (1995) generated strains of mice that expressed 109-fold more LCAT than the control mice. Plasma lipid profile data also showed hyperalphalipoproteinaemia, which is proposed to be anti-atherogenic. A rabbit transgenic strain has been developed as a better model for lipoprotein metabolism in humans (Brousseau et al., 1996; Hoeg et al., 1996b). In these animals, LCAT activity was increased up to 3.1-fold above the background strain. Total cholesterol and HDL-cholesterol were increased by 1.5- and 2.5-fold, respectively. This alteration in lipoprotein profile was attributed to the increased apoE rich HDL-1 particles. It was further suggested that overexpression of LCAT in the presence of CETP led to hyperalphalipoproteinaemia and reduction in atherogenic lipoproteins. However, further study of these transgenic animals has shown that in response to a high cholesterol diet, 0.3% cholesterol, after 11 weeks, the degree of atherosclerosis remained the same for both the transgenic and control groups despite elevated HDL in the transgenic group. This study concluded that despite maintenance of elevated HDL in the LCAT transgenic group on a high cholesterol diet, the progression of atherosclerosis was unaffected (Hoeg et al., 1996a). Co-expression of human apoA-I and apoA-II (hu AI and hu AII), with LCAT has shed some light into the in vivo relationship between LCAT and these plasma apoproteins. Moderate increase in LCAT activity (1.6-fold), in hu AI and hu AI/AII mice resulted in elevation of total cholesterol by 4- and 2-fold, respectively. In hu AI/LCAT and hu AI/AII/LCAT animals, HDL particles were reported to be larger in size and richer in cholesteryl esters (up to 2-fold). In addition, increased LCAT activity in hu AI/LCAT and hu AI/AII/LCAT was closely
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associated with reduction in apoA-I ratio in the prebeta HDL fraction. The study concluded that expression of LCAT had a significant impact on plasma lipid metabolism and that human LCAT has a significant preference for HDL which contain human apoA-I (Francone et al., 1995). Although mouse and rabbit transgenic models have improved our knowledge of in vivo LCAT function, there are major concerns regarding the interpretation of the data. All transgenic models have achieved human LCAT overexpression, however, the physiological relevance of this approach is highly questionable because there is a great deal of evidence that there are differences in substrate specificity and enzyme activation among human, mouse and rabbit species. Furthermore, extremely large quantities of LCAT expressed in these models render the data obtained uninterpretable as they represent extreme, not physiological states. Future studies perhaps will address and control for more physiologically relevant variables. LCAT knock-out model Targeted disruption of the mouse LCAT gene has allowed generation of LCAT deficient mice (Ng et al., 1997; Sakai et al., 1997). Despite the similarities in most biochemical parameters with human familial LCAT deficiency, mouse models do not develop the severe clinical complications present in FLD (i.e., corneal opacity, renal insufficiency). The only remarkable characteristics are elevated triglyceride levels (Ng et al., 1997; Sakai et al., 1997) and upregulation of the adrenal SR-BI mRNA due to severe depletion of adrenal lipid stores. These observations suggest that LCAT may play a role in catabolism of triglyceride rich apo-B containing lipoproteins and that the reduction in plasma HDL-cholesterol may have an impact on the cholesterol flux to the adrenal via a selective uptake pathway (Ng et al., 1997).
LCAT DEFICIENCY SYNDROMES It is now well established that either familial LCAT deficiency (FLD) or fish-eye disease (FED) are caused by mutations in the human LCAT gene which are both inherited in an apparently autosomal recessive manner. We will only briefly illustrate the predominant phenotypic characteristics of these disorders, since these LCAT deficiency syndromes have been extensively reviewed (McIntyre, 1988; Kuivenhoven et al., 1997; Assmann et al., 1991; Glomset et al., 1989).
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Familial LCAT Deficiency As described earlier, this disorder was first reported in a Norwegian family (Norum and Gjone, 1967; Gjone and Norum, 1968a). Since then, a number of unrelated families diagnosed with FLD have been identified. There are major clinical and biochemical findings that are in common to this disorder (Kuivenhoven et al., 1997). Ocular changes The most striking physical characteristic in these patients is the unusual appearance of their cornea. All patients present with corneal opacities in early childhood. They are best described as several minute, grayish dots spread throughout the entire stroma resulting in the cloudy appearance of the cornea. Histological analysis of the corneal sections has indicated the presence of electron dense membranous deposits and increased concentrations of unesterified cholesterol and phosphoplipids (Glomset et al., 1989). Corneal opacities are not unique to this condition. They have been noted in other genetic disorders affecting HDL metabolism such as apoA-1 deficiency (Funke et al., 1991b), Tangier disease (Chu et al., 1979), fish eye disease (Carlson, 1982) and a combined deficiency of apoA-I and apoC-III (Norum et al., 1982). Haematological findings These patients present with normochromic anaemia, which has been suggested to be due to moderate anaemia with weak compensatory erythropoiesis (Chevet et al., 1978). Furthermore, erythrocytes display abnormalities in appearance and lipid composition (Gjone et al., 1968). A compositional analysis of red blood cells revealed nearly double the normal levels of unesterified cholesterol and phosphatidylcholine with a marked reduction in sphingomyelin and phosphatidylethanolamine levels (Chevet et al., 1978; Godin et al., 1978; Yawata et al., 1984). In addition, the erythrocytes in LCAT deficiency are associated with a significant reduction in sodium influx and a decrease activity of acetylcholinesterase (Knipping et al., 1986). Although it is unclear whether other cells are affected, platelet function and lipid composition appears to be normal (Nordoy and Gjone, 1971). Cellular analysis of bone marrow has revealed target cells and a small number of foam cells. In addition, sea-blue histocytes have been identified in bone marrow and spleen using Giemsa stain (Jacobsen et al., 1972). Composition analysis of histocyte granules has revealed membranes in a lamellar arrangement (Hovig and Gjone, 1973a). It is suggested that these membranes contain unesterified cholesterol and
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phosphatidylcholine, as the levels of both lipids are elevated in the spleen and liver (Stokke et al., 1974). Renal complications While in most patients proteinuria is detected early in life, a small number do not develop renal complications (Gjone et al., 1974a; Borysiewicz et al., 1982; Murayama et al., 1984). Serum albumin concentrations are slightly reduced, but can rapidly decrease due to an increase in proteinuria and subsequent renal failure. Although renal function and blood pressure may remain normal for many years, hypertension and renal insufficiency may develop unexpectedly (Gjone et al., 1974a). Morphologic analysis of the kidney has revealed various structural changes assessed by light and electron microscopy (Stokke et al., 1974; Magil et al., 1982; Hovig and Gjone, 1973b; Hovig and Gjone, 1973a; Imbasciati et al., 1986). Plasma lipoprotein abnormalities Plasma lipoprotein analyses of LCAT deficient patients show a multitude of lipoprotein abnormalities with a great deal of variability in plasma lipoprotein levels. The common characteristics, however, are high plasma levels of unesterified cholesterol and phosphatidylcholine, and residual levels of cholesteryl esters and lyso-phosphatidylcholine (Glomset and Norum, 1973). The small quantities of cholesteryl esters may be explained by the action of the interacellular enzyme, acyl coenzyme A: cholesterol acyltransferase (ACAT) in the intestine. This is consistent with abnormally high proportion of palmitic and oleic acids instead of linoleic acid in the detected cholesteryl esters. A number of methods have been employed to isolate and study various lipoprotein fractions. These techniques include ultracentrifugation, gel filtration, electron microscopy and gel electrophoresis. (Glomset and Norum, 1973). Study of VLDL showed abnormal beta mobility of particles which had higher concentrations of unesterified cholesterol and phosphatidylcholine relative to triglyceride and protein. In addition, protein analysis showed an increase in apoC-I and apoE, but a reduction in apoC-II and apoC-III resulting in reduction of electronegative proteins, which can explain the unusual electrophoretic mobility (Glomset et al., 1980). Particle size distribution analysis revealed that a large number of particles in the fraction had a diameter greater than 90 nm in overnight fasting samples. However, these particles were significantly reduced when the patients consumed low fat diets the night before sample collection, which suggested that they were chylomicrons (Glomset et al., 1975). Intermediate density lipoproteins (IDL, 1.006
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an abnormal size heterogeneity demonstrated by gel filtration. These particles have been shown to be rich in triglyceride and unesterified cholesterol as the composition of the normal sized particles were shown to be the intermediate between VLDL and LDL particles (Glomset et al., 1973). It has been suggested that reduced hepatic lipase (HL) activity may contribute to an increase in triglyceride content of these particles due to decreased ability of HL to hydrolyse their triglyceride (Murano et al., 1987). Abnormalities of lipoprotein particles are also present in LDL fraction (Glomset et al., 1973). The elution profile from the gel filtration of the LDL particles resulted in three well-defined components, while there is only one in normal individuals. The first subfraction, which eluted in the void volume was of 90 nm diameter size which does not appear in normal LDL fractions. This particle was composed of multilamellar structures containing an unusually high ratio (1:2) of phosphatidylcholine and unesterified cholesterol both of which contribute to the lamellar structure. The protein content of this subfraction was mainly limited to small quantities of albumin. The total concentrations of these particles were proportional to large VLDL particles yet they varied among patients. Furthermore, similar to larger VLDL particles, levels of the larger LDL particles could be altered by the fat content of the patients diet (Glomset et al., 1975). The second relatively distinct subfraction was eluted just after the void volume and consisted of abnormal particles 30–80 nm in diameter. Similar to the previous fractions these particle were not observed in normal plasma. The structure of some of these particles closely resembled Lp-X which is an abnormal lipoprotein found in cholestasis (Hamilton et al., 1971). Electron microscopy has revealed a discoidal structure that formed stacks. These particles were composed of phosphatidylcholine, unesterified cholesterol and apoC-I. This fraction also contained some spherical particles resembling normal remnants of VLDL and chylomicrons (Ritland and Gjone, 1975). The third subfraction of patient LDL has normal physical characteristics. These particles have a diameter ranging between 20 and 22 nm. Although these spherical particles resemble normal LDL, their triglyceride content is much higher than normal (13 fold) while the cholesteryl ester levels are lower (1.5–3 fold). However the proteinapoB100-to core lipid ratio has been shown to be similar to the normal particles. In addition, the apoB concentration was shown to be low suggesting that normal LDL particles comprise a small fraction of the total LDL pool in these patients (Norum et al., 1971). HDL also display a very heterogeneous distribution of size and shape, consistent with the other lipoprotein classes. The HDL are predominantly disc shaped with a minor component of spherical particles of very small size (6nm). Phosphatidylcholine and unesterified
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cholesterol are the major components of these discs, which may also contain a mixture of apoA-I, apoA-IV and C apolipoproteins (Torsvik, 1972, Soutar et al., 1982). Some discs contain apoE (Mitchell et al., 1980). The small spherical HDL particles are composed of unesterified cholesterol, phosphatidylcholine, small amount of core lipid and 2 molecules of apoA-I. The concentration of these particles is also dependent on the fat content of each individual’s diet (Chen et al., 1984). Fish-Eye Disease Two Swedish families were the original cases of FED reported (Carlson and Philipson, 1979; Carlson, 1982). These cases were characterised by severe HDL deficiency and extensive corneal opacities which was responsible for the unusual name of the disorder, since the eyes of the affected individuals resembled that of boiled fish. The number of reported cases with FED is much smaller than that of FLD. There have only been 11 cases of FED reported in 6 families from Algeria, Germany, the Netherlands and Sweden. Furthermore, there have been a number of cases which share some biochemical characteristics with FED (Frohlich et al., 1987). Ocular changes Corneal opacities have been reported as early as the second decade of life (Carlson and Philipson, 1979; Carlson, 1982). The appearance of corneal changes are similar to those described in FLD which consist of minute grayish dots throughout the entire stroma while increasing in density at the periphery. Visual acuity in these patients is often impaired at an earlier age than that of FLD. This may be attributed to some reports of greater opacification in FED in comparison to FLD. Composition analysis of the vacuoles in the stroma revealed high concentration of unesterified cholesterol and membrane like material in the periphery (Carlson, 1982). Plasma lipoprotein abnormalities The original Swedish families have been the major source of information on the composition and structure of the plasma lipoproteins in FED (Carlson, 1982; Forte and Carlson, 1984). Unlike normal plasma, the VLDL fraction of these patients (subjects) presented a great deal of size heterogeneity. The concentration of these particles increased five-fold despite the normal triglyceride to cholesteryl ester ratio. The triglyceride content of IDL and LDL is also increased, especially in the LDL fraction, in which the triglyceride to cholesteryl
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ester ratio is 10 times greater than normal. These particles are of very homogeneous round structure with a smaller mean diameter than normal LDL. However, there are occasional occurrences of particles with diameters ranging from 50 to 85 nm. These large particles are absent from the normal LDL fraction. In FED patients, HDL fractions show the greatest abnormalities. This fraction is reduced to 10% of the normal while the unesterified cholesterol level is about three times more than control HDL. Furthermore, these particles are enriched in apoE while the A apolipoprotein levels are drastically reduced (90%) (Carlson, 1982). Structurally, the HDL fraction presents two major subclasses. These are small, round particles with a diameter of 7.6 nm and discoidal particles with a thickness of 4.4 nm and an average diameter of 17.4 nm (Forte and Carlson, 1984).
ATHEROSCLEROSIS IN LCAT DEFICIENCY SYNDROMES There is controversial evidence on the role of LCAT in atherosclerosis. The role of LCAT in the reverse cholesterol transport (RCT) pathway has been clearly established, and it is well accepted that high esterification rates will promote the efflux of cholesterol from periphery. Thus, LCAT appears to play an important role in maturation of HDL (Glomset et al., 1989). However, patients who lack mature HDL, due to dysfunctional LCAT protein, do not present with premature atherosclerotic vascular disease (Glomset et al., 1989; Assmann et al., 1991; Funke et al., 1993). In FED patients, a partially functional RCT pathway has been suggested to protect against premature atherosclerosis. However, this does not account for the absence of CAD in FLD patients whose RCT is completely impaired. This issue has been addressed by another hypothesis suggesting that the residual HDL in FLD can be an excellent acceptor for peripheral cholesterol (Glomset et al., 1970). The recent findings by Ohta et al. (1994), on the other hand, indicates that these particles are not as good acceptors of unesterified cholesterol as originally suggested. Kinetic analysis of HDL subfractions in both FLD and FED patients has provided further insight into the metabolic basis of the absence of CAD in LCAT deficiency syndromes (Rader et al., 1994). It was shown that HDL particles containing both apoA-I and apoA-II (LpA-I:A-II) were cleared much faster than the particles containing apoA-I alone (LpA-I). Thus, it was concluded that the low plasma HDL level promoted accelerated catabolism of apoA-II and LpA-I:A-II. The remaining LpAI fraction may have a protective potential against atherogenesis (Stampfer et al., 1991; Parra et al., 1992; Puchois et al., 1990). The
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investigators proposed that the remaining LpA-I could maintain an effective RCT pathway. Furthermore, it was hypothesised that hypercatabolism of LpA-I:A-II and the presence of apoA-I and LpA-I contribute to a relative absence of risk of premature CAD in both FED and FLD. However, the subsequent studies with macrophage foam cells showed that LpA-I and LpA-I:A-II of FLD patients were poor acceptors of intercellular cholesterol and it appeared that LCAT activity was required to remove cholesterol form the foam cells by LpA-I (Ohta et al., 1992; Ohta et al., 1994). Thus, it was concluded that factors other than RCT mediated by apoA-I should be considered in order to explain the apparent protection of FLD patients from premature CAD. The next hypothesis postulated that a reduction in cholesteryl ester-rich LDL could explain the reduced number of CAD cases described. Although the incidence of premature CAD in FLD is very limited, there is some evidence of early coronary atherosclerosis in a few cases and first family members (Miller et al., 1995; Vergani et al., 1983). However, 6 out of 19 known FED patients from 5 families have been reported to suffer from premature atherosclerosis (Funke et al., 1991a; Kuivenhoven et al., 1995; Kuivenhoven et al., 1996a). It is noteworthy to mention that all the patients with premature CAD are male. The difference in risk of premature CAD between FED and FLD requires further investigation. In addition, this difference may be related to the presence of LCAT activity associated with LDL particles in FED (Figure 12.2). It is our contention that the referral biases and small sample size prevent any meaningful conclusion. However, it must be stressed that despite a general absence of premature CAD in LCAT deficiency syndromes, there is growing evidence for the higher risk of premature atherosclerosis in several cases of male patients with FED.
CLASSIFICATION OF LCAT GENE DEFECTS In a recent review, our laboratory proposed a new scheme for the classification of natural mutations of the LCAT gene (Kuivenhoven et al., 1997). This method of classification is based on the specific nature of each mutation and avoids the major complications with classification of these defects based on their biochemical phenotype alone. First, biochemical analysis of many gene defects in vitro has resulted in discrepancies in characterisation of the gene defect. This has been partly attributed to dissimilarities in methodologies for LCAT activity and mass assessments (Hill, 1994; Hill et al., 1993b; Qu et al., 1995; Klein et al., 1995). Furthermore, use of multiple expression systems has been shown to result in variations in protein processing due to the differences in the nature and source of the cells and their protein processing machinery (Qu et al., 1995; Klein et al., 1995; O et al.,
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1993b; Hill et al., 1993a; Kuivenhoven et al., 1995; Hill et al., 1993b; Kuivenhoven et al., 1996a; Kuivenhoven et al., 1996b; Klein et al., 1993a; Miettinen et al., 1995; Steyrer et al., 1995; Moriyama et al., 1995; Miller et al., 1995). Second, not all defects can be identified in the homozygous state. Some LCAT gene defects have manifested in combination with another defect-resulting in compound heterozygous state. This has posed a difficulty in establishing a clear classification for some gene defects. Lastly, some defective gene products display an intermediate biochemical phenotype which
Table 12.1 Mutations that underlie LCAT deficiency syndromes
Defect
Exon Clinical Phenotype References a FLD FED
Homozygous mutations (Bujo et al., 1991, McLean, 1992; Murano et al., 1987; Ohta et al., 1986)
1.
C-insertion (codons 9, 10)b1
2.
P10L
1
(Carlson and Phillipson, 1979; Carlson, 1982; Skretting and Prydz, 1992)
3.
G30S
2
Owen et al., 1996; Bron et al., 1975; Yang et al., 1997)
4.
Y83-STOP
3
(Funke et al., 1983)
5.
A93T
3
(Funke et al., 1983; Assmann et al., 1991; Hill et al., 1993b)
6.
R99C
3
(Blanco-Vaca et al., 1997)
6.
T123I
4
(Kastelein et al., 1992; Funke et al., 1991a; O et al., 1993b)
7.
N131D
4
(Kuivenhoven et al., 1995)
8.
R140H
4
(Steyrer et al., 1995)
9.
G 141-insertion
4
(Gotoda et al., 1991)
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10. L209P
5
(Funke et al., 1993; Assmann et al., 1991)
11. N228K
6
(Gotoda et al., 1991)
12. G230R
6
(Miettinen, 1998)
13. R244G
6
(McLean, 1992; Vrabec et al., 1988)
14. M252I
6
(Gjone et al., 1974b; Gjone et al., 1981 Skretting et al., 1992; Norum and Gjone, 1967)
15. M293
6
(Maeda et al., 1991; McLean, 1992; Gotoda et al., 1991; Sakuma et al., 1982; Albers et al., 1982; Albers et al., 1985)
16. L300-deletion
6
(Klein et al., 1993b; Cierc et al., 1991)
17. T321M
6
(Utermann et al., 1972; Utermann et al., 1981; Funke et al., 1993)
18. G344S
6
(Moriyama et al., 1995)
19. G-deletion (codon 6 264)b
(Moriyama et al., 1995)
Heterozygous mutations 1.
P10Q R135Q
1, 4
(Kuivenhoven et al., 1996a)
2.
L32P T321M
2, 6
(McLean, 1992)
3.
G33R 30bp Insertion (codn 4)
1, 2
(Wiebusch et al., 1995)
4.
Y83- Y156N stop
3, 5
(Klein et al., 1993a)
5.
T123I
4, 4
(Contacos et al., 1996)
6.
T123I Intron 4 defect
4 intron
(Kuivenhoven et al., 1996b)
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(IVS4:T22C) 7.
T123I T347M
4, 6
8.
T123I Unknown 4, ?
9.
R135W A-insertion (codon 376)b
(Klein et al., 1992) (Winder, 1993) 4, 6
(Frohlich et al., 1982; Frohlich et al., 1978; Funke et al., 1993; Assmann et al., 1991)
10. R147W Unknown
4, ?
(Assmann et al., 1991; Vergani et al., 1983; Taramelli et al., 1990)
11. R147W Y171-stop
4, 5
(Guerin et al., 1997)
12. G183S A-T substitution/ C- 6, deletion (codon 5 120)b
(Bethell et al., 1975; McLean, 1992)
13. M252K N391S
6, 6
(Hill et al., 1993a)
14. T321M C-deletion (codon 168)b
5, 6
(Miller et al., 1995)
15. R399C C-insertion (codon 9, 10)b
6, 1
(Miettinen et al., 1995)
a References include biochemical, clinical and genetic reports on the LCAT gene defects presented. b Premature truncation as a result of a frameshift.
shares specific characteristics from both FED and FLD (Hill, 1994; Hill et al., 1993a; Kuivenhoven et al., 1995; Klein et al., 1993b). The following section describes our classification of the natural mutants of the LCAT gene using a combination (where possible) of chemical, biochemical and genetic data. Class 1 A variety of null mutations of the LCAT gene which result in a total loss of catalytic activity and which have the clinical phenotype of FLD in homozygous subjects are placed in this class. These defects can arise directly from the introduction of a stop codon, (Klein et al., 1993a; Funke et al., 1993; Guerin et al., 1997) defective splicing of LCAT mRNA (Kuivenhoven et al., 1996b), or production of a truncated/nonsense protein form an underlying frameshift (Miettinen et al., 1995;
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Moriyama et al., 1995; Miller et al., 1995; Funke et al., 1993; Bujo et al., 1991; McLean, 1992; Assmann et al., 1991). Two additional reports have documented insertions of one or more nucleotides (Gotoda et al., 1991; Wiebusch et al., 1995). There are also mutations that are difficult to classify, such as a specific 3bp deletion at codon 300 (Klein et al., 1993b). As an illustration of the features characteristic of class 1 mutations, a single nucleotide substitution in intron 4 of the LCAT gene has been recently described. (Kuivenhoven et al., 1996b). As a result, there is lack of normal expression of the affected allele associated with half normal LCAT concentrations and activity, and half the levels of HDL cholesterol in heterozygous subjects. Class 2 Class 2 contains a collection of missense mutations that cause a complete or nearly complete loss of catalytic activity of LCAT. As a result, the measurement of cholesterol esterification, using heatinactivated or artificial substrates, is very low to
Table 12.2 Classes of LCAT gene mutations
Class/Definition
Defect
References a
1. Null mutations causing FLD Total loss of catalytic activity.
30 Bp Insertion (Wiebusch et al., 1995) (codon 4)
LCAT mass: (virtually absent).
C-insertion (codon 9, 10)
(Miettinen et al., 1995; Bujo et al., 1991; McLean, 1992)
Y83-stop
(Klein et al., 1993a; Funke et al., 1993)
G141-insertion (Gotoda et al., 1991) Intron 4 defect (IVS4:T-22C)
(Kuivenhoven et al., 1996b)
A-T substitution/
(McLean, 1992)
C-deletion (codon 120) C-deletion (codon 168)
(Miller et al., 1995)
Y171-stop
(Guerin et al., 1997)
G-deletion (codon 264)
(Moriyama et al., 1995)
Plasma lipids and their role in disease
A-insertion (codon 376)
316
(Assmann et al., 1991; Funke et al., 1993)
2. Missense mutations causing FLD Total loss of catalytic activity.
G30S
(Owen et al., 1996; Yang et al., 1997)
LCAT mass: normal/reduced/ absent.
L32P
(Qu et al., 1995; McLean, 1992)
G33R
(Wiebusch et al., 1995)
A93T
(Assmann et al., 1991; Hill et al., 1993b; Qu et al., 1995; Funke et al., 1993)
R135W
(Assmann et al., 1991; Qu et al., 1995; Funke et al., 1993; Hill, 1994)
R135Q
(Kuivenhoven et al., 1996a)
R140H
(Steyrer et al., 1995)
R147W
(Steyrer et al., 1995; Assmann et al., 1991; Qu et al., 1995; Klein et al., 1995)
Y156N
(Klein et al., 1995; Klein et al., 1993a)
G183S
(McLean, 1992)
L209P
(Assmann et al., 1991; Qu et al., 1995; Funke et al., 1993; Hill, 1994)
N228K
(52, 64)
G23OR
(Miettinen et al., 1998)
R244G
(McLean, 1992)
M252K
(Skretting et al., 1992; Hill et al., 1993a; Hill, 1994)
T321M
(Qu et al., 1995; Miller et al., 1995; Funke et al., 1993; McLean 1992)
G344S
(Moriyama et al., 1995)
T347M
(Hill, 1994, Qu et al., 1995; Klein et al., 1993a; Klein et al., 1992)
R399C
(Miettinen et al., 1995)
3. Missense mutations and minor deletions causing FED
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Partial loss of activity against
R99C
(Blanco-Vaca et al., 1997)
(a) LDL, or (b) both HDL and LDL
N131D
(Kuivenhoven et al., 1995)
LCAT mass: reduced.
L300 deletion
(Klein et al., 1993a; Klein et al., 1993b; Hill, 1994)
N391S
(Hill, 1994; Hill et al., 1993a)
4. Missense mutations causing FED Partial loss of activity against HDL only. LCAT mass: reduced.
P10L
(Hill, 1994; Qu et al., 1995; Klein et al., 1995; Skretting and Prydz, 1992)
P10Q
(Kuivenhoven et al., 1996a)
T123I (Hill, 1994; Funke et al., 1991a; O et al., 1993b; Kuivenhoven et al., 1996b; Qu et al., 1995; Klein et al., 1995; Klein et al., 1992; Contacos et al., 1996) 5. Unclassified mutations Y144C (Contacos et al., 1996) M293I (Klein et al., 1995; Gotoda et al., 1991; Sakuma et al., 1982; Maeda et al., 1991) R158C (Assmann et al., 1991; Luc, 1994; Duverger, 1993; Hill et al., 1993b; Qu et al., 1995; Klein et al., 1995) a The references are reports describing the identification and functional assessment (when available) for the LCAT gene defects.
absent. The plasma LCAT concentrations of homozygous subjects may be normal (Owen et al., 1996) or reduced (most cases) relative to the virtual absence of LCAT activity. This absence causes the clinical and biochemical characteristics of FLD. Up until now, a great number of mutations have been shown to be associated with this phenotype (Table 12.2). A specific single amino acid substitution, the previously described M252K (Skretting et al., 1992), is a good example for this class of defects. It has been shown that the defect results in a complete loss of LCAT activity and is associated with FLD in homozygous subjects (Hill, 1994). Class 3 In the third class, the mutations in the LCAT gene result in proteins that produce a phenotype in between the description of those in classes 1, 2
Plasma lipids and their role in disease
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and class 4. These proteins arise from either missense mutations (Hill, 1994; Kuivenhoven et al., 1995) or minor deletions (Klein et al., 1993b). As a result, there is a selective loss of activity using LDL as substrate (Klein et al., 1993b), or a joint partial loss of activity against both HDL and LDL (Hill, 1994; Kuivenhoven et al., 1995) substrate. In addition, in vivo, homozygous subjects may have LCAT concentrations half of normal and have a moderate reduction in cholesterol esterification rate. Phenotypically, they present the same as FED. Only four mutations, up until now, have been reported with the class 3 phenotype. A near complete loss of activity against HDL (5% of wild type recombinant LCAT) in conjunction with a 75% reduction in activity towards LDL as substrate (as compared to wild type) was first reported in the N131D mutation upon functional assessment (Kuivenhoven et al., 1995). Secondly, the L300Del and R99C produced the clinical phenotype of FED in homozygotes and upon biochemical examination, the LCAT was reported to have altered function towards both HDL and LDL (Klein et al., 1993b; Blanco-Vaca et al., 1997). The fourth and final mutation that meets the criteria for class 3 mutations is the N391S defect. It was found in a compound heterozygous patient who suffered from FED (Hill, 1994; Frohlich et al., 1987). Similarly to the above two, this mutation displayed a reduced activity in vitro against HDL (18% of wild type) and LDL (53% of wild type). Class 4 Class 4 mutations are simply defined as defects that cause the typical FED phenotype. This phenotype (Carlson and Holmquist, 1985) is described as a missense mutations that lead to the specific loss of LCAT activity against HDL particles while the activity against LDL and VLDL is largely unaltered. The unaffected activity against apoB containing lipoproteins results in near normal esterification of cholesterol in heat-inactivated plasma. In classic FED, there is a specific loss of LCAT activity towards HDL substrate in vitro in homozygous subjects yet the cholesterol esterification rate is either normal to moderately reduced. There have only been three defects identified thus far that meet the class 4 criteria. The first mutation to present with this phenotype is the P10L defect (Hill, 1994). This mutation was identified in the original FED patients (Carlson, 1982; Skretting and Prydz, 1992) from Sweden. A nucleotide exchange— resulting in a change of proline to glutamine (P10Q)—within the same codon was the second occurrence. When characterised, it was shown to maintain similar properties (Kuivenhoven et al., 1996a). The final and most common FED mutation is T123I (Funke et al., 1991a; Kastelein et al., 1992; Klein et al., 1993a) which has demonstrated loss of
Familial lecithin
319
specificity in activity towards HDL (Hill, 1994; O et al., 1993b). Patients display near normal levels of LCAT in plasma which has been supported in tissue culture in which rLCAT has been shown to be secreted at near normal levels. Class 5 There are three defects, thus far, which do not fit into any of the above classes. Therefore, the fifth and final category contains unclassified LCAT mutations. All homozygous and compound heterozygous mutations resulting in FED subjects have been characterised in vitro with the exception of the Y144C defect. This mutation was identified in a compound heterozygous subject who also carried the FED-associated T1231 mutation (Contacos et al., 1996). Due to the fact that the carriers of two FED alleles cannot be distinguished from carriers of one FLD and one FED allele, it is difficult to deduce the effect of the Y144C mutation on LCAT function. Without the proper in vitro expression data, this mutation cannot be classified and it is therefore included in the category of unclassified mutations (Table 12.2). Important Considerations It is clear from Table 12.2 that the region within LCAT residues 123 and 156 is of importance. It has been shown that the loss of positively charged arginine residues—135, 140 and 147—in this region results in complete loss of LCAT activity, i.e., (Assmann et al., 1991; Steyrer et al., 1995; Kuivenhoven et al., 1996a). In addition, other mutations (T123I and N131D) of this small region can also lead to a milder FED phenotype. Based on this information, one may postulate that this region is important in the interaction of the enzyme with lipoproteins and plays a key role in substrate specificity. Work carried out by Klein et al. (1995) provides some indirect evidence for this notion. Specifically, the catalytic activity of LCAT, using water- soluble substrates, has been shown to be unaffected by a number of LCAT defects, including R146W. Furthermore, in vitro expression of human LCAT and its mutants has suggested that the activity of LCAT towards HDL analogues is more easily disrupted, when compared to its reactivity toward native LDL. Naturally, this is true for class 4 mutants, but it also is valid for class 2 and class 3 defects, to a lesser degree. To better illustrate this point, data from representative defects from each group is plotted in Figure 12.1.
Plasma lipids and their role in disease
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Figure 12.1 This graph represents the LCAT activity corresponding to wild type (WT) LCAT and LCATs from a representative member of each class. LCAT activity was assessed using both HDL-analogues (proteoliposomes) and endogenous LDL particles. Results are expressed as a percentage of activity corresponding to WT LCAT.
CONCLUSIONS AND FUTURE DIRECTION The in vitro characterisation of natural mutants of LCAT in conjunction with a better understanding of its molecular pathology has led to a new classification system for the genetic defects of LCAT. Although this new classification scheme has addressed a number of sources of ambiguity in identification, assessment, and smanagement of this multifaceted disorder, there are still some confounding issues surrounding a small number of defects. In our opinion, further research and understanding of the nature of the interaction and binding of the protein to its substrate particles will provide more effective tools to identify and define very specific regions of the protein. An improved knowledge of the biochemical and molecular consequences of natural
Familial lecithin
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defects of LCAT in combination with its physical properties will allow us to better identify individual defects. The most crucial step in achieving the above goal is the determination of the three-dimensional structure of the protein in its crystal form, which is currently being undertaken through a number of collaborative efforts in our laboratory.
Figure 12.2 This figure illustrates the biochemical presentation of various classes of LCAT gene defect. Class 1 —Null mutations causing FLD; there is a total loss of LCAT activity and LCAT mass is virtually absent. Class 2—Missense mutation causing FLD; there is a total loss of LCAT activity and LCAT mass can vary from normal to absent. Class 3—Missense mutations and minor deletions causing FED; there is a partial loss of activity against HDL or both LDL and HDL; LCAT mass is reduced. Class 4 — Missense mutations causing FED. There is a partial loss of LCAT activity against HDL only and LCAT mass is reduced. UC, unesterified cholesterol. CE, cholesteryl esters.
The role of LCAT and its related disorders in progression or protection from premature atherosclerosis is still unclear. Recent studies using various transgenic models have yielded more controversial observations contributing to the current state of uncertainty. However, our current work on prospective studies of
Plasma lipids and their role in disease
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family(s) with LCAT deficiency syndromes will provide a more definitive understanding of the role of natural defects of LCAT in atherosclerosis. References Albers, J.J., Adolphson, J., Chen, C.H., Murayama, N., Honma, S. and Akanuma, Y. (1985). Defective enzyme cause lecithin-cholesterol acyltransferease deficiency in a Japanese kindred. Biochimica et Biophysica Acta , 835 , 253–7. Albers, J.J., Adolphson, J.L. and Chen, C.H. (1981). Radioimmunoassay of human plasma lecithin-cholesterol acyltransferase. Journal of Clinical Investigation , 67 , 141–8. Albers, J.J., Chen, C.H., Adolphson, J., Sakuma, M., Kodama, T. and Akanuma, Y. (1982). Familial lecithin-cholesterol acyltransferase deficiency in a Japanese family: evidence for functionally defective enzyme in homozygotes and obligate heterozygotes. Human Genetics , 62 , 82–5. Albers, J.J. and Utermann, G. (1981). Genetic control of lecithincholesterol acyltransferase (LCAT): measurement of LCAT mass in a large kindred with LCAT deficiency. American Journal of Human Genetics , 33 , 702–8. Assmann, G., von Eckerdstein, A. and Funke, H. (1991). Lecithin: cholesterol acyltransferae deficiency and fish eye disease. Current Opinion in Lipidology , 2 , 110–117. Bethell, W., McCulloch, C. and Ghosh, M. (1975). Lecithin cholesterol acyl transferase deficiency. Light and electron microscopic finding from two corneas. Canadian Journal of Ophthalmology , 10 , 494– 501. Blanco-Vaca, F., Qu, S.J., Fiol, C., Fan, H.Z., Pao, Q., MarzalCasacuberta, A., Albers, J.J., Hurtado, I., Gracia, V., Pinto, X., Marti, T. and Pownall, H.J. (1997). Molecular basis of fish-eye disease in a patient from Spain. Characterization of a novel mutation in the LCAT gene and lipid analysis of the cornea. Arteriosclerosis, Thrombosis & Vascular Biology , 17 , 1382–91. Borysiewicz, L.K., Soutar, A.K., Evans, D.J., Thompson, G.R. and Rees, A.J. (1982). Renal failure in familial lecithin: cholesterol acyltransferase deficiency. Quarterly Journal of Medicine , 51 , 411– 26. Bron, A.J., Lloyd, J.K., Fosbrooke, A.S., Winder, A.F. and Tripathi, R.C. (1975). Letter: Primary L.C.A.T.-deficiency disease. Lancet , 1 , 928–9. Brousseau, M.E., Santamarina-Fojo, S., Zech, L.A., Berard, A.M., Vaisman, B.L., Meyn, S.M., Powell, D., Brewer, H.B., Jr. and Hoeg, J.M. (1996). Hyperalphalipoproteinemia in human lecithin cholesterol acyltransferase transgenic rabbits. In vivo apolipoprotein A-I catabolism is delayed in a gene dose-dependent manner. Journal of Clinical Investigation , 97 , 1844–51. Bujo, H., Kusunoki, J., Ogasawara, M., Yamamoto, T., Ohta, Y.,
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Shimada, T., Saito, Y. and Yoshida, S. (1991). Molecular defect in familial lecithin: cholesterol acyltransferase (LCAT) deficiency: a single nucleotide insertion in LCAT gene causes a complete deficient type of the disease. Biochemical & Biophysical Research Communications , 181 , 933–40. Carlson, L.A. (1982). Fish eye disease: a new familial condition with massive corneal opacities and dyslipoproteinaemia. European Journal of Clinical Investigation , 12 , 41–53. Carlson, L.A. and Holmquist, L. (1985). Evidence for deficiency of high density lipoprotein lecithin: cholesterol acyltransferase activity (alpha-LCAT) in fish eye disease . Acta Medica Scandinavica , 218 , 189–96. Carlson, L.A. and Philipson, B. (1979). Fish-eye disease. A new familial condition with massive corneal opacities and dyslipoproteinaemia. Lancet , 2 , 922–4. Chen, C., Applegate, K., King, W.C., Glomset, J.A., Norum, K.R. and Gjone, E. (1984). A study of the small spherical high density lipoproteins of patients afflicted with familial lecithin: cholesterol acyltransferase deficiency. Journal of Lipid Research , 25 , 269–82. Chevet, D., Ramee, M.P., Le Pogamp, P., Thomas, R., Garre, M. and Alcindor, L.G. (1978). Hereditary lecithin cholesterol acyltransferase deficiency. Report of a new family with two afflicted sisters. Nephron , 20 , 212–9. Chu, F.C., Kuwabara, T., Cogan, D.G., Sceafer, E.J. and Brewer, H.P.J. (1979). Ocular manifestations of familial high-density lipoprotein deficiency (Tangier disease). Archives of Ophthalmology , 97 , 1926–8. Clerc, M., Dumon, M.F., Sess, D., Freneix-Clerc, M., Mackness, M. and Conri, C. (1991). A “Fish-eye disease” familial condition with massive corneal opacities and hypoalphalipoproteinaemia: clinical, biochemical and genetic features. European Journal of Clinical Investigation , 21 , 616–24. Contacos, C., Sullivan, D.R., Rye, K.A., Funke, H. and Assmann, G. (1996). A new molecular defect in the lecithin: cholesterol acyltransferase (LCAT) gene associated with fish eye disease. Journal of Lipid Research , 37 , 35–44. Duverger, N., Klein, H.G., Luc, G., Fruchart, L.C., Albers, J.J. and Brewer, H.B. Jr. (1993). Identification of a novel mutation in the LCAT gene resulting in fish eye disease with alpha-LCAT activity Circulation , 88 , I-423 #2274 (Abstract). Forte, T.M. and Carlson, L.A. (1984). Electron microscopic structure of serum lipoproteins from patients with fish eye disease. Arteriosclerosis , 4 , 130–7. Francone, O.L., Evangelista, L. and Fielding, C.J. (1993). Lecithincholesterol acyltransferase: effects of mutagenesis at N-linked oligosaccharide attachment sites on acyl acceptor specificity. Biochimica et Biophysica Acta , 1166 , 301–4. Francone, O.L., Gong, E.L., Ng, D.S., Fielding, C.J. and Rubin, E.M. (1995). Expression of human lecithin-cholesterol acyltransferase in
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183 , 107–12 MH—Adult MH—Blood Protein Electrophoresis MH— Blood Volume Determination MH—Bone Marrow Examination MH—*Cholesterol/bl Blood]. Gjone, E. and Norum, K.R. (1968b). Familial serum cholesterol ester deficiency. Clinical study of a patient with a new syndrome. Acta Medica Scandinavica , 183 , 107–12. Gjone, E., Skarbovik, A.J., Blomhoff, J.P. and Teisberg, P. (1974b). Familial lecithin: cholesterol acyltransferase deficiency. Report of a third Norwegian family with two afflicted members. Scandinavian Journal of Clinical & Laboratory Investigation—Supplement, 137 , 101–5. Gjone, E., Torsvik, H. and Norum, K.R. (1968). Familial plasma cholesterol ester deficiency. A study of the erythrocytes. Scandinavian Journal of Clinical & Laboratory Investigation , 21 , 327–32. Glomset, J.A., Applegate, K., Forte, T., King, W.C., Mitchell, C.D., Norum, K.R. and Gjone, E. (1980). Abnormalities in lipoproteins of d<1.006 g/ml in familial lecithin: cholesterol acyltransferase deficiency. Journal of Lipid Research , 21 , 1116–27. Glomset, J.A., Assmann, G., Gjone, E. and Norum, K.R. (1989). In The Metabolic and Molecular Bases of Inherited Disease , (ed., Scriver, C.R., A.L.B., Sly, W.S., Standbury, J.B., Wyngaarden, J.B. and Fredrickson, D.S.) New York: McGraw-Hill Inc., pp. 1933–1951. Glomset, J.A., Nichols, A.V., Norum, K.R., King, W. and Forte, T. (1973). Plasma lipoproteins in familial lecithin: cholesterol acyltransferase deficiency. Further studies of very low and low density lipoprotein abnormalities. Journal of Clinical Investigation , 52 , 1078–92. Glomset, J.A. and Norum, K.R. (1973). The metabolic role of lecithin: cholesterol acyltransferase: perspectives form pathology. Advances in Lipid Research , 11 , 1–65. Glomset, J.A., Norum, K.R. and King, W. (1970). Plasma lipoproteins in familial lecithin: cholesterol acyltransferase deficiency: lipid composition and reactivity in vitro . Journal of Clinical Investigation , 49 , 1827–37. Glomset, J.A., Norum, K.R., Nichols, A.V., King, W.C., Mitchell, C.D., Applegate, K.R., Gong, E.L. and Gjone, E. (1975). Plasma lipoproteins in familial lecithin: cholesterol acyltransferase deficiency: effects of dietary manipulation. Scandinavian Journal of Clinical & Laboratory Investigation—Supplement , 142 , 3–30. Godin, D.V., Gray, G.R. and Frohlich, J. (1978). Erythrocyte membrane alterations in lecithin: cholesterol acyltransferase deficiency. Scandinavian Journal of Clinical & Laboratory Investigation—Supplement , 150 , 162–7. Gotoda, T., Yamada, N., Murase, T., Sakuma, M., Murayama, N., Shimano, H., Kozaki, K., Albers, J.J., Yazaki, Y. and Akanuma, Y. (1991). Differential phenotypic expression by three mutant alleles in familial lecithin: cholesterol acyltransferase deficiency (see comments) Lancet , 338 , 778–81.
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Guerin, M., Dachet, C, Goulinet, S., Chevet, D., Dolphin, P.J., Chapman, M.J. and Rouis, M. (1997). Familial lecithin: cholesterol acyltransferase deficiency: molecular analysis of a compound heterozygote: LCAT (Arg 147 Trp) and LCAT (Tyr 171 Stop). Atherosclerosis , 131 , 85–95. Hamilton, R.L., Havel, R.J., Kane, J.P., Blaurock, A.E. and Sata, T. (1971). Cholestasis: lamellar structure of the abnormal human serum lipoprotein. Science , 172 , 475–8. Hill, J. (1994). In Department of Pathology and Laboratory Medicine . University of British Columbia, Vancouver, p. 145. Hill, J.S., O, K., Wang, X., Paranjape, S., Dimitrijevich, D., Lacko, A.G. and Pritchard, P.H. (1993a). Expression and characterization of recombinant human lecithin: cholesterol acyltransferase. Journal of Lipid Research , 34 , 1245–51. Hill, J.S., O, K., Wang, X. and Pritchard, P.H. (1993b). Lecithin: cholesterol acyltransferase deficiency: identification of a causative gene mutation and a co-inherited protein polymorphism. Biochimica et Biophysica Acta , 1181 , 321–3. Hoeg, J.M., Santamarina-Fojo, S., Berard, A.M., Cornhill, J.F., Herderick, E.E., Feldman, S.H., Haudenschild, C.C., Vaisman, B.L., Hoyt, R.F., Jr., Demosky, S.J., Jr., Kauffman, R.D., Hazel, C.M., Marcovina, S.M. and Brewer, H.B., Jr. (1996a). Overexpression of lecithin: cholesterol acyltransferase in transgenic rabbits prevents diet-induced atherosclerosis. Proceedings of the National Academy of Sciences of the United States of America , 93 , 11448–53. Hoeg, J.M., Vaisman, B.L., Demosky, S.J., Jr., Meyn, S.M., Talley, G.D., Hoyt, R.F., Jr., Feldman, S., Berard, A.M., Sakai, N., Wood, D., Brousseau, M.E., Marcovina, S., Brewer, H.B., Jr. and Santamarina-Fojo, S. (1996b). Lecithin: cholesterol acyltransferase overexpression generates hyperalpha-lipoproteinemia and a nonatherogenic lipoprotein pattern in transgenic rabbits. Journal of Biological Chemistry , 271 , 4396–402. Hovig, T. and Gjone, E. (1973a). Familial plasma lecithin: cholesterol acyltransferase (LCAT) deficiency. Ultrastructural aspects of a new syndrome with particular reference to lesions in the kidneys and the spleen. Acta Pathologica et Microbiologica Scandinavica—Section A, Pathology , 81 , 681–97. Hovig, T. and Gjone, E. (1973b). Ultrastructural aspects of familial lecithin-cholesterol acyltransferase deficiency. Nutrition & Metabolism , 15 , 89–96. Imbasciati, E., Paties, C., Scarpioni, L. and Mihatsch, M.J. (1986). Renal lesions in familial lecithin-cholesterol acyltransferase deficiency. Ultrastructural heterogeneity of glomerular changes. American Journal of Nephrology , 6 , 66–70. Jacobsen, C.D., Gjone, E. and Hovig, T. (1972). Sea-blue histiocytes in familial lecithin: cholesterol acyltransferase deficiency. Scandinavian Journal of Haematology , 9 , 106–13. Jonas, A. (1991). Lecithin-cholesterol acyltransferase in the metabolism of high-density lipoproteins Biochimica et Biophysica
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Acta , 1084 , 205–20. Kastelein, J.J., Pritchard, P.H., Erkelens, D.W., Kuivenhoven, J.A., Albers, J.J. and Frohlich, J.J. (1992). Familial high-densitylipoprotein deficiency causing corneal opacities (fish eye disease) in a family of Dutch descent. Journal of Internal Medicine , 231 , 413– 9. Klein, H.G., Duverger, N., Albers, J.J., Marcovina, S., Brewer, H.B., Jr. and Santamarina-Fojo, S. (1995). In vitro expression of structural defects in the lecithin-cholesterol acyltransferase gene. Journal of Biological Chemistry , 270 , 9443–7. Klein, H.G., Lohse, P., Duverger, N., Albers, J.J., Rader, D.J., Zech, L.A. Santamarina-Fojo, S. and Brewer, H.B., Jr. (1993a). Two different allelic mutations in the lecithin: cholesterol acyltransferase (LCAT) gene resulting in classic LCAT deficiency: LCAT (tyr 83 stop) and LCAT (tyr 156 asn) Journal of Lipid Research , 34 , 49–58. Klein, H.G., Lohse, P., Pritchard, P.H., Bojanovski, D., Schmidt, H. and Brewer, H.B., Jr. (1992). Two different allelic mutations in the lecithin-cholesterol acyltransferase gene associated with the fish eye syndrome. Lecithin-cholesterol acyltransferase (Thr123- - -Ile) and lecithin-cholesterol acyltransferase (Thr347- - -Met). Journal of Clinical Investigation , 89 , 499–506. Klein, H.G., Santamarina-Fojo, S., Duverger, N., Clerc, M., Dumon, M.F., Albers, J.J., Marcovina, S. and Brewer, H.B., Jr. (1993b). Fish eye syndrome: a molecular defect in the lecithin-cholesterol acyltransferase (LCAT) gene associated with normal alpha-LCATspecific activity. Implications for classification and prognosis. Journal of Clinical Investigation , 92 , 479–85. Knipping, G., Birchbauer, A., Steyrer, E., Groener, J., Zechner, R. and Kostner, G.M. (1986). Studies on the substrate specificity of human and pig lecithin: cholesterol acyltransferase: role of low-density lipoproteins . Biochemistry , 25 , 5242–9. Kuivenhoven, J.A., Pritchard, H., Hill, J., Frohlich, J., Assmann, G. and Kastelein, J. (1997). The molecular pathology of lecithin: cholesterol acyltransferase (LCAT) deficiency syndromes. Journal of Lipid Research , 38 , 191–205. Kuivenhoven, J.A., Stalenhoef, A.F., Hill, J.S., Demacker, P.N., Errami, A., Kastelein, J.J. and Pritchard, P.H. (1996a). Two novel molecular defects in the LCAT gene are associated with fish eye disease. Arteriosclerosis, Thrombosis & Vascular Biology , 16 , 294– 303. Kuivenhoven, J.A., van Voorst tot Voorst, E.J., Wiebusch, H., Marcovina, S.M., Funke, H., Assmann, G., Pritchard, P.H. and Kastelein, J.J. (1995). A unique genetic and biochemical presentation of fish-eye disease. Journal of Clinical Investigation , 96 , 2783–91. Kuivenhoven, J.A., Weibusch, H., Pritchard, P.H., Funke, H., Benne, R., Assmann, G. and Kastelein, J.J. (1996b). An intronic mutation in a lariat branchpoint sequence is a direct cause of an inherited human disorder (fish-eye disease). Journal of Clinical Investigation , 98 ,
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358–64. Luc, G., L.Elkhalil, Z.Majd, R.Bakir, P.Poulain, B.La-croix, N.Duhal, and J.C.Fruchart (1994). Fish eye dis-ease: structural and metabolic abnormalities of high density lipoproteins. Atherosclerosis , 109 , 121 (Abstract). Maeda, E., Naka, Y., Matozaki, T., Sakuma, M., Akanuma, Y., Yoshino, G. and Kasuga, M. (1991). Lecithin-cholesterol acyltransferase (LCAT) deficiency with a missense mutation in exon 6 of the LCAT gene. Biochemical & Biophysical Research Communications , 178 , 460–6. Magil, A., Chage, W. and Frohlich, J. (1982). Unusual renal biopsy findings in a patient with familial lecithin: cholesterol acyltransferase deficiency. Human Pathology , 13 , 183. McIntyre, N. (1988). Familial LCAT deficiency and fish-eye disease Journal of Inherited Metabolic Disease , 11 , 45–56. McLean, J., Fielding, C, Drayna, D., Dieplinger, H., Baer, B., Kohr, W., Henzel, W. and Lawn, R. (1986a). Cloning and expression of human lecithin-cholesterol acyltransferase cDNA. Proceedings of the National Academy of Sciences of the United States of America , 83 , 2335–9. McLean, J., Wion, K., Drayna, D., Fielding, C. and Lawn, R. (1986b). Human lecithin-cholesterol acyltransferase gene: complete gene sequence and sites of expression Nucleic Acids Research , 14 , 9397– 406. McLean, J.W. (1992). In High Density Lipoproteins and Atherosclerosis III (Ed, Tall, N.E.M. a. A.R.) pp. 59–65, Elsevier Science Publishers B.V., Amsterdam. Mehlum, A., Staels, B., Duverger, N., Tailleux, A., Castro, G., Fievet, C., Luc, G., Fruchart, J.C., Olivecrona, G., Skretting, G. et al. (1995). Tissue-specific expression of the human gene for lecithin: cholesterol acyltransferase in transgenic mice alters blood lipids, lipoproteins and lipases towards a less atherogenic profile. European Journal of Biochemistry , 230 , 567–75. Miettinen, H., Gylling, H., Ulmanen, I., Miettinen, T.A. and Kontula, K. (1995). Two different allelic mutations in a Finnish family with lecithin: cholesterol acyltransferase deficiency Arteriosclerosis, Thrombosis & Vascular Biology , 15 , 460–7. Miettinen, H.E., Gylling, H., Tenhunen, J., Virtamo, J., Jauhiainen, M., Huttunen, J.K., Kantola, I., Miettinen, T.A. and Kontula, K. (1998). Molecular genetic study of Finns with hypoalphalipoproteinemia and hyperalphalipoproteinemia: a novel Gly230 Arg mutation (LCAT [Fin]) of lecithin: cholesterol acyltransferase (LCAT) accounts for 5% of cases with very low serum HDL cholesterol levels Arteriosclerosis, Thrombosis & Vascular Biology , 18 , 591–8. Miller, M., Zeller, K., Kwiterovich, P.C., Albers, J.J. and Feulner, G. (1995). Lecithin: cholesterol acyltransferase deficiency: identification of two defective alleles in fibroblast cDNA. Journal of Lipid Research , 36 , 931–8. Mitchell, C.D., King, W.C., Applegate, K.R., Forte, T., Glomset, J.A.,
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Norum, K.R. and Gjone, E. (1980). Characterization of apolipoprotein E-rich high density lipoproteins in familial lecithin: cholesterol acyltransferase deficiency. Journal of Lipid Research , 21 , 625–34. Moriyama, K., Sasaki, J., Arakawa, F., Takami, N., Maeda, E., Matsunaga, A., Takada, Y., Midorikawa, K., Yanase, T., Yoshino, G. et al. (1995). Two novel point mutations in the lecithin: cholesterol acyltransferase (LCAT) gene resulting in LCAT deficiency: LCAT (G873 deletion) and LCAT (Gly344 Ser). Journal of Lipid Research , 36 , 2329–43. Murano, S., Shirai, K., Saito, Y., Yoshida, S., Ohta, Y., Tsuchida, H., Yamamoto, S., Asano, G., Chen, C.H. and Albers, J.J. (1987). Impaired intermediate-density lipoprotein triglyceride hydrolysis in familial lecithin: cholesterol acyltransferase (LCAT) deficiency. Scandinavian Journal of Clinical & Laboratory Investigation , 47 , 775–83. Murayama, N., Asano, Y., Kato, K., Sakamoto, Y., Hosoda, S., Yamada, N., Kodama, T., Murase, T. and Akanuma, Y. (1984). Effects of plasma infusion on plasma lipids, apoproteins and plasma enzyme activities in familial lecithin: cholesterol acyltransferase deficiency European Journal of Clinical Investigation , 14 , 122–9. Ng, D.S., Francone, O.L., Forte, T.M., Zhang, J., Haghpassand, M. and Rubin, E.M. (1997). Disruption of the murine lecithin: cholesterol acyltransferase gene causes impairment of adrenal lipid delivery and up-regulation of scavenger receptor class B type I . Journal of Biological Chemistry , 272 , 15777–81. Nordoy, A. and Gjone, E. (1971). Familial plasma lecithin: cholesterol acyltransferase deficiency. A study of the platelets. Scandinavian Journal of Clinical & Laboratory Investigation , 27 , 263–8. Norum, K.R. and Gjone, E. (1967). Familial serum-cholesterol esterification failure. A new inborn error of metabolism. Biochimica et Biophysica Acta , 144 , 698–700. Norum, K.R. and Gjone, E. (1968). The effect of plasma transfusion on the plasma cholesterol esters in patients with familial plasma lecithin: cholesterol acyltransferase deficiency. Scandinavian Journal of Clinical & Laboratory Investigation , 22 , 339–42. Norum, K.R., Glomset, J.A., Nichols, A.V. and Forte, T. (1971). Plasma lipoproteins in familial lecithin: cholesterol acyltransferase deficiency: physical and chemical studies of low and high density lipoproteins. Journal of Clinical Investigation , 50 , 1131–40. Norum, R.A., Lakier, J.B., Goldstein, S., Angel, A., Goldberg, R.B., Block, W.D., Noffze, D.K., Dolphin, P.J., Edelglass, J., Bogorad, D.D. and Alaupovic, P. (1982). Familial deficiency of apolipoproteins A-I and C-III and precocious coronary-artery disease. New England Journal of Medicine , 306 , 1513–9. O, K., Hill, J.S., Wang, X., McLeod, R. and Pritchard, P.M. (1993a). Lecithin: cholesterol acyltransferase: role of N-linked glycosylation in enzyme function. Biochemical Journal , 294 , 879–84. O, K., Hill, J.S., Wang, X. and Pritchard, P.H. (1993b). Recombinant lecithin: cholesterol acyltransferase containing a Thr 123 Ile
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mutation esterifies cholesterol in low density lipoprotein but not in high density lipoprotein. Journal of Lipid Research , 34 , 81–8. Ohta, T., Nakamura, R., Ikeda, Y., Frohlich, J., Takata, K., Saito, Y., Horiuchi, S. and Matsuda, I. (1994). Evidence for impaired cellular cholesterol removal mediated by apo A-I containing lipoproteins in patients with familial lecithin: cholesterol acyltransferase deficiency. Biochimica et Biophysica Acta , 1213 , 295–301. Ohta, T., Nakamura, R., Ikeda, Y., Shinohara, M., Miyazaki, A., Horiuchi, S. and Matsuda, I. (1992). Differential effect of subspecies of lipoprotein containing apolipoprotein A-I on cholesterol efflux from cholesterol-loaded macrophages: functional correlation with lecithin: cholesterol acyltransferase. Biochimica et Biophysica Acta , 1165 , 119–28. Ohta, Y., Yamamoto, S., Tsuchida, H., Murano, S., Saitoh, Y., Tohjo, S. and Okada, M. (1986). Nephropathy of familial lecithincholesterol acyltransferase deficiency: report of a case. American Journal of Kidney Diseases , 7 , 41–6. Owen, J.S., Wiebusch, H., Cullen, P., Watts, G.F., Lima, V.L., Funke, H. and Assmann, G. (1996). Complete deficiency of plasma lecithincholesterol acyltransferase (LCAT) activity due to a novel homozygous mutation (Gly-30-Ser) in the LCAT gene. Human Mutation , 8 , 79–82. Parra, H.J., Arveiler, D., Evans, A.E., Cambou, J.P., Amouyel, P., Bingham, A., McMaster, D., Schaffer, P., Douste-Blazy, P., Luc, G. et al. (1992). A case-control study of lipoprotein particles in two populations at contrasting risk for coronary heart disease. The ECTIM Study Arteriosclerosis & Thrombosis , 12 , 701–7. Pritchard, P.H. (1997). In Hand Book of Lipoprotein Testing , Vol. 1 (ed. Rifai N., G.R.W., Dominiczak, M.H.) Washington: AACC Press, pp. 401–14. Puchois, P., Ghalim, N., Zylberberg, G., Fievet, P., Demarquilly, C. and Fruchart, J.C. (1990). Effect of alcohol intake on human apolipoprotein A-I-containing lipoprotein subfractions. Archives of Internal Medicine , 150 , 1638–41. Qu, S.J., Fan, H.Z., Blanco-Vaca, F. and Pownall, H.J. (1993). Effects of site-directed mutagenesis on the N-glycosylation sites of human lecithin: cholesterol acyltransferase. Biochemistry , 32 , 8732–6. Qu, S.J., Fan, H.Z., Blanco-Vaca, F. and Pownall, H.J. (1995). In vitro expression of natural mutants of human lecithin: cholesterol acyltransferase. Journal of Lipid Research , 36 , 967–74. Rader, D.J., Ikewaki, K., Duverger, N., Schmidt, H., Pritchard, H., Frohlich, J., Clerc, M., Dumon, M.F., Fairwell, T., Zech, L. et al. (1994). Markedly accelerated catabolism of apolipoprotein A-II (ApoA-II) and high density lipoproteins containing ApoA-II in classic lecithin: cholesterol acyltransferase deficiency and fish-eye disease. Journal of Clinical Investigation , 93 , 321–30. Ritland, S. and Gjone, E. (1975). Quantitative studies of lipoprotein-X in familial lecithin: cholesterol acyltransferase deficiency and during cholesterol esterification. Clinica Chimica Acta , 59 , 109–19.
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Sakai, N., Vaisman, B.L., Koch, C.A., Hoyt, R.F. Jr., Meyn, S.M., Talley, G.D., Paiz, J.A., Brewer, H.B., Jr. and Santamarina-Fojo, S. (1997). Targeted disruption of the mouse lecithin: cholesterol acyltransferase (LCAT) gene. Generation of a new animal model for human LCAT deficiency. Journal of Biological Chemistry , 272 , 7506–10. Sakuma, M., Akanuma, Y., Kodama, T., Yamada, N., Murata, S., Murase, T., Itakura, H. and Kosaka, K. (1982). Familial plasma lecithin: cholesterol acyltransferase deficiency. A new family with partial LCAT activity. Acta Medica Scandinavica , 212 , 225–32. Skretting, G., Blomhoff, J.P., Solheim, J. and Prydz, H. (1992). The genetic defect of the original Norwegian lecithin: cholesterol acyltransferase deficiency families FEBS Letters , 309 , 307–10. Skretting, G. and Prydz, H. (1992). An amino acid exchange in exon I of the human lecithin: cholesterol acyltransferase (LCAT) gene is associated with fish eye disease (published erratum appears in Biochem Biophys Res Commun 1992 Apr 15; 184(1):549). Biochemical & Biophysical Research Communications , 182 , 583–7. Soutar, A.K., Knight, B.L. and Myant, N.B. (1982). The characterization of lipoproteins in the high density fraction obtained from patients with familial lecithin: cholesterol acyltransferase deficiency and their interaction with cultured human fibroblasts. Journal of Lipid Research , 23 , 380–90. Stampfer, M.J., Sacks, P.M., Salvini, S., Willett, W.C. and Hennekens, C.H. (1991). A prospective study of cholesterol, apolipoproteins, and the risk of myocardial infarction (see comments). New England Journal of Medicine , 325 , 373–81. Steyrer, E., Haubenwallner, S., Horl, G., Giessauf, W., Kostner, G.M. and Zechner, R. (1995). A single G to A nucleotide transition in exon IV of the lecithin: cholesterol acyltransferase (LCAT) gene results in an Arg140 to His substitution and causes LCAT-deficiency. Human Genetics , 96 , 105–9. Stokke, K.T., Bjerve, K.S., Blomhoff, J.P., Oystese, B., Flatmark, A., Norum, K.R. and Gjone, E. (1974). Familial lecithin: cholesterol acyltransferase deficiency. Studies on lipid composition and morphology of tissues. Scandinavian Journal of Clinical & Laboratory Investigation—Supplement , 137 , 93–100. Taramelli, R., Pontoglio, M., Candiani, G., Ottolenghi, S., Dieplinger, H., Catapano, A., Albers, J., Vergani, C. and McLean, J. (1990). Lecithin cholesterol acyl transferase deficiency: molecular analysis of a mutated allele. Human Genetics , 85 , 195–9. Teisberg, P., Gjone, E. and Olaisen, B. (1975). Genetics of LCAT (lecithin: cholesterol acyltransferase) deficiency. Annals of Human Genetics , 38 , 327–31. Torsvik, H. (1972). Studies on the protein moiety of serum high density lipoprotein from patients with familial lecithin: cholesterol acyltransferase deficiency. Clinical Genetics , 3 , 188–200. Torsvik, H., Gjone, E. and Norum, K.R. (1968). Familial plasma cholesterol ester deficiency. Clinical studies of a family. Acta
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13 Apolipoprotein E and Human Disease Yadong Huang 1 and Robert W.Mahley 1 , 2 1
Gladstone Institute of Cardiovascular Disease, Cardiovascular Research Institute, and 2 Departments of Pathology and Medicine, University of California, PO Box 419100 San Francisco, California 94141– 9100
Apolipoprotein (apo) E, a 299-amino acid glycoprotein with a molecular weight of 34,200, has a central role in lipid metabolism and redistribution and thus is involved in both physiological and pathophysiological processes. It is a protein constituent of several plasma lipoproteins, including chylomicrons, chylomicron remnants, very low density lipoproteins (VLDL), intermediate density lipoproteins, and a subclass of high density lipoproteins (HDL). Apolipoprotein E serves as a high-affinity ligand for several hepatic receptors, including the low density lipoprotein (LDL) receptor and the LDL receptor-related protein, and for cellsurface heparan sulfate proteoglycans (HSPG). By interacting with these receptors or with HSPG, apoE mediates the clearance of chylomicrons, VLDL, and their remnants from the circulation. Absence or structural mutations of apoE cause significant disorders in lipid metabolism, such as type III hyperlipoproteinaemia. Apolipoprotein E also has significant functions in the central nervous system. Expression of apoE mRNA in the brain is second only to the liver in abundance, and apoE-containing HDL are major cerebrospinal lipoproteins. Apolipoprotein E takes up lipids released during neuron degeneration and delivers them to cells requiring lipids for proliferation or repair. Furthermore, apoE4, one of the three common alleles of apoE, has been
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implicated as a risk factor for Alzheimer’s disease. Genetic and epidemiologic studies in a variety of populations worldwide have demonstrated that apoE4 is overrepresented in late-onset Alzheimer’s disease subjects compared with age- and sex-matched controls. Thus, apoE may also play an important role in neurobiology via its local lipid transport function and possibly via nonlipid transport functions. KEYWORDS: Type III hyperlipoproteinaemia, Alzheimer’s disease, cholesterol, lipid metabolism, remnant lipoprotein.
GENETICS, STRUCTURE, AND FUNCTION OF APOLIPOPROTEIN E Apolipoprotein E Gene and Sites of Synthesis The apolipoprotein (apo) E gene is located on chromosome 19 (Olaisen et al., 1982; Das et al., 1985), where it is closely linked to the genes for apoC-I, pseudo-apoC-I, apoC-II, and apoC-IV (Jackson et al., 1984; Lauer et al., 1988; Allan et al., 1995). The apoE gene is 3.7kb in length and contains four exons (Das et al., 1985; Paik et al., 1985) that code for an mRNA of 1163 nucleotides (McLeans et al., 1984; Zannis et al., 1984). The primary translation product of apoE mRNA comprises 317 amino acids, with 18 amino acids serving as a signal peptide at its amino terminus. Thus, the mature apoE is secreted as a 299-amino acid protein with a molecular weight of 34,200. Apolipoprotein E is synthesised by different cells in many tissues, including the liver, brain, spleen, lung, adrenal, ovary, kidney, and muscle (for review, see Blue et al., 1983; Elshourbagy et al., 1985; Lin et al., 1986; Mahley 1988). Among them, the liver parenchymal cells synthesise and secrete the majority of plasma apoE. The second largest quantity is found in the brain, where astrocytes provide the major source (Pitas et al., 1987a). Tissue macrophages are likely to account for the major production of apoE in other organs. In addition, macrophages in culture and in atherosclerotic lesions can synthesise and secrete large amounts of apoE (Basu et al., 1981; Basu et al., 1982). The production and secretion of apoE by macrophages is regulated by intracellular cholesterol content (Mazzone et al., 1987; Mazzone et al., 1989; Mazzone and Basheeruddin, 1991). Polymorphism of Apolipoprotein E and Its Impact on Lipid Metabolism The three common isoforms of the polymorphic apoE, apoE2, apoE3,
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and apoE4 (Figure 13.1), can be separated by isoelectric focusing electrophoresis (Utermann et al., 1980; Zannis and Breslow, 1981; Utermann et al., 1982). The three major apoE isoforms are products of the same gene that exists as three alleles (ε2, ε3, and ε4) at a single gene locus (Zannis et al., 1981). Therefore, three homozygous phenotypes (apoE2/2, E3/3, and E4/4) and three heterozygous phenotypes (apoE3/2, E4/3, and E4/2) are created from the expression of any two of the three alleles. Studies of apoE phenotypes and ε allelic frequencies among various populations around the world
Allelic frequency:
15%
77%
8%
Arg112 Arg 158
Cys112 Arg 158
Cys112 Cys 15
Receptor binding:
100%
100%
2%
Lipoprotein preference:
VLDL
HDL
HDL
Sequence differences:
Associated disorder:
Alzheimer’s Disease “Normal” Type III Hyperlipo
Figure 13.1 Apolipoprotein E isoforms and their properties. E2, apoE2; E3, apoE3; E4, apoE4.
have demonstrated that the apoE3/3 phenotype is the most common (typically ~ 50–70% of the population) and the ε3 allele accounts for ~ 70–80% of the apoE gene pool (Figure 13.1). The ε4 and ε2 alleles account for only ~ 10–15% and ~ 5–10%, respectively, of the apoE gene pool, making the apoE4/4, E4/2, and E2/2 phenotypes relatively rare in most populations (for review, see Mahley and Rall, 1995). The molecular basis for apoE polymorphism has been elucidated by analysis of the amino acid sequences of the three isoforms (Weisgraber et al., 1981; Rall et al., 1982a; Rall et al., 1982b), which differ from each other by single amino acid substitutions at two sites in the protein, residues 112 and 158 (Figure 13.1). The apoE3 isoform has cysteine at residue 112 and arginine at residue 158. The apoE4 isoform differs from apoE3 only at residue 112, where arginine is substituted for the normally occurring cysteine. The apoE2 isoform differs from apoE3 only at residue 158, where cysteine is substituted for the normally occurring arginine. Apolipoproteins E3 and E4 bind normally to the low density lipoprotein (LDL) receptor, while apoE2 is very defective in receptor binding (Figure 13.1) (for review, see Mahley, 1988; Mahley and Rall, 1995). Apolipoprotein E polymorphism is one of the common genetic factors responsible for interindividual differences in plasma lipid and lipoprotein levels in humans (for review, see Utermann, 1985;
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Davignon et al., 1988; Dallongeville et al., 1992; Mahley and Rall, 1995). The ε2 allele is associated with decreased levels of plasma total cholesterol and LDL cholesterol and with higher levels of plasma triglycerides (Figure 13.2) (Utermann et al., 1979a; Robertson and Cumming, 1985; Sing and Davignon, 1985) The effect of apoE2 on plasma lipid levels has been confirmed in apoE2 transgenic mice on wild-type, mouse apoE-null, or LDL receptor-null
Figure 13.2 The effect of apoE alleles on various lipid and lipoprotein parameters. HDL-C, high density lipoprotein cholesterol; LDL-B, low density lipoprotein apoB; LDL-C, LDL cholesterol; TC, total cholesterol; TG, triglycerides; VLDL-C, very low density lipoprotein cholesterol. , ε2; , ε3; , ε4. (Adapted from Sing, C.F. and Davignon, J. (1985) Role of the apolipoprotein E polymorphism in determining normal plasma lipid and lipoprotein variation. Am. J.Hum. Genet., 37, 268–285 and published by permission.)
background, in which moderate expression of apoE2 led to decreased total and LDL cholesterol levels but increased triglyceride levels (Huang et al., 1998; Huang et al., 1996; Huang et al., 1997a). On the other hand, the ε4 allele is associated with increased levels of plasma total cholesterol and LDL cholesterol (Utermann et al., 1979a; Robertson and Cumming, 1985; Sing and Davignon, 1985). Furthermore, the ε2 allele is also associated with increased apoE and decreased apoB plasma levels, whereas the ε4 allele has the opposite effect (Utermann, 1985). Interestingly, it also has been suggested that ε4 is associated with a higher incidence of coronary heart disease (for
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review, see Cumming and Robertson, 1984; Davignon et al., 1988; Kuusi et al., 1989; Eichner et al., 1993; Luc et al., 1994; Mahley and Rall, 1995; Stengård et al., 1996), probably due either to higher plasma cholesterol and LDL cholesterol levels or to potentially different roles of apoE isoforms within the atherosclerotic lesion (Cumming and Robertson, 1984; Mahley, 1988; Kuusi et al., 1989; Hixson and the Pathobiological Determinants of Atherosclerosis in Youth (PDAY) Research Group, 1991). In contrast, the presence of the ε2 allele appears to reduce the risk for coronary heart disease, probably due to lower plasma and LDL cholesterol levels, in subjects without type III hyperlipoproteinaemia (HLP) (for review, see Davignon et al., 1988; Eichner et al., 1993; Luc et al., 1994; Mahley and Rall, 1995). In fact, those apoE2 homozygous subjects that develop type III HLP are at increased risk for both coronary and peripheral artery disease (for review, see Mahley and Rall, 1995). Relationship of Structure and Function in Apolipoprotein E Biochemical and crystallographic studies have established that apoE contains two structural domains that differ in function—an aminoterminal (residues ~ 1–165) domain and a carboxyl-terminal (residues ~ 200–299) domain—that are connected by a region of random structure (residues 165–200) (for review, see Aggerbeck et al., 1988; Wetterau et al., 1988; Weisgraber, 1994; Mahley and Rall, 1995). The carboxylterminal domain with its strong amphipathic -helical region includes the major lipid-binding region. The amino-terminal domain, occurring as a four-helix bundle, contains the region responsible for interaction with the LDL receptor, the LDL receptor-related protein (LRP), and heparan sulfate proteoglycans (HSPG). By using several different experimental approaches, the receptorbinding domain of apoE has been mapped to residues 136–150, a region enriched in basic amino acids (six arginines, two lysines, and one histidine) (for review, see Mahley, 1988; Mahley et al., 1990; Weisgraber, 1994; Mahley and Rall, 1995). The crystal structure of apoE reveals that these residues form a 20-A region of positively charged amino acids on the surface of helix 4 and appear to interact directly with the acidic amino acid residues in the ligand-binding domain of the LDL receptor (Wilson et al., 1991). The LDL receptor possesses seven cysteine-enriched repeated segments at its amino terminus that contain the critical acidic amino acids (aspartate and glutamate) responsible for ligand binding (for review, see Brown et al., 1991). The LRP, a member of the LDL receptor gene family, contains 31 domains homologous to the ligand-binding region of the LDL receptor and thus binds multiple apoE molecules (for review, see
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Brown et al., 1991; Mahley, 1996). The binding of apoE to heparin or to HSPG is also mediated by an ionic interaction between the key basic amino acid residues in the 136–150 region of apoE and the negatively charged sulfated glycosaminoglycan side chains on the HSPG molecule. The importance of the basic amino acids in the 136–150 region of apoE for receptor binding is highlighted by screening the naturally occurring variants of apoE and by site-directed mutagenesis (for review, see Lalazar et al., 1988; Mahley, 1988; Lalazar et al., 1989; Weisgraber, 1994; Mahley and Rall, 1995). Substitution of neutral amino acids for the basic residues results in defective receptor binding (Table 13.1) and defective HSPG binding. Apolipoprotein E2 is one exception. With cysteine substituted for arginine at residue 158 outside the receptor-binding region, it has the most defective receptor-binding activity, but nearly normal HSPG-binding activity. This substitution affects receptor binding secondarily by altering the conformation of the 136–150 region as demonstrated by both biochemical studies and elucidation of the crystal structure of apoE3 and apoE2. A second exception is apoE3-Leiden, which has a seven-amino acid insertion that represents a tandem repeat of residues 121–127 (Wardell et al., 1989). Although residues 121–127 lie outside the receptor-binding region, this mutation leads to defective receptor binding (25% of apoE3) (Wardell et al., 1989). The apoE3-Leiden mutation may affect receptor binding secondarily by altering the conformation of the 136–150 region (Wardell et al., 1989). However, unlike apoE2, apoE3-Leiden is also defective in HSPG binding (Ji et al., 1994b). The secondary versus the direct effect of these apoE variants on receptor binding, together with their different effects on HSPG binding, could have significant impact on the mode of inheritance of type III HLP (see below). The structural properties of apoE are also an important determinant of its distribution in the various plasma lipoproteins (Figure 13.1) (for review, see Weisgraber, 1994; Mahley and Rall, 1995). Apolipoproteins E2 and E3 preferentially bind to high density lipoproteins (HDL), while apoE4 is preferentially associated with large, triglyceride-rich very low density lipoproteins (VLDL). The preference of apoE4 for VLDL is determined by the interaction of the aminoterminal and carboxyl-terminal domains (Weisgraber, 1990; Wilson et al., 1991; Dong et al., 1994; Dong and Weisgraber, 1996). The arginine-112 of apoE4—cysteine occurs in both apoE2 and apoE3— likely displaces arginine-61 from its position in apoE2 and apoE3, allowing the formation of a salt bridge between arginine-61 and glutamic acid-255. The resulting conformational change causes apoE4 to be preferentially associated with the larger, triglyceride-rich VLDL. Interestingly, only human apoE has arginine at residue 61, whereas the other nine species in which the apoE gene has been sequenced all have
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threonine at this position (Weisgraber, 1994). The lipoprotein preference of the apoE isoforms has a significant impact on the expression of type III HLP (see below).
Table 13.1 Naturally occurring apoE variants associated with type III hyperlipoproteinaemia
Mutation a
Mode of LDL Heparin- VLDL inheritance receptor binding preference binding defect Reference b (s)
Arg158 Cys Recessive (apoE2)
2%
No
No
Weisgraber et al., 1982; Ji et al., 1994b
Lys146
Gln Dominant
40%
Yesc
–
Rall et al., 1983
Lys 146
Glu Dominant
<5%
Yes
–
Mann et al., 1989
Arg145
Cys Dominant
45%
Yes
–
Rall et al., 1982a
Arg145
Cys Dominant
20%
Yes
Yes
Rall et al., 1989
40%
–
–
Wardell et al., 1987
–
–
–
Feussner et al., 1994
25%
Yes
Yes
Wardell et al., 1989
Arg136 Ser Arg136
Dominant
Cys Dominant
7-amino acid Dominant insertion of residues 121– 127
a Changes compared to apoE3 sequence. b Percentage of apoE3-binding activity. c Ji, Z.S.
and Mahley, R.W., unpublished observation.
Role of Apolipoprotein E in Remnant Lipoprotein Metabolism Apolipoprotein E plays a key role in plasma lipoprotein catabolism, especially for chylomicron and VLDL remnants (Figure 13.3) (for review, see Mahley, 1988). Synthesised in the intestine, chylomicrons transport dietary lipids to peripheral tissues and to the liver (for review,
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see Havel et al., 1980a; Mahley, 1988; Mahley and Hussain, 1991). These triglyceride-rich lipoproteins contain primarily apoB48, apoA-I, and apoA-IV and essentially no apoE. Upon entering the circulation, the core triglycerides in these particles are hydrolysed, primarily by endothelial cell-bound lipoprotein lipase (LPL) and to a lesser extent by hepatic lipase in the liver. During the lipolytic process, they gradually loose apoA-I and apoA-IV and receive apoE from other lipoproteins and are finally converted to cholesterol- and apoE-enriched chylomicron remnants. The chylomicron remnants are then rapidly cleared from the circulation by the liver, primarily via an apoEmediated process. The liver-derived VLDL, also triglyceride-rich lipoproteins, undergo lipolytic processing similar to that of chylomicrons (for review, see Havel et al., 1980a). However, the lipolytic cascade of VLDL generates an intermediate product, intermediate density lipoproteins (IDL), and an end product, LDL, in a precursor-
Figure 13.3 General scheme showing the role of apoE in plasma lipoprotein metabolism. Apolipoprotein E mediates the clearance of chylomicron remnants, VLDL, and their remnants from the plasma.
product manner. During the processing of VLDL to IDL, the particles become cholesterol- and apoE-enriched, representing remnants of the VLDL. After further processing, the IDL are depleted in apoE and other apolipoproteins, and finally become LDL, which contain only apoB100. The catabolism of particles along this cascade is complex: a portion of the particles generated at any stage of the cascade can be removed from circulation by a receptor-mediated process, whereas others (~ 50%) proceed completely through the cascade to become LDL. Again, apoE serves as the ligand for the clearance of the remnants generated in this cascade.
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The key role of apoE in remnant lipoprotein catabolism has been established by numerous in vitro and in vivo studies (for review, see Mahley, 1988; Brown et al., 1991; Mahley and Hussain, 1991; Herz and Willnow, 1995). An initial study indicated that the intravenous infusion of apoE markedly reduces plasma lipid levels in cholesterolfed rabbits, which have severe hyperlipidaemia and an accumulation of remnant lipoproteins (called -VLDL) (Mahley et al., 1989). Second, the addition of excess apoE to chylomicrons markedly stimulates their clearance from the plasma of normal rabbits, specifically by the liver (Hussain et al., 1989a; Hussain et al., 1989b). Third, patients with defective or absent apoE develop severe hyperlipidaemia with a marked accumulation of remnant lipoproteins ( -VLDL) in plasma (for review, see Schaefer et al., 1986; Mabuchi et al., 1989; Mahley and Rall, 1995). Fourth, apoE knockout mice display severe hypercholesterolaemia characterised by remnant accumulation (Plump et al., 1992; Zhang et al., 1992). Finally, both transgenic mice and rabbits expressing high levels of defective apoE, such as apoE(Arg142 Cys), apoE-Leiden, or apoE2(Arg158 Cys), also have hyperlipidaemia associated with increased remnant lipoproteins (Fazio et al., 1993; van den Maagdenberg et al., 1993; Huang et al., 1996; Huang et al., 1997b). Thus, apoE is critical for mediating remnant lipoprotein clearance. Several steps may be involved in the plasma clearance and uptake of remnant lipoproteins by the liver (Figure 13.4) (for review, see Mahley and Hussain, 1991; Mahley and Rall, 1995; Mahley, 1996). The first step appears to be sequestration of remnant lipoproteins within the space of Disse, where apoE accumulates after it is secreted by hepatocytes. When remnant lipoproteins enter the space of Disse, the particles can acquire apoE and become apoE-enriched. The apoEenriched remnants appear to bind to HSPG (Ji et al., 1993; Ji et al., 1994a; Ji et al., 1995), which are also abundant in the space of Disse (Stow et al., 1985). This step probably accounts for the rapid initial clearance of remnant lipoproteins from the plasma. A second step may involve further processing of the remnant lipoproteins by lipases in the space of Disse. Both hepatic lipase (Borensztajn et al., 1988; Diard et al., 1994; Ji et al., 1994e; Shafi et al., 1994; Ji et al., 1997a), a heparinbinding protein produced in the liver, and LPL (Beisiegel et al., 1991; Chappell et al., 1993), which can attach to the remnant lipoproteins and be transferred into the liver from peripheral endothelial cells, have been suggested to process remnant lipoproteins. In addition, in vitro studies have shown that both hepatic lipase and LPL can act as ligands for mediating the binding of remnant lipoproteins to cell-surface HSPG (Eisenberg et al., 1992; Ji et al., 1994e; Ji et al., 1997a). Finally, the LDL receptor and the LRP appear to mediate the uptake of remnant lipoproteins by hepatocytes (Beisiegel et al., 1989; Herz et al., 1990;
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Choi et al., 1991; Hussain et al., 1991; Willnow et al., 1994). In vitro
Figure 13.4 A three-step model of apoE-mediated clearance of remnant lipoproteins by the liver. The first step is sequestration of apoE-enriched remnants by binding to HSPG in the space of Disse; the second step is further processing of the remnant particles by hepatic lipase and lipoprotein lipase; the third step is uptake of the remnants mediated by both the LDL receptor and the LRP. (Modified from Mahley, R.W. and Rall, S.C., Jr. (1995) Type III hyperlipoproteinemia (dysbetalipoproteinemia): The role of apolipoprotein E in normal and abnormal lipoprotein metabolism. In The Metabolic and Molecular Bases of Inherited Disease, edited by Scriver, C.R., Beaudet, A.L., Sly, W.S. and Valle, D. 7th edn, pp. 1953–1980. New York: McGraw-Hill, with permission.) E, apoE.
studies suggest that HSPG-bound remnants may be transferred to the LRP for internalisation or that HSPG and the LRP may form a complex that is internalised with the remnants (Ji et al., 1993; Ji et al., 1994a; Ji et al., 1994b). Furthermore, HSPG alone may serve as a receptor mediating remnant lipoprotein uptake by cells (Ji et al., 1997a; Ji et al., 1997b). These results are consistent with other studies showing that
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HSPG mediates a slow uptake of lipoproteins (Williams and Fuki, 1997). Since the clearance of remnant lipoproteins from the plasma involves multiple steps, disruption at any point in the cascade would impair clearance and cause the accumulation of remnants in the plasma.
APOLIPOPROTEIN E AND TYPE III HYPERLIPOPROTEINAEMIA Phenotype and Pathogenesis Type III HLP is a genetic disorder of lipid metabolism in humans, occurring with a frequency of about 1–5 per 5000 in the general population (Utermann, 1985). It is characterised by both hypercholesterolaemia and hypertriglyceridaemia and predisposes the affected subjects to premature development of atherosclerosis. The hyperlipidaemia is caused by the accumulation in plasma of abnormal lipoproteins, namely -VLDL, which are a biochemical hallmark of type III HLP. The -VLDL are cholesterol-enriched remnant lipoproteins generated by lipolytic processing of triglyceride-rich lipoproteins from both the intestine and liver (for review, see Mahley and Rall, 1995). Type III HLP can be caused either by the expression of any of several receptor binding-defective variants of apoE (for review, see Mahley, 1988; Mahley and Rall, 1995) or by apoE deficiency (Ghiselli et al., 1981; Schaefer et al., 1986; Mabuchi et al., 1989; Kurosaka et al., 1991). As described above, apoE normally functions as a ligand mediating remnant uptake by the lipoprotein receptors, mainly in the liver (Mahley, 1988; Mahley and Hussain, 1991). In type III HLP patients, defective apoE causes impaired receptor-mediated remnant lipoprotein catabolism that leads to accumulation of -VLDL in the plasma. Type III HLP can be transmitted in either a recessive or a dominant mode, depending on the specific mutation of apoE (for review, see Rall and Mahley, 1992; Mahley and Rall, 1995). As described above, apoE has three main functions in modulating remnant lipoprotein catabolism—LDL receptor binding, HSPG binding, and lipoprotein preference. Thus, the occurrence of a particular mode of inheritance of type III HLP results from the changes in one or more of the properties that result from the specific apoE mutations. Recessive Mode of Inheritance Most commonly, type III HLP occurs in homozygous carriers of apoE2, which has cysteine at both residues 112 and 158 and which binds defectively to the LDL receptor (<2% of normal apoE3-binding
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activity) (for review, see Mahley, 1988; Mahley and Rall, 1995). Because virtually no individuals heterozygous for apoE2 have type III HLP or high levels of -VLDL (Utermann et al., 1977), the disorder appears to be recessive. Furthermore, although 1 % of the population is homozygous for apoE2 and although all apoE2 homozygotes have VLDL (primary dysbetalipoproteinaemia) (Utermann et al., 1979b), only about 1–10% of these individuals ever develop overt hyperlipidaemia (type III HLP). This extremely low degree of penetrance for type III HLP indicates that other genetic or environmental factors are necessary for expression of the hyperlipidaemia (Utermann, 1985). In fact, most subjects homozygous for apoE2 have hypocholesterolaemia due to low levels of LDL cholesterol (Davignon et al., 1988) and, to a lesser extent, low levels of HDL cholesterol (Havel et al., 1980b; Zhao et al., 1994). Thus, homozygosity for apoE2 seems to sensitise these individuals to secondary factors that disrupt remnant lipoprotein metabolism. Recently, a transgenic mouse model displaying lipid changes similar to those seen in typical recessive type III HLP has been established, in which both hypo- and hyperlipidaemia developed, depending on the expression levels of apoE2 (Huang et al., 1996). The hypolipidaemic apoE2 mice provide the opportunity to study genetic and environmental factors that may precipitate or exacerbate type III HLP. Factors responsible for normal or lowcholesterol levels in most apoE2 homozygotes Several possibilities have been suggested to explain the discrepancy between the severe LDL receptor-binding defect of apoE2 and its association with normal or even lowcholesterol levels in most apoE2 homozygotes. First, although apoE2 shows defective binding to the LDL receptor in in vitro assays, the binding activity of this variant can be modulated from very poor to almost normal under a variety of conditions, including altered lipid composition of the lipoprotein particles in response to dietary changes (Innerarity et al., 1984; Innerarity et al., 1986). The variability in LDL receptor-binding activity of apoE2 may be explained by the fact that this variant appears to disrupt receptor binding secondarily by altering the conformation of the 136–150 region of apoE. Therefore, this variant may not always display a severe receptor-binding defect in vivo. Second, although apoE2 binds defectively to the LDL receptor, it possesses significant binding activity for cell-surface HSPG (~ 50–80% of normal apoE3). In fact, it has greater binding affinity for HSPG than all other apoE variants that have been studied to date (Ji et al., 1993; Ji et al., 1994b). As described above, interaction of apoE with cell-surface HSPG involves an important initial step of remnant clearance (sequestration).
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Thus, the retention of near normal HSPG-binding affinity of apoE2 might keep remnant clearance at a relatively normal level since, after sequestration and processing, remnant particles can be internalised by both the LDL receptor and the LRP. Apolipoprotein E2 also possesses significant binding activity to the LRP. A third factor is probably the expression level of apoE2 in plasma. Recently, a dose-dependent effect of apoE2 on plasma cholesterol and triglyceride levels has been demonstrated in apoE2 transgenic mice lacking endogenous mouse apoE (Figure 13.5). Plasma cholesterol levels in these mice decreased gradually with an increase in apoE2 ranging from ~ 4–25 mg/dl. At an apoE2 concentration of ~ 20 mg/dl, plasma cholesterol levels in these mice were nearly normal, indicating that increasing the amount of apoE2 up to a critical level may compensate for the lower LDL receptor-binding activity of apoE2. Actually, plasma apoE2 levels are always higher than apoE3 and apoE4 in humans (Utermann, 1985). There are several hypotheses to explain the LDL cholesterollowering effect of apoE2 (Davignon et al., 1988; Mahley and Rall, 1995). One hypothesis is that the lower rate of transport of cholesterolrich remnant lipoproteins into the liver due to defective LDL receptor binding of apoE2 leads to an upregulation of hepatic LDL receptors, which in turn causes an accelerated clearance of plasma LDL (for review, see Davignon et al., 1988; Mahley and Rall, 1995). However, some experimental evidence argues against this hypothesis and suggests that the number of hepatic LDL receptors is not increased in either apoE2 transgenic rabbits or in apoE-null mice (Woollett et al., 1995; Huang et al., 1997b). A second hypothesis to explain the LDLlowering effect of apoE2 is that apoE2-containing VLDL compete poorly with apoB-containing LDL for binding to the hepatic LDL receptors, causing a faster clearance of LDL and leading to a decrease in plasma LDL (Woollett et al., 1995). A third hypothesis suggests that apoE2 impairs lipolytic conversion of VLDL to LDL (Chung and Segrest, 1983; Ehnholm et al., 1984) by directly inhibiting LPL activity (Ehnholm et al., 1984). Recently, we have demonstrated that the LDL cholesterol-lowering effect of apoE2 occurred regularly in apoE2 transgenic mice lacking LDL receptors, indicating that low LDL cholesterol does not depend on the upregulation of the LDL receptor. Instead, in vitro lipolytic studies indicate that low LDL cholesterol is caused by the impairment of LPL-mediated lipolysis of triglyceriderich lipoproteins (Huang et al., 1998). Factors precipitating type III hyperlipoproteinaemia in apoE2 homozygotes Several genetic or environmental factors have been suggested to be responsible for precipitating the hyperlipidaemia in normo- or hypo-
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cholesterolaemic apoE2 homozygotes (for review, see Davignon et al., 1988; Mahley, 1988; Mahley and Rall, 1995). One factor that may contribute to remnant accumulation is overproduction of apoBcontaining lipoproteins, which can be caused either by a high-fat diet (Grundy and Denke, 1990) or by the coexistence of separate genes for hyperlipidaemia, such as familial combined hyperlipidaemia (Hazzard et al., 1981). Recently, this hypothesis has been confirmed in human apoB and apoE2 double-transgenic mice, in which overproduction of apoB-containing lipoproteins results in an apoE2-induced accumulation of remnant lipoproteins ( -VLDL) (Huang et al., 1997a). In all likelihood, the increased production of apoB-containing lipoproteins further stresses the catabolic pathway for VLDL, IDL, and remnant lipoprotein catabolism already disturbed by the presence of the defective apoE2. Another secondary factor that has been implicated in the exacerbation of type III HLP is low LDL receptor activity (for review, see Davignon et al., 1988; Mahley and Rall, 1995). The expression of hepatic LDL receptors is rapidly and readily regulated by diet, drugs, and some hormones (Havel et al., 1980a; Angelin et al., 1983; Brown and Goldstein, 1983). Several studies support the hypothesis that expression of type III HLP is influenced by the level of hepatic LDL receptors. First, LDL receptor number decreases with age (for review, see Angelin, 1995), and apoE2-associated type III HLP usually manifests itself during adulthood. Second, in families with heterozygous LDL receptor deficiency, the occurrence of a single apoE2 allele markedly increases the prevalence of type III HLP (Hopkins et al., 1991). Third, diet-induced expression of type III HLP may also be related to LDL receptor expression (for review, see Grundy and Denke, 1990). Increased dietary cholesterol downregulates hepatic LDL receptors (Havel et al., 1980a; Angelin et al., 1983; Brown and Goldstein, 1983). Finally, crossing apoE2 transgenic mice with LDL receptor-null mice has resulted in mice with reduced or no LDL receptors and conversion of the hypolipidaemic apoE2 mice to a type III HLP phenotype (Huang et al., 1997a), directly indicating a regulatory role of the LDL receptor in the expression of type III HLP in apoE2 carriers. A third factor responsible for precipitating type III HLP is oestrogen status (for review, see Mahley and Rall, 1995). Type III HLP caused by apoE2 homozygosity occurs predominantly in males; almost all affected women are postmenopausal and oestrogen treatment reversed the hyperlipidaemia in several clinical cases (Kushwaha et al., 1977; Falko et al., 1979), suggesting that oestrogen status may modulate the expression of this disorder in humans. In addition, apoE2 transgenic rabbits also show a significant gender difference in the expression of type III HLP, with females being more resistant to apoE2-induced
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hyperlipidaemia and to the development of spontaneous atherosclerosis (Huang et al., 1997b). Interestingly, oestrogen treatment markedly reduced the hyperlipidaemia in male apoE2 transgenic rabbits and converted the lipoprotein profile to a normal pattern. Conversely, ovariectomy increased the hyperlipidaemia in female apoE2 rabbits and induced a more overt type III HLP phenotype (Huang et al., 1997b). The effect of oestrogen in modulating the expression of type III HLP probably involves two principal mechanisms. First, oestrogen profoundly stimulates the expression of hepatic LDL receptors (Kovanen et al., 1979; Ma et al., 1986; Huang et al., 1997b). Second, oestrogen stimulates both LPL and hepatic lipase activities, leading to enhanced lipolysis (Demacker et al., 1991; Huang et al., 1997b). Thus, low oestrogen status may precipitate overt type III HLP through a combination of effects: impairment of remnant clearance due secondarily to low LDL receptor activity and impairment of lipolysis due to low levels of lipolytic processing. Finally, increased cholesteryl ester transfer protein (CETP) activity has also been suggested as a secondary factor that may trigger the hyperlipidaemia in apoE2 homozygous patients (Tall et al., 1987). It has been demonstrated that CETP activity is increased several fold in type III HLP patients (Tall et al., 1987; McPherson et al., 1991). CETP transfers cholesterol esters from HDL to lower-density lipoproteins, including VLDL and IDL, and probably remnant lipoproteins as well. Thus, increased CETP activity would enrich the VLDL and IDL (probably also remnants) in cholesteryl esters, which could further enhance the formation of -VLDL in apoE2 homozygous patients who have little tolerance for remnant catabolism. In addition to genetic factors, some environmental factors may also influence the expression of type III HLP (for review, see Davignon et al., 1988; Mahley and Rall, 1995). One factor commonly associated with type III HLP is obesity (Fredrickson et al., 1967), and a correspondence between clinical expression of type III HLP and weight has been reported (Morganroth et al., 1975). Clinical glucose intolerance and diabetes are also common in type III HLP patients, and their presence exacerbates the hyperlipidaemia (Morganroth et al., 1975). Hypothyroidism also exacerbates the hyperlipidaemia in type III HLP subjects, probably due to decreased LDL receptor activity (Hazzard and Bierman, 1972; Thompson et al., 1981). Mechanism of hypertriglyceridaemia caused by apoE2 Population studies have demonstrated that apoE2 is associated with increased plasma triglyceride levels (Utermann et al., 1984; Dallongeville et al., 1992) and that it precipitates hypertriglyceridaemia, in addition to hypercholesterolaemia, when type
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III HLP occurs (Mahley and Rall, 1995). This phenomenon has been ascribed to defective LDL receptor binding of apoE2, which impairs the clearance of triglyceride-rich lipoproteins from the circulation (Dallongeville et al., 1992; Mahley and Rall, 1995). However, the normal or only mildly elevated triglyceride levels observed in apoEnull mice that develop very severe hypercholesterolaemia argues against this hypothesis (Plump et al., 1992; Zhang et al., 1992). This discrepancy is also true for humans with apoE deficiency, whose hypercholesterolaemia is generally much more severe than their hypertriglyceridaemia (Ghiselli et al., 1981; Mabuchi et al., 1989; Kurosaka et al., 1991). These results suggest that apoE2 might affect plasma triglyceride metabolism independent of its receptor bindingdefective property. Early on; in vivo studies suggested that apoE2 impairs lipolytic conversion of VLDL to LDL in type III HLP subjects (Chait et al., 1977; Chait et al., 1978). A similar conclusion was also reached from more recent in vitro studies (Chung and Segrest, 1983; Ehnholm et al., 1984). This concept has now been confirmed and expanded in apoE2 transgenic mice lacking endogenous mouse apoE. Intermediate (20–30 mg/dl) and high (40–50 mg/dl) expression of apoE2 on the mouse apoE-null background increased plasma triglyceride levels 2–4-fold compared to the apoE-null-only mice (Figure 13.5). This indicates that, as far as triglyceride metabolism is concerned, having defective apoE2 is worse than having no apoE at all (Huang et al., 1997a). In line with this in vivo observation, in vitro lipolytic studies demonstrate that the presence of apoE2 in VLDL or IDL impairs LPL-mediated lipolysis by ~ 80% and ~ 70%, respectively (Huang et al., 1998). On the other hand, since plasma total cholesterol levels tend in the opposite direction in the apoE2 transgenic mice lacking mouse apoE (i.e., decreased compared with the apoE-null-only mice) (Figure 13.5), it seems that the hypercholesterolaemia and hypertriglyceridaemia associated with apoE2 occurs at least in part via two separate mechanisms: one caused by defective binding of apoE2 to the LDL receptor, and the other caused mostly by impaired lipolysis of triglyceride-rich lipoproteins by apoE2. Of course, these two apoE2associated blockages on remnant lipoprotein metabolism will exacerbate each other. Defective receptor binding of apoE2 causes its accumulation in remnant particles, which in turn leads to impaired lipolysis. On the other hand, impaired lipolysis tends to increase triglyceride-rich lipoproteins that are poorly removed from plasma and further acquire or retain more apoE2, and thus are more defective in receptor binding.
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Figure 13.5 Correlation of plasma apoE2 and lipids in apoE2 transgenic mice lacking endogenous mouse apoE (hE2+/0, mE−/−). A, apoE2 versus total cholesterol. B, apoE2 versus triglycerides. Plasma total cholesterol and triglyceride levels in apoE-null-only mice are 400±45 and 91±40mg/dl, respectively. hE2, human apoE2; mE, mouse apoE.
Dominant Mode of Inheritance Several rare variants of apoE that are associated with type III HLP have also been described (Table 13.1) (for review, see Mahley, 1988; Rall and Mahley, 1992; Mahley and Rall, 1995). In most of these cases, heterozygosity for the rare apoE variants is sufficient for expression of type III HLP, and secondary factors are not required, indicating a dominant mode of transmission. For example, two rare apoE variants identified earlier, apoE(Arg142 Cys) and apoE-Leiden, were described in families with type III HLP (Havel et al., 1983; Rall et al., 1989; de Knijff et al., 1991). All affected family members are heterozygous for either apoE(Arg142 Cys) or apoE-Leiden (Rall et al., 1989; de Knijff et al., 1991). Recently, it has been demonstrated in transgenic mice that high expression of apoE(Arg142 Cys) leads to spontaneous hyperlipidaemia, accumulation of -VLDL in plasma, and increased susceptibility to high fat diet-induced atherosclerosis (Fazio et al., 1993; Fazio et al., 1994). Transgenic mice expressing high levels of apoE-Leiden also develop spontaneous hyperlipidaemia and increased susceptibility to atherosclerosis (van den Maagdenberg et al., 1993; van Vlijmen et al., 1994). These transgenic mice are yielding important information for our understanding of the mechanisms of dominant type III HLP. Almost all of the identified apoE variants that are associated with the dominant mode of inheritance of type III HLP involve substitutions of
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neutral or acidic amino acid residues for basic ones in the 136–150 region of the apoE molecule (Table 13.1) that interacts directly with the LDL receptor. Thus, these dominant mutations represent defective LDL receptor binding (~5–45% of normal apoE3 binding activity) by directly affecting the ionic interaction with the receptor. One exception is apoE-Leiden, which has a seven-amino acid duplication of residues 121–127 (van den Maagdenberg et al., 1989; Wardell et al., 1989). This insertion occurs outside the receptor-binding region of apoE and, thus, probably indirectly affects its binding to the receptor by altering the conformation of the receptor-binding region of apoE. However, the impaired LDL receptor binding cannot completely explain the dominantly expressed type III HLP caused by these rare apoE variants, since all of them possess somewhat higher LDL receptor-binding activity than the recessive apoE2 variants (Table 13.1). Thus, changes in other properties of apoE, together with the receptor-binding defect, must account for the dominant expression of type III HLP in patients with these rare apoE variants. One of the changes is probably the heparin (HSPG)-binding affinity. The heparinbinding site of apoE is coincident with its LDL receptor-binding site, i.e., residues 136–150 (see above). Thus, substitution of neutral or acidic amino acids for the basic ones in this region could also cause a heparin (HSPG)-binding defect, as demonstrated for most of the rare apoE variants in Table 13.1. Since interaction of apoE with HSPG in the space of Disse is an important initial step for remnant clearance, defective HSPG binding would block remnant clearance at a very early stage of the catabolic cascade, and together with the defective LDL receptor binding, lead to type III HLP. Another possibility that may explain the dominant transmission of type III HLP is the lipoprotein preference property of some apoE variants. It is known that two of the variants associated with the dominant mode of type III HLP, apoE (Arg142 Cys) and apoE-Leiden, are derived from apoE4 (i.e., with arginine at residue 112); thus, both possess a preference for triglyceride-rich lipoproteins. In fact, subjects heterozygous for these variants show a markedly increased ratio of variant to normal apoE in their -VLDL (~3:1 for apoE(Arg142 Cys) and ~7:1 for apoE-Leiden) (de Knijff et al., 1991; Horie et al., 1992). Recently, a similar higher ratio has also been demonstrated in both apoE(Arg142 Cys) and apoE-Leiden transgenic mice that develop dominant type III HLP (Fazio et al., 1993; van den Maagdenberg et al., 1993). Probably a much higher amount of variant apoE relative to normal apoE in VLDL or remnant lipoproteins would lead to a dominant negative effect of the variant apoE resulting in impaired clearance of the remnant lipoproteins. However, a VLDL preference is not characteristic of all the variants displaying a dominant inheritance pattern. Alternatively, accumulation of the variant apoE in triglyceride-rich lipoproteins
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would impair lipolytic processing of the particles, which, in turn, causes hypertriglyceridaemia directly and an impaired clearance of the remnants secondarily to inefficient lipolysis. In line with the latter hypothesis, the hypertriglyceridaemia occurs in subjects with either apoE(Arg142 Cys) or apoE-Leiden is actually more severe than that observed in apoE2-associated type III HLP (Havel et al., 1983; Rall et al., 1989; de Knijff et al., 1991). More severe hypertriglyceridaemia is also observed in both apoE(Arg142 Cys) and apoE-Leiden transgenic mice compared with apoE2 transgenic mice (Fazio et al., 1993; van den Maagdenberg et al., 1993; Huang et al., 1996).
APOLIPOPROTEIN E AND ALZHEIMER’S DISEASE Role of Apolipoprotein E in Neurobiology Increasing evidence obtained from both in vitro and in vivo studies has suggested an important role of apoE in neurobiology (for review, see Mahley, 1988; Weisgraber et al., 1994a; Weisgraber et al., 1994b; Mahley et al., 1995; Weisgraber and Mahley, 1996). First, the brain is second only to the liver in abundance of apoE mRNA (Elshourbagy et al., 1985). Immunocytochemical and metabolic labelling studies have demonstrated that apoE is synthesised and secreted primarily by astrocytes in the brain (Boyles et al., 1985; Pitas et al., 1987a). Second, apoE-containing HDL are found in cerebrospinal fluid (CSF) and account for the major portion of lipoproteins in CSF (Pitas et al., 1987b). Third, apoE levels are increased ~ 250–350-fold following injury of peripheral nerves in a rat model (Skene and Shooter, 1983; Müller et al., 1985; Ignatius et al., 1986; Müller et al., 1986; Boyles et al., 1989). A lesser accumulation of apoE is induced by injury to the central nervous system (Boyles et al., 1989). Finally, apoE, together with a source of cholesterol ( -VLDL, CSF lipoproteins, and others), markedly stimulates neurite extension in cultured dorsal root ganglion and neuroblastoma (Neuro-2a) cells (Handelmann et al., 1992; Nathan et al., 1994a; Nathan et al., 1994b). Apolipoprotein E appears to acquire lipids released by neuron degeneration and to redistribute them to the cells needing them for proliferation, membrane repair, or remyelination of the new axons (Ignatius et al., 1987; Boyles et al., 1989). Furthermore, the receptors for apoE, including HSPG, the LDL receptor, and the LRP, are also highly expressed in the brain (Pitas et al., 1987b; Rebeck et al., 1993). Pathological Features of Alzheimer’s Disease Alzheimer’s disease (AD) is an irreversible neurodegenerative disorder
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characterised by progressive dementia. There are three clinical categories for AD: early-onset familial AD occurring before 50 years of age, late-onset familial AD occurring after 65 years of age, and sporadic late-onset AD occurring after 65 years of age without significant familial association. The neuropathological markers of AD are neuritic amyloid plaques and neurofibrillary tangles in the brain (Figure 13.6) (for review, see Selkoe, 1991; Crowther, 1993; Roses, 1994). Neuritic plaques are extracellular deposits of amyloid (Figure 13.6). The major component of the deposits is the amyloid beta (A ) peptide, which is also present in plasma and CSF (Seubert et al., 1992). A peptide, ranging from 39–42 residues in length, is a proteolytic product of the amyloid protein precursor (APP) that is present on the surface of different cells, including neurons (Haass and Selkoe, 1993). If not cleared effectively, A peptides tend to aggregate. In contrast to amyloid deposits, neurofibrillary tangles are primarily intracellular in neurons (Figure 13.6). Appearing as paired helical filaments, their major component is a highly phosphorylated form of the tau protein (for review, see Crowther, 1993). The tau protein is a member of the microtubule-associated protein family that aids microtubule assembly by stabilising its structure. Abnormal phosphorylation of tau protein reduces its binding affinity for microtubules, leading to microtubule destabilisation and neuron degeneration.
Figure 13.6 The neuropathological lesions of Alzheimer’s disease and their association with apoE. Apolipoprotein E is immunohistochemically localised in both the intracellular neurofibrillary tangles (composed of paired helical filaments) and the extracellular neuritic amyloid plaques (composed of amyloid beta (A ) peptide). APP, amyloid protein precursor. (Reproduced with permission from Weisgraber, K.H., Pitas, R.E. and Mahley, R.W. (1994a) Lipoproteins, neurobiology, and Alzheimer’s disease: Structure and function of apolipoprotein E.Curr. Opin.
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Struct. Biol., 4, 507–515.)
Genetic Linkage of Apolipoprotein E and Late-Onset Alzheimer’s Disease The first evidence suggesting that apoE might be involved in the pathogenesis of AD was the observation that apoE is colocalised with two neuropathological markers of AD (i.e., extracellular amyloid deposits and intracellular neurofibrillary tangles) (Figure 13.6) (Namba et al., 1991; Wisniewski and Frangione, 1992; Strittmatter et al., 1993a). Additional evidence came from the observation that the apoE4 allele was overrepresented in late-onset familial AD patients (0.50) compared with age- and sex-matched controls (0.16) (Strittmatter et al., 1993a). Since then, association of the apoE4 allele with the development of late-onset familial AD has been confirmed in several populations around the world (for review, see Simonds et al., 1991; Roses, 1996). The concept was later extended to include late-onset sporadic AD (Saunders et al., 1993). Furthermore, a gene dosedependent effect of apoE4 on the risk and age of onset of AD has been demonstrated. The risk for the development of AD in families with late-onset AD increased from 20% to 90% and the mean age of onset decreased from 84 to 68 years as the number of apoE4 alleles increased from 0 to 1 to 2 (Corder et al., 1993). Therefore, it is clear from the genetic and epidemiological studies that the apoE4 allele is a major risk factor or susceptibility gene for the development of late-onset AD. In contrast, the apoE2 allele seems to delay the age of onset and decrease the risk of AD (Corder et al., 1994; Lippa et al., 1997), suggesting that the apoE2 allele probably protects against the development of AD. Role of Apolipoprotein E in Alzheimer’s Disease Isoform-specific interactions of apolipoproteins E3 and E4 with the A peptide Consistent with apoE’s association with neurite amyloid plaques in vivo, lipid-free apoE3 and apoE4 can form an SDS- and guanidine hydrochloride-stable complex with the A peptide in vitro, with apoE4 being more rapid and effective in the formation of the complex (Strittmatter et al., 1993b). Interestingly, addition of reducing agents, such as dithiothreitol or -mercaptoethanol, prevents the formation of the SDS-stable complex, suggesting that oxidation of apoE may be required (Strittmatter et al., 1993a). Furthermore, experiments using various apoE fragments identified residues 244–272 as the apoE domain responsible for binding to the A peptide (Strittmatter et al., 1993b), the same domain responsible for binding to lipoprotein
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particles (see above). However, incubation of the lipidated apoE3 or apoE4, which were isolated from apoE3 and apoE4 stably transfected cells, respectively, with A peptide demonstrated a different response (LaDu et al., 1994; LaDu et al., 1995; LaDu et al., 1997). Lipidated apoE3 bound with a 20-fold greater affinity than lipidated apoE4 to the A peptide. Thus, it has been suggested that avid binding of apoE3 to the A peptide could lead to enhanced clearance of the complex, thus preventing the conversion of A into a neurotoxic species (LaDu et al., 1995; LaDu et al., 1997). The physiological relevance of these observations in the context of AD pathology awaits further in vivo studies. Morphological studies demonstrated that prolonged incubation (several days) of apoE with the A peptide forms insoluble, highmolecular-weight complexes that precipitate as fibres. Again, compared with apoE3, apoE4 forms a denser, more extensive matrix of amyloid monofibrils and acts more rapidly and effectively (Sanan et al., 1994). Immunogold labelling revealed that apoE associates with these fibrils along their entire length, suggesting that apoE may help to form a hybrid fibril (Sanan et al., 1994). This observation has been confirmed by studies in which the formation of fibrils associated with either apoE3 or apoE4 was quantified by fibril counting or by thioflavin fluorescence (Ma et al., 1994; Wisniewski et al., 1994). Taken together, these studies demonstrate that apoE3 and apoE4 bind the A peptide differently and suggest a mechanism to explain the association of apoE4 and AD. Increased amyloid fibril formation associated with apoE4 may trigger or exacerbate the development of AD. Isoform-specific interaction of apolipoproteins E3 and E4 with tau protein The interaction of apoE with the tau protein in vitro is also isoform specific. In this case, apoE3 forms an SDS-stable complex with the tau protein in a 1:1 ratio, while apoE4 does not form a significant complex (Strittmatter et al., 1994b). Phosphorylation of the tau protein by a crude brain extract inhibits the interaction of apoE3 with tau protein (Strittmatter et al., 1994b), suggesting that apoE3 binds to unphosphorylated residues of the tau protein. The binding utilises the amino-terminal domain of apoE3 (Strittmatter et al., 1994b) and is irreversible to the microtubule-binding repeat regions of the tau protein (Strittmatter et al., 1994a). In neurons, the tau protein normally binds to and stabilises the microtubules and stimulates the assembly of microtubules by polymerising tubulin. Microtubules are necessary for neurite extension and for the transport of materials along the axon and dendrites (for review, see Goedert et al., 1991). In the brains of AD patients, tau
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protein becomes abnormally phosphorylated by an unknown mechanism and self-assembles into the pathological paired helical filaments, thereby forming neurofibrillary tangles (Biernat et al., 1992; Goedert et al., 1992). Thus, binding of apoE3 to the tau protein may protect it from hyperphosphorylation and preserve the ability of the tau protein to stabilise microtubules. In contrast, the association of apoE4 with the development of AD may be due to a lack of the protective function of apoE4, since it does not bind well to the tau protein. Isoform-specific effect of apoE3 and apoE4 on cytoskeletal function and neurite extension As described above, in the presence of a source of lipid (e.g., cholesterol-rich -VLDL or CSF lipoproteins), apoE significantly stimulated neurite extension (Handelmann et al., 1992). Recently, this observation has been expanded to an isoform-specific effect of apoE3 and apoE4 (Nathan et al., 1994b; Holtzman et al., 1995; Nathan et al., 1995a). Apolipoprotein E3 plus -VLDL significantly stimulated neurite extension, whereas apoE4 plus -VLDL markedly inhibited neurite branching and extension in both dorsal root ganglion neurons and neuroblastoma (Neuro-2a) cells (Figure 13.7) (Nathan et al., 1994b; Nathan et al., 1995a). Furthermore, apoE3-transfected Neuro-2a cells growing in medium containing either -VLDL or CSF-HDL showed greater neurite extension than did the apoE4-transfected Neuro2a cells (Bellosta et al., 1995). Thus, apoE3 and apoE4 have different effects on neurite extension, with apoE3 stimulating neurite extension and apoE4 inhibiting neurite extension. The apoE4-associated inhibition of neurite extension is probably due to its effect on microtubule stability (Nathan et al., 1995a). Incubation of Neuro-2a cells with apoE4 and -VLDL leads to significantly increased soluble tubulin and decreased polymerised tubulin levels, while apoE3 plus -VLDL had the opposite effect. Immunocytochemical analysis of tubulin in the Neuro-2a cells incubated with apoE4 plus -VLDL reveals diffuse tubulin staining and few well-organised microtubules compared with the cells incubated with apoE3 plus -VLDL (Nathan et al., 1995a). These results suggest that apoE4 may depolymerise or destabilise the microtubules, leading to disruption of the cytoskeletal structure and, thus, impaired neurite extension. Interestingly, the isoform-specific effects of apoE3 and apoE4 on neurite extension were abolished by addition of an antibody against the receptor-binding domain of apoE or by reductive methylation of critical lysine residues, suggesting that the effect of apoE is mediated by lipoprotein receptors on the cell surface (Nathan et al., 1994b). Furthermore, treatment of cells with heparinase to remove cell-surface
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Figure 13.7 Isoform-specific effects of apoE3 and apoE4 on neurite extension in murine Neuro-2a cells. A, cells incubated with apoE3 (30 g/ml) plus rabbit -VLDL (40 g cholesterol/ml) display enhanced neurite extension. B, cells incubated with apoE4 (30 g/ml) plus rabbit -VLDL (40 g cholesterol/ml) show little neurite extension. Scale bar=20 m. (Reproduced with permission from Mahley, R.W., Nathan, B.P., Bellosta, S. and Pitas, R.E. (1995) Apolipoprotein E: Impact of cytoskeletal stability in neurons and the relationship to Alzheimer’s disease. Curr. Opin. Lipidol., 6, 86–91.)
HSPG blocked both apoE3-induced neurite extension and apoE4-
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induced inhibition of neurite extension. Likewise, treatment of the cells either with the 39-kDa protein, which blocks the binding of ligand to the LRP, or with an anti-LRP antibody also abolished the isoformspecific effects of apoE3 and apoE4. Thus, these results implicate the HSPG/LRP pathway as responsible for the isoform-specific effects of apoE3 and apoE4 on neurite extension (Bellosta et al., 1995; Holtzman et al., 1995; Nathan et al., 1995b). Differential accumulation of apoE3 and apoE4 in neurons When Neuro-2a cells were incubated with -VLDL plus either apoE3 or apoE4, apoE3 accumulated in much higher concentrations in the cells than apoE4 (Nathan et al., 1994a). Immunofluorescent staining revealed that apoE3 distributes in the cell bodies around the nucleus and diffusely throughout the neurites, whereas apoE4 accumulates mostly in the cell bodies (Nathan et al., 1994a). Recently, this observation has been confirmed and extended by quantitatively determining intracellular accumulation of apoE in neurons incubated with -VLDL together with either 125I-labelled apoE3 or apoE4 (Ji et al., 1997b). In this case, accumulation of 125I-apoE3 in the neurons is twofold greater than that of 125I-apoE4. Furthermore, cell-surface HSPG appear to play a primary role in both the retention of apoE and the differential accumulation of apoE3 versus apoE4 (Ji et al., 1997b). Although the intracellular fate of apoE remains to be determined, the differential accumulation of apoE3 versus apoE4 may be a key point that determines the specific apoE isoform effects in neurobiology.
CONCLUSION Apolipoprotein E is a multifunctional protein, with very important roles in both lipid metabolism and neurobiology. The three common isoforms of apoE—apoE2, apoE3, and apoE4—have different impacts on both lipid metabolism and neurobiology. Among them, apoE3, the most common isoform, functions “normally”. Apolipoprotein E2 binds defectively to the LDL receptor and is associated with a recessive mode of inheritance of type III HLP. With its unique intramolecular domain interaction, apoE4 is associated with the development of AD. Future studies at the cellular and molecular levels are required to understand fully the physiological and pathophysiological effects of apoE in lipid metabolism and in neurobiology. Determination of the three-dimensional structure of the intact apoE molecule will provide further insight into the mechanisms whereby apoE interacts with lipids and lipoprotein receptors. Transgenic animals expressing different apoE variants will provide useful tools to study the mechanisms
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whereby these variants give rise to the recessive versus dominant expression of type III HLP. Finally, further studies are needed to determine the precise mechanisms by which apoE affects normal and abnormal biological processes within the nervous system. Acknowledgments We thank John Carroll and Amy Corder for assistance with graphics, Gary Howard and Stephen Ordway for editorial assistance, and Sylvia Richmond for help with manuscript preparation. This work was supported in part by National Institutes of Health Program Project Grants HL41633 and HL47660. References Aggerbeck, L.P., Wetterau, J.R., Weisgraber, K.H., Wu, C.-S.C. and Lindgren, F.T. (1988). Human apolipoprotein E3 in aqueous solution. II. Properties of the amino- and carboxyl-terminal domains. J.Biol Chem. , 263 , 6249–6258. Allan, C.M., Walker, D., Segrest, J.P. and Taylor, J.M. (1995). Identification and characterization of a new human gene (APOC4) in the apolipoprotein E, C-I, and C-II gene locus . Genomics , 28 , 291– 300. Angelin, B. (1995). Studies on the regulation of hepatic cholesterol metabolism in humans. Eur. J.Clin. Invest. , 25 , 215–224. Angelin, B., Raviola, C.A., Innerarity, T.L. and Mahley, R.W. (1983). Regulation of hepatic lipoprotein receptors in the dog. Rapid regulation of apolipoprotein B, E receptors, but not of apolipoprotein E receptors, by intestinal lipoproteins and bile acids. J.Clin. Invest. , 71 , 816–831. Basu, S.K., Brown, M.S., Ho, Y.K., Havel, R.J. and Goldstein, J.L. (1981). Mouse macrophages synthesize and secrete a protein resembling apolipoprotein E. Proc. Natl. Acad. Sci. USA , 78 , 7545– 7549. Basu, S.K., Ho, Y.K., Brown, M.S., Bilheimer, D.W., Anderson, R.G.W. and Goldstein, J.L. (1982). Biochemical and genetic studies of the apoprotein E secreted by mouse macrophages and human monocytes. J.Biol. Chem. , 257 , 9788–9795. Beisiegel, U., Weber, W. and Bengtsson-Olivecrona, G. (1991). Lipoprotein lipase enhances the binding of chylomicrons to low density lipoprotein receptor-related protein. Proc. Natl. Acad. Sci. USA , 88 , 8342–8346. Beisiegel, U., Weber, W., Ihrke, G., Herz, J. and Stanley, K.K. (1989). The LDL-receptor-related protein, LRP, is an apolipoprotein Ebinding protein. Nature , 341 , 162–164. Bellosta, S., Nathan, B.P., Orth, M., Dong, L.-M., Mahley, R.W. and Pitas, R.E. (1995). Stable expression and secretion of apolipoproteins E3 and E4 in mouse neuroblastoma cells produces differential effects
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60 , 344–351. Utermann, G., Vogelberg, K.H., Steinmetz, A., Schoenborn, W., Pruin, N., Jaeschke, M. et al. (1979b). Polymorphism of apolipoprotein E. II. Genetics of hyperlipoproteinemia type III. Clin. Genet. , 15 , 37– 62. van den Maagdenberg, A.M.J.M., de Knijff, P., Stalenhoef, A.F.H., Gevers Leuven, J.A., Havekes, L.M. and Frants, R.R. (1989). Apolipoprotein E*3-Leiden allele results from a partial gene duplication in exon 4. Biochem. Biophys. Res. Commun. , 165 , 851– 857. van den Maagdenberg, A.M.J.M., Hofker, M.H., Krimpenfort, P.J.A., de Bruijn, I., van Vlijmen, B., van der Boom, H. et al. (1993). Transgenic mice carrying the apolipoprotein E3-Leiden gene exhibit hyperlipoproteinemia. J.Biol. Chem. , 268 , 10540–10545. van Vlijmen, B.J.M., van den Maagdenberg, A.M.J.M., Gijbels, M.J.J., van der Boom, H., HogenEsch, H., Frants, R.R. et al. (1994). Dietinduced hyperlipoproteinemia and atherosclerosis in apolipoprotein E3-Leiden transgenic mice. J.Clin. Invest. , 93 , 1403–1410. Wardell, M.R., Brennan, S.O., Janus, E.D., Fraser, R. and Carrell, R.W. (1987). Apolipoprotein E2-Christchurch (136 Arg Ser). New variant of human apolipoprotein E in a patient with type III hyperlipoproteinemia. J.Clin. Invest. , 80 , 483–490. Wardell, M.R., Weisgraber, K.H., Havekes, L.M. and Rall, S.C., Jr. (1989). Apolipoprotein E3-Leiden contains a seven-amino acid insertion that is a tandem repeat of residues 121–127. J.Biol. Chem. , 264 , 21205–21210. Weisgraber, K.H. (1990). Apolipoprotein E distribution among human plasma lipoproteins: Role of the cysteine-arginine interchange at residue 112. J.Lipid Res. , 31, 1503–1511. Weisgraber, K.H. (1994). Apolipoprotein E: Structure-function relationships. Adv. Protein Chem. , 45 , 249–302. Weisgraber, K.H., Innerarity, T.L. and Mahley, R.W. (1982). Abnormal lipoprotein receptor-binding activity of the human E apoprotein due to cysteine-arginine interchange at a single site. J.Biol. Chem. , 257 , 2518–2521. Weisgraber, K.H. and Mahley, R.W. (1996). Human apolipoprotein E: The Alzheimer’s disease connection. FASEB J. , 10 , 1485–1494. Weisgraber, K.H., Pitas, R.E. and Mahley, R.W. (1994a). Lipoproteins, neurobiology, and Alzheimer’s disease: Structure and function of apolipoprotein E. Curr. Opin. Struct. Biol. , 4 , 507–515. Weisgraber, K.H., Rall, S.C., Jr. and Mahley, R.W. (1981). Human E apoprotein heterogeneity. Cysteine-arginine interchanges in the amino acid sequence of the apo-E isoforms. J.Biol. Chem. , 256 , 9077–9083. Weisgraber, K.H., Roses, A.D. and Strittmatter, W.J. (1994b). The role of apolipoprotein E in the nervous system. Curr. Opin. Lipidol. , 5 , 110–116. Wetterau, J.R., Aggerbeck, L.P., Rall, S.C., Jr. and Weisgraber, K.H. (1988). Human apolipoprotein E3 in aqueous solution. I. Evidence
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for two structural domains. J.Biol. Chem. , 263 , 6240–6248. Williams, K.J. and Fuki, I.V. (1997). Cell-surface heparan sulfate proteoglycans: Dynamic molecules mediating ligand catabolism. Curr. Opin. Lipidol. , 8 , 253–262. Willnow, T.E., Sheng, Z., Ishibashi, S. and Herz, J. (1994). Inhibition of hepatic chylomicron remnant uptake by gene transfer of a receptor antagonist. Science , 264 , 1471–1474. Wilson, C, Wardell, M.R., Weisgraber, K.H., Mahley, R.W. and Agard, D.A. (1991). Three-dimensional structure of the LDL receptorbinding domain of human apolipoprotein E. Science , 252 , 1817– 1822. Wisniewski, T., Castaño, E.M., Golabek, A., Vogel, T. and Frangione, B. (1994). Acceleration of Alzheimer’s fibril formation by apolipoprotein E in vitro. Am . J.Pathol. , 145 , 1030–1035. Wisniewski, T. and Frangione, B. (1992). Apolipoprotein E: A pathological chaperone protein in patients with cerebral and systemic amyloid. Neurosci. Lett. , 135 , 235–238. Woollett, L.A., Osono, Y., Herz, J. and Dietschy, J.M. (1995). Apolipoprotein E competitively inhibits receptor-dependent low density lipoprotein uptake by the liver but has no effect on cholesterol absorption or synthesis in the mouse. Proc. Natl. Acad. Sci. USA , 92 , 12500–12504. Zannis, V.I. and Breslow, J.L. (1981). Human very low density lipoprotein apolipoprotein E isoprotein polymorphism is explained by genetic variation and posttranslational modification. Biochemistry , 20 , 1033–1041. Zannis, V.I., Just, P.W. and Breslow, J.L. (1981). Human apolipoprotein E isoprotein subclasses are genetically determined. Am. J.Hum. Genet. , 33 , 11–24. Zannis, V.I., McPherson, J., Goldberger, G., Karathanasis, S.K. and Breslow, J.L. (1984). Synthesis, intracellular processing, and signal peptide of human apolipoprotein E. J.Biol. Chem. , 259 , 5495–5499. Zhang, S.H., Reddick, R.L., Piedrahita, J.A. and Maeda, N. (1992). Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E. Science , 258 , 468–471. Zhao, S.-P., Smelt, A.H.M., Van den Maagdenberg, A.M.J.M., Van Tol, A., Vroom, T.F.F.P., Leuven, J.A.G. et al. (1994). Plasma lipoproteins in familial dysbetalipoproteinemia associated with apolipoproteins E2(Arg158 Cys), E3-Leiden, and E2(Lys146 Gln), and effects of treatment with simvastatin. Arterioscler. Thromb. , 14 , 1705–1716.
14 Role of the Plasma Phospholipid Transfer Protein in Plasma Lipid Transport Matti Jauhiainen and Christian Ehnholm Department of Biochemistry, National Public Health Institute, Mannerheimintie 166, FIN-00300 Helsinki, Finland
Lipid transfer proteins include the cholesteryl ester transfer protein (CETP) and the phospholipid transfer protein (PLTP), which play an essential role in the intravascular dynamics of lipids between lipoprotein classes and mediate lipid transfer processes. Although PLTP has been known for almost 15 years it has, unlike CETP, been investigated quite little, especially with regard to its physiological function. PLTP facilitates the transfer and exchange of phospholipids, but not neutral lipids, between lipoproteins. It is also capable of converting HDL to larger and smaller particles in vitro. Small particles are similar to primary cholesterol acceptors from peripheral cells in a reverse cholesterol transport process. Future studies will offer more data on the physiological role of PLTP in this process, with tools for such studies being offered by recent elucidation of the PLTP complementary DNA sequence and the organisation of its gene structure. Furthermore, the molecular genetic approaches now available will also offer a new starting point for further clinical research at the molecular level. An interesting new observation is that PLTP is a member of a gene family comprising lipopolysaccharide-binding protein, bactericidal permeability-increasing protein and CETP genes. Future studies will show whether the interaction of PLTP with lipopolysaccharide has any physiological relevance. KEYWORDS: PLTP, lipid transfer, HDL, preßHDL, reverse cholesterol transport, HDL-conversion.
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INTRODUCTION High density lipoproteins (HDL) exist in plasma as multiple forms. Populations of HDL that differ in density, composition, electrophoretic mobility and apolipoprotein profile have been identified. These HDL populations differ not only in physicochemical properties but also in their physiological function. Epidemiological studies have provided strong evidence for an inverse relationship between plasma HDL cholesterol levels and coronary heart disease (CHD). The mechanisms by which HDL exerts their antiatherogenic effects have not been conclusively elucidated. However, cell culture studies (Castro & Fielding, 1988; Johnson et al., 1991) and animal models (Rubin et al., 1991; Paszty et al., 1994) have suggested that the antiatherogenic mechanism of HDL involves HDL-mediated removal of cholesterol from cells of the arterial wall and the subsequent transport of cholesterol to the liver for excretion. This process, named reverse cholesterol transport (RCT), limits the accumulation of cholesterol in the arterial wall and is probably responsible for the decreased CHD risk. HDL are continuously remodelled during their move through the plasma compartment and this affects their composition, size, and plasma level. In addition, this remodelling affects their antiatherogenic potential. HDL modulation is caused by various enzymes and functional proteins such as the phospholipid transfer protein, (PLTP). The recent literature on the structure and function of PLTP and its possible role in atherosclerosis is reviewed below.
PHOSPHOLIPID TRANSFER PROTEIN (PLTP) STRUCTURE AND GENE ORGANISATION Molecular Characteristics of PLTP Phospholipid transfer protein in human plasma is functionally and genetically different from the other plasma lipid transfer protein CETP (Tall et al., 1983a; Brown et al., 1990). PLTP complementary DNA (cDNA) encodes a 17 amino acid signal peptide and a mature polypeptide of 476 amino acids (Day et al., 1994). The mature protein has a predicted molecular weight of about 55,000 Da. Upon sodium dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, PLTP migrates with an apparent molecular mass of about 81 kDa. The difference between the calculated molecular weight and the mass based on SDS-PAGE estimation is probably due to the high degree of glycosylation of the protein. PLTP has six
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potential N-glycosylation sites and several potential O-glycosylation sites (Day et al., 1994). The deduced PLTP sequence contains four cysteines suggesting the potential to form intra-chain disulfide bonds. Predictions of PLTP secondary structure suggest that these disulfide bonds can be constructed between cysteines 5 and 129 and cysteines 168 and 318 (Albers et al., 1996b). PLTP has a high content of hydrophobic residues concentrated in the C-terminal half of the protein. More than 40% of the amino acids in the PLTP sequence are hydrophobic. Based on secondary structure predictions it has been suggested that PLTP has two potential transmembrane regions (Albers et al., 1996b). In addition to the human protein, PLTP has also been characterised in two other mammalian species, the mouse and the pig. Mature mouse PLTP has 493 amino acids (including signal sequence) and shares 83% amino acid sequence identity with the human protein (Albers et al., 1995; Jiang and Bruce 1995). Pig PLTP contains 496 amino acids (including signal sequence) and is 93% identical with the human counterpart (Pussinen et al., 1997a). Organisation of the PLTP Gene and its Promoter The PLTP gene has been mapped to chromosome 20, the 20q12–q13.1 region (Whitmore et al., 1995). The gene is 13.3kb in length. The gene is interrupted by 15 introns, varying from 79 bp up to 2350 bp in size. The size of the exons varies from 43 bp to 404 bp. The PLTP exon 1 codes for the 5′-untranslated region and is interrupted by intron 1, which is located 11 bp upstream of the translation initiating site. Exon 2 encodes for the entire signal peptide and the first 16 amino acids of the mature, secreted protein. A putative heparin binding domain is encoded by exon 12 (residues 372–392). The last exon 16 contains the end of the coding region and the 3′-untranslated region (Whitmore et al., 1995). Identification of the functional promoter region of PLTP indicates multiple sites for transcription initiation (Tu et al., 1995a). Several potential binding sites for the transcription factors glucocorticoid receptor (GR), AP-2 and Sp1 are spread over the entire functional promoter, and possibly in synergy drive the basal transcription of the human PLTP gene (Tu et al., 1997). The presence of a glucocorticoid receptor binding element in the PLTP promoter indicates that the transcription may be activated together with the glucocorticoidinducible genes known to be upregulated in inflammation and sepsis. Expression of PLTP in Various Cells and Tissues A wide variety of cell types are known to secrete phospholipid transfer
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activity, to secrete the actual PLTP, or to express the PLTP mRNA. Northern blot analysis of human, mouse and pig tissues indicates that PLTP mRNA is widely distributed (Day et al., 1994; Jiang & Bruce 1995; Pussinen et al., 1997a). The highest mRNA levels in human tissues were found in the thymus, ovary and placenta, whereas moderate levels were detected in the pancreas, small intestine, testis, lung and prostate. Only very low levels were found in the heart, colon, skeletal muscle, leucocytes and brain. As in humans, mouse peripheral tissues displayed a higher level of PLTP mRNA than the central organs. The order of PLTP mRNA expression was as follows: lung>adipose tissue, placenta, testis>brain>muscle, heart and liver (Jiang and Bruce, 1995). Tissue expression of pig PLTP mRNA was examined by a method based on reverse transcription-polymerase chain reaction (RT-PCR) and solid-phase minisequencing in nine pig tissues (Pussinen et al., 1997a). The highest levels were detected in the pancreas, brain, lung and liver. Transfection of PLTP cDNA and its expression has been carried out in many cell lines including HepG2, COS-7, CHO (Tu et al., 1995a), BHK (Albers et al., 1995) and COS-1 (Jiang and Bruce, 1995; Huuskonen et al., 1996). In each case the levels of secreted activity have been different, and the quantity of PLTP protein secreted has been difficult to determine because no mass determination method based on specific immunoassay has yet been described. PLTP as a Member of the Lipid Transfer/Lipopolysaccharide Binding Protein Gene Family The PLTP gene as well as the CETP gene seem to have evolved from an ancient family of genes encoding lipopolysaccharide (LPS) binding proteins (Tu et al., 1995b; Gray et al., 1989). Human PLTP protein is homologous to members of the LPS binding proteins including the plasma LPS binding protein (LBP) and the neutrophil bactericidal permeability increasing protein (BPI) (Lusis et al., 1987). Furthermore, the PLTP, LBP, and BPI genes are localised to the same region on chromosome 20. LBP and BPI are mapped to the 20q11.23-q12 region in close vicinity of the PLTP gene (Gray et al., 1989). However, CETP which belongs to the same gene family, has been localised to chromosome 16q12-q21 (Lusis et al., 1987). The PLTP protein shares 24% identical amino acids with human BPI and 26% with human LBP (Day et al., 1994).
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FUNCTIONS OF PLTP Phospholipid Transfer Activity Plasma PLTP is able to mediate the net transfer of phospholipids from vesicles to HDL (Tall et al., 1983a, b; Damen et al., 1982). PLTP can also enhance the net transfer and exchange of phospholipids between VLDL and HDL (Tall et al., 1985; Lagrost et al., 1994). During lipoprotein lipase-catalysed chylomicron and VLDL hydrolysis, release of surface remnants containing phospholipids occurs. Net transfer of these phospholipids to HDL is enhanced by PLTP. Therefore both the transfer and exchange activities of PLTP are stimulated by lipolysis (Tall et al., 1985). In human plasma PLTP is responsible for about half of the overall phospholipid exchange activity between lipoproteins, the other half being due to CETP (Brown et al., 1990). The phospholipid substrate specificity of PLTP has been studied by determining its acyl chain and its phospholipid species preferences by using a fluorometric assay (Huuskonen et al., 1996; Massey et al., 1985; Rao et al., 1997). PLTP mediated equally the transfer of the various phospholipid species with the exception of phosphatidylethanolamine, which was transferred 2–3-fold more slowly. Neither the length of the acyl chain (from 6 to 14 carbons), nor the acyl chain position, either sn-1 or sn-2, significantly affected the transfer. Plasma-derived and recombinant PLTP produced in COS-1 cells showed identical specificity. In a study by Rao et al., (1997) the lipoprotein specificity of recombinant PLTPmediated phospholipid transfer was studied using VLDL, LDL and HDL as pyrene-lipid labelled donors and unlabelled lipoproteins as acceptors. The highest rate of phospholipid transfer was detected between donor HDL and acceptor HDL. It was shown that PLTP transfers a variety of lipids with two acyl chains and a polar head group; exchanging sn-1/sn-2 acyl chains had only minor effects (see also Huuskonen et al., 1996). Regarding specificity it is noteworthy that PLTP mediates, in addition to phospholipids, the transfer of diacylglycerides. This observation is interesting in the context of the observation that HDL particles carry a substantial amount of diacylglycerides (Vien et al., 1996). Rao et al., (1997) demonstrated that, in contrast to spontaneous transfer, PLTP mediates the accumulation of PC in small rHDL particles. This suggests that one role of PLTP may be to deliver phosphatidylcholine to small, nascent HDL which are known to be the initial acceptors of cholesterol from membranes of peripheral cells (Fielding and Fielding, 1995). The molecular mechanism of PLTP-mediated PL-transfer is not known at the moment. Although a true lipid carrier mechanism may be involved in the transfer (Nishida and Nishida, 1997), the role of
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specific collision complexes between PLTP and the acceptor and donor lipoproteins cannot be excluded (Rao et al., 1997). Cholesterol Transfer Activity Several studies have shown that PLTP facilitates the transfer and exchange of phospholipids but not neutral lipids (Tall et al., 1983a; Albers et al., 1984; Tu et al., 1993). Nishida and Nishida (1997) recently demonstrated that plasma derived PLTP enhances the transfer of cholesterol from single bilayer vesicles containing phosphatidylcholine and cholesterol to HDL3. The maximal rate of cholesterol transfer was observed with vesicles containing 20–25 mol % cholesterol. Scatchard analysis indicated that PLTP has many binding sites for cholesterol. The apparent binding capacity was 13 mol of cholesterol/mol PLTP. However, PLTP showed a higher affinity and binding capacity for PC than for cholesterol. The physiological relevance of the PLTP-mediated cholesterol transfer remains to be clarified. The
-Tocopherol Transfer Activity
Vitamin E or -tocopherol is an important anti-oxidant of plasma lipoproteins and cell membranes (Kayden and Traber, 1993). The role of antioxidants in the protection of LDL from autoxidation has been under intense research since oxidised LDL has been suggested to be an important initiating factor in atherosclerosis (Steinberg et al., 1989). Alpha-tocopherol has been shown to move actively between lipoproteins of different density classes (Traber et al., 1992). Recently, Kostner et al., (1995) demonstrated that PLTP catalyses -tocopherol exchange between different lipoprotein classes and particularly between HDL and cell membranes. PLTP has also been shown to enhance transfer of -tocopherol from liposomes to HDL. The physiological significance of this observation remains to be elucidated. Lipopolysaccharide Transfer Activity Human PLTP shares 26% identical amino acids with the lipopolysaccharide (LPS) binding protein (LBP) (Day et al., 1994). LPS or endotoxin is a membrane lipid of gram-negative bacteria that mediates an inflammatory response in mammals (Ulevitch and Tobias, 1995). LBP first transfers an LPS monomer to a soluble plasma protein, CD14. The LPS-CD14 complex may then transfer LPS to cells thus triggering an inflammatory reaction. LBP also can transfer LPS to HDL which leads to the neutralisation of LPS (Wurfel et al., 1994). It was recently shown that LBP transfers not only LPS, but also several phospholipids (Yu et al., 1997). Structural and functional similarity of
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PLTP with LBP was recently observed by Hailman et al., (1996) who showed that incubation of recombinant PLTP with LPS resulted in the inhibition of the ability of LPS to stimulate a response in neutrophils and in cytokine production. PLTP was able to mediate the exchange of LPS between micelles and the transfer of LPS to rHDL. PLTP is the first protein described which is able to transfer LPS but does not transfer it to CD14 and therefore does not enhance a cellular response to bacterial LPS. The HDL Conversion Activity The relationships between the subpopulations of HDL have not been completely elucidated, although there is evidence that some of them are interconvertible. The plasma factors modulating the HDL composition and size include enzymes, such as lecithin-cholesterol acyltransferase (LCAT), hepatic triglyceride lipase (HL) and lipoprotein lipase (LPL), as well as the lipid transfer proteins CETP and PLTP (Nichols et al., 1992). Originally the function of PLTP in the remodelling of HDL was described by Rye and Barter (1984). This factor was then called the putative HDL-conversion factor. After the isolation of this factor it was shown to modulate the particle size of HDL by converting a homogeneous particle population into new populations of particles, some smaller and others larger than in the original HDL population (Rye and Barter, 1986; Barter et al., 1988). This activity was first connected with the function of CETP (Barter et al., 1990; Lagrost et al., 1990). It was later shown that highly purified PLTP has the ability to convert HDL3 into populations of large and small HDL particles and was thus identified as a conversion factor (Tu et al., 1993; Jauhiainen et al., 1993). In a recent study comparing the ability of purified CETP and PLTP to induce HDL conversion it was shown that CETP facilitates preferentially the formation of small HDL subpopulations, whereas PLTP mediates the formation of both the large and small particles (Lagrost et al., 1996). Both human and mouse recombinant PLTP are able to convert human or mouse HDL into larger and smaller particles (Albers et al., 1995). Likewise, incubation of human HDL3 or pig HDL in the presence of purified pig PLTP induced a conversion of the homogeneous HDL into larger and smaller particles (Pussinen et al., 1995). Furthermore, human plasma PLTP appears to act, not only on HDL3, but also on HDL2 generating both large and small particles (Marques-Vidal et al., 1997).
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The Mechanism and Regulation of PLTP-Mediated HDL Conversion The mechanism(s) behind the PLTP-mediated HDL conversion has not been clarified in detail. To study the issue, Lusa et al., (1996) used reconstituted HDL (rHDL) particles containing either pyrene-labelled phosphatidylcholine as a surface marker or pyrene-labelled cholesterol ester as a core marker. Dilution of the pyrenyl compound, whether due to lipid exchange or particle fusion, was followed by monitoring the excimer to monomer intensities (E/M ratio). These data suggested that particle fusion rather than net lipid transfer or particle aggregation is responsible for the PLTP-mediated HDL conversion. A model for the PLTP-mediated HDL conversion is shown in Figure 14.1. According to this model, PLTP first interacts with HDL and mediates a rapid transfer of phospholipid molecules between particles. This early phospholipid transfer step does not correlate with the size change and possibly predisposes the particles to fusion. Recently, Rao and his colleagues (1997) confirmed the finding that no change in HDL size occurs in 1hr, indicating that the predominant activity of PLTP over a short time span is phospholipid transfer and not HDL fusion. It is possible that a net transfer of phospholipids to an HDL subpopulation leads to an increase in surface pressure, which causes the release of apoA-I-phospholipid complexes from the particle surface. The importance of apoA-I release in the fusion process is supported by the finding that rHDL reconstituted with apoA-I as the only protein was converted in the presence of PLTP, while rHDL with apoA-II alone was not (Lusa et al., 1996). Further support for the role of apoA-I was obtained from the study where human apoA-II was incorporated into pig HDL and the hybrid HDL particles formed were then used to study the conversion process (Pussinen et al., 1997b). Both the formation of large particles and the release of apoA-I were inhibited by increasing concentrations of apoA-II in the HDL particle. PLTP in this model could also cause the apoA-I displacement by directly interacting with apoA-I. Indeed, there is evidence that PLTP can bind to apoA-I and that the PLTP binding domain of apoA-I resides in the aminoterminal region of the protein (Pussinen et al., 1998). However, regardless of the actual mechanism of apoA-I release, the HDL particles become unstable after dissociation of apoA-I since such dissociation leads to a shortage of amphiphilic surface to cover the hydrophobic lipid core (Figure 14.1). The unstable particles then fuse to form a more stable particle with an increased diameter. The primary fusion particle can possibly undergo additional cycles of fusion. In this context it is interesting to note the recent observation that the CETPmediated reduction in reconstituted HDL size involves particle fusion (Rye et al., 1997). According to the mechanism postulated, CETP
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sequentially binds to two rHDL to form a ternary complex, after which fusion of the rHDL occurs. The fusion product is unstable and subsequently reorganises into three smaller particles. Taken together,
Figure 14.1 PLTP-mediated HDL conversion, based on the model proposed by Lusa et al. (1996).
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these data provide evidence that both plasma lipid transfer proteins, PLTP and CETP, may mediate the fusion of HDL particles. Details of the mechanisms of these fusion reactions remain to be demonstrated.
DISTRIBUTION AND REGULATION OF PLTP IN PLASMA In plasma the lipid transfer proteins are complexed with lipoproteins. Several studies have shown that most of the CETP is associated with HDL when lipoproteins are separated by gel filtration (Tall, 1995), whereas only a few reports have shown association of PLTP with lipoproteins. However, during density gradient ultra-centrifugation, PLTP is recovered in the smaller, denser HDL fraction (major activity between density 1.18–1.25g/ml); upon prolonged ultracentrifugation PLTP dissociates into the lipoprotein-free fraction similar to CETP (Tall et al., 1983b). In a study by Speijer et al., (1991) in which plasma was fractionated by gel filtration, the PLTP activity coeluted with a fraction of HDL particles with the size of human HDL2. In this study, CETP activity coeluted mainly with relatively small HDL particles. Information on the regulation of plasma PLTP activity is scarce. PLTP activity has been shown to correlate positively with the waist:hip circumference ratio, body mass index, the ratio of VLDL/LDL cholesterol ester concentration and alcohol consumption (Lagrost et al., 1996). It correlated with the amount of stearic acid in the diet (Aro et al., 1997). PLTP activity significantly increases during a high fat and cholesterol diet, whereas endotoxin or LPS injection reduces PLTP activity in C57BL/6 mice (Jiang and Bruce, 1995).
ANIMAL MODELS OF PLTP FUNCTION The role of PLTP in lipoprotein metabolism in vivo is poorly understood. Although in vitro studies suggest that PLTP influences HDL size and composition and transfers phospholipids among lipoproteins, the physiological function of PLTP has remained unclear. In order to extend in vitro studies to more physiological model systems, transgenic mice that express human PLTP have been generated in two studies. In the study of Jiang et al., (1996) plasma PLTP activity was increased by 29% in the PLTP transgenic mice. However, analysis of plasma lipoproteins revealed no significant changes in the plasma lipoprotein lipids or apolipoproteins when compared to control animals. When the human PLTP transgenic mice were cross-bred with human apoA-I transgenics, PLTP activity in this background increased by 47%, coupled with an increase in HDL phospholipids and apoA-I. A
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major increase of apoA-I (56%) was observed in the pre -mobile HDL fraction and a small increase in -HDL. The size distribution of HDL particle species did not change. These observations suggest that PLTP is involved in increasing the amount of potentially antiatherogenic pre -HDL particles. In another study (Albers et al., 1996a), there was no significant difference in PLTP activity between PLTP transgenic and non-transgenic mice. However, the HDL cholesterol/non-HDL cholesterol ratio increased significantly in the PLTP transgenics. Even though these studies do not give conclusive answers to the question of PLTP function in vivo, they indicate that PLTP is closely connected to the metabolism of HDL.
PLTP AND ATHEROSCLEROSIS A number of studies have demonstrated that PLTP can promote the conversion of HDL particles into new populations of large and small particles (see above). The small particles contain apoA-I as the only apolipoprotein and phospholipids and have a diameter of about 7.5nm (Jauhiainen et al., 1993; Pussinen et al., 1995). Further characterisation of the small apoA-I containing particles indicates that they have a high sphingomyelin:phosphatidylcholine ratio and they display pre mobility in agarose gel electrophoresis (Marques-Vidal et al., 1997; Pussinen et al., 1997b). Taken together, the characteristics of the small HDL particles produced by PLTP resemble those of the small pre HDL particles described previously (Castro and Fielding, 1988; Fielding and Fielding, 1995; Barrans et al., 1996). Recent data on cholesterol efflux from cultured fibroblasts suggests that PLTP-derived pre -HDL-like particles enhance the ability of plasma to stimulate cholesterol efflux from cells (Von Eckardstein et al., 1996). Furthermore, plasma PLTP activity has been shown to be a highly significant predictor of cholesterol efflux from Fu5AH rat hepatoma cells (Syvänne et al., 1996). These studies suggest a central role for PLTP in regenerating the primary acceptors of peripheral cholesterol. The function of PLTP in the reverse cholesterol transport pathway is shown in Figure 14.2. The overall role of PLTP in atherogenesis probably depends on the metabolic context in each subject. Although several factors involved in the reverse cholesterol transport pathway (LCAT, CETP, hepatic lipase) have been shown to be
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Figure 14.2 Function of phospholipid transfer protein in reverse cholesterol transport. Reverse cholesterol transport consists of four main steps: I. Efflux of free cholesterol (C) from peripheral cell membranes to primary acceptors, pre -HDL, in the extravascular space; II. Esterification of free cholesterol in pre -HDL by lecithin-cholesterol acyltransferase (LCAT); III. Transfer of cholesteryl esters (CE) in exchange of triglycerides (Tg) from HDL to other plasma lipoproteins (VLDL, IDL, LDL) by cholesteryl
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ester transfer protein (CETP); IV. Delivery of the cholesteryl esters to the liver via LDL and HDL receptors. The conversion of HDL by PLTP generates the primary acceptors of cell membrane cholesterol, pre -HDL. This pre -HDL regeneration process maintains the ability of plasma to stimulate cholesterol efflux from peripheral cell membranes. Large HDL particles formed in PLTP-mediated HDL-conversion can be delivered to the liver via the HDL receptor.
functionally important, the interplay between PLTP and these other factors has not been characterised and is therefore a stimulating topic for future work. Further progress in understanding the relationship of PLTP to human atherosclerosis will be achieved by studies on the relationships of genetic PLTP deficiency to coronary heart disease, in vivo studies in PLTP transgenic animals and analysis of the effects of dietary factors on PLTP. An important direction in future studies of PLTP is to elucidate the basic molecular mechanisms of PLTPmediated lipid transfer and HDL conversion. Acknowledgements The authors would like to express their gratitude to Drs. Vesa Olkkonen and Jarkko Huuskonen for their helpful suggestions concerning this chapter. References Albers, J.J., Tollefson, J.H., Chen, C-H. and Steinmetz, A. (1984). Isolation and characterization of human plasma lipid transfer proteins. Arteriosclerosis , 4 , 49–58. Albers, J.J., Tu, A-Y., Paigen, B., Chen, H., Cheung, M.C. and Marcovina, S.M. (1996a). Transgenic mice expressing human phospholipid transfer protein have increased HDL/non-HDL cholesterol ratio. International Journal of Clinical and Laboratory Research , 26 , 262–267. Albers, J.J., Tu, A-Y., Wolfbauer, G., Cheung, M.C and Marcovina, S.M. (1996b). Molecular biology of phospholipid transfer protein Current Opinion of Lipidology , 7 , 88–93. Albers, J.J., Wolfbauer, G., Cheung, M.C., Day, J.R., Ching, A.F.T. and Tu, A-Y. (1995). Functional expression of human and mouse plasma phospholipid transfer protein: effect of recombinant and plasma PLTP on HDL subspecies. Biochimica et Biophysica Acta , 1258 , 27–34. Aro, A., Jauhiainen, M., Partanen, R., Salminen, I. and Mutanen, M. (1997). Stearic acid, trans fatty acids, and dairy fat: effect on serum
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Nichols, A.V., Gong, E.L. and Tall, A.R. (1992). Modulation of HDL precursor structure and metabolism. In High Density Lipoproteins and Atherosclerosis III , edited by Miller, N.E. and Tall, A.R. Amsterdam: Excerpta Medica, pp. 79–87. Nishida, H.I. and Nishida, T. (1997). Phospholipid transfer protein mediates transfer of not only phosphatidylcholine but also cholesterol from phosphatidylcholine-cholesterol vesicles to high density lipoproteins. Journal of Biological Chemistry , 272 , 6959– 6964. Paszty, C., Maeda, N., Verstuyft, J. and Rubin, E.M. (1994). Apolipoprotein A-I transgene corrects apolipoprotein E-deficiency induced atherosclerosis in mice. Journal of Clinical Investigation , 94 , 899–903. Pussinen, P.J., Jauhiainen, M. and Ehnholm, C. (1997b). ApoAII/apoA-I molar ratio in the HDL particle influences phospholipid transfer protein-mediated HDL interconversion . Journal of Lipid Research , 38 , 12–21. Pussinen, P.J., Jauhiainen, M., Metso, J., Pyle, E., Marcel, Y.L., Fidge, N.H. et al. (1998). Binding of phospholipid transfer protein (PLTP) to apolipoproteins A-I and A-II. Location of a PLTP binding domain in the amino terminal region of apoA-I. Journal of Lipid Research , 39 , 152–161. Pussinen, P.J., Jauhiainen, M., Metso, J., Tyynelä, J. and Ehnholm, C. (1995). Pig plasma phospholipid transfer protein facilitates HDL interconversion. Journal of Lipid Research , 36 , 975–985. Pussinen, P.J., Olkkonen, V.M., Jauhiainen, M. and Ehnholm, C. (1997a). Molecular cloning and functional expression of cDNA encoding the pig plasma phospholipid transfer protein. Journal of Lipid Research , 38 , 1473–1481. Rao, R., Albers, J.J., Wolfbauer, G. and Pownall, H.J. (1997). Molecular and macromolecular specificity of human plasma phospholipid transfer protein. Biochemistry , 36 , 3645–3653. Rubin, E.M., Krauss, R.M., Spangler, E.A., Verstuyft, J.G., and Clift, S.M. (1991). Inhibition of early atherosclerosis in transgenic mice by human apolipoprotein A-I. Nature , 353 , 265–267. Rye, K-A. and Barter, P.J. (1986). Changes in the size and density of human high-density lipoproteins promoted by a plasma-conversion factor. Biochimica et Biophysica Acta , 875 , 429–438. Rye, K-A. and Barter, P.J. (1984). Evidence of the existence of a highdensity lipoprotein transformation factor in pig and rabbit plasma. Biochimica et Biophysica Acta , 795 , 230–237. Rye, K-A., Hime, N.J. and Barter, P.J. (1997). Evidence that cholesteryl ester transfer protein-mediated reductions in reconstituted high density lipoprotein size involve particle fusion. Journal of Biological Chemistry , 272 , 3953–3960. Speijer, H., Groener, J.E.M., van Ramhorst, E. and van Tol, A. (1991). Different locations of cholesteryl ester transfer protein and phospholipid transfer protein activities in plasma. Atherosclerosis , 90 , 159–168.
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15 Oxidation of Plasma Lipids and Lipoproteins J.M.Upston and R.Stocker Biochemistry Group, The Heart Research Institute, 145 Missenden Road, Camperdown, NSW 2050, Australia
According to the “oxidation theory” of atherosclerosis (Steinberg et al., 1989) the oxidative modification of low density lipoprotein (LDL) increases its atherogenicity and thus contributes to atherogenesis. Despite much research however, in vivo “oxidised LDL” remains largely undefined and there remains little direct evidence for a causative role of “oxidised LDL” to atherogenesis. By contrast, more progress has been made in our understanding of the molecular mechanisms underlying the oxidative modifications of LDL. In this chapter we propose to define the categories of oxidative modifications of LDL and other lipoproteins, and the role vitamin E plays in this. We also address the site of oxidation and the relationship between oxidised lipids in different lipoproteins in plasma and intima versus disease. KEYWORDS: Antioxidants, atherosclerosis, high density lipoproteins, lipid peroxidation, peroxidase, vitamin E.
INTRODUCTION Atherosclerosis remains the leading cause of death in many Western societies and plasma lipids emerge from epidemiology studies as significant risk factors. Thus, a strong correlation exists between plasma concentration of low density lipoprotein (LDL) and the incidence of coronary heart disease (CHD). In addition, abundant evidence implicates oxidatively modified LDL (referred to here as “oxidised LDL”) in initiating and/or promoting atherosclerosis in vivo. This chapter will discuss how LDL may become oxidised in vivo and
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preventative strategies that exist to modulate or remove oxidised lipids/lipoproteins. In addition, reasons for the apparent inverse relationship between high density lipoprotein (HDL) and CHD will be discussed. It should be noted that despite a clear association between oxidised lipoproteins (specifically LDL) and atherosclerosis, there is, as yet, no in vivo evidence for a causal role of such damage in this disease. Retention of LDL in the artery wall is an early and important event in atherosclerosis (see e.g., Schwenke and Carew, 1989). Characteristically, early lesions contain macrophages excessively loaded with lipid (foam cells) beneath an intact endothelium. High levels of LDL cholesterol normally downregulate the plasma membrane receptor for LDL in cells, including macrophages, which do not uncontrollably take up native LDL in vitro (Goldstein et al., 1983). However excessive cellular accumulation of LDL can be achieved in vitro by chemically modifying LDL (Goldstein et al., 1979) and such uptake is mediated by scavenger receptors. LDL incubated with various cells found in the arterial wall is also a substrate for scavenger receptors, and can induce foam cell formation in vitro (Henriksen et al., 1981). Importantly, lipid peroxidation is the common feature of the modified LDL that is a scavenger receptor substrate and added or contaminating transition metals are required for these cell-induced LDL modifications (Steinbrecher et al., 1984). From these early studies it has been proposed that oxidised LDL promotes cellular accumulation of lipids and foam cell formation in vivo and this contributes to the early stages of atherosclerosis (Steinberg et al., 1989; Ylä-Herttuala et al., 1989; Steinbrecher et al., 1990; Ross, 1993; Berliner and Heinecke, 1996). In addition to the above, the “oxidation theory of atherosclerosis” (Steinberg et al., 1989) is strengthened by findings that “oxidised LDL” exerts many pro-atherogenic activities in vitro (reviewed in (Berliner and Heinecke 1996)), and lipoprotein-like particles with oxidative modifications and oxidised apolipoprotein B100 (apo B) (Ylä-Herttuala et al., 1989; Hazell et al., 1996), lipids (Smith et al., 1967; Hoff and Hoppe, 1995) and oxidised lipid-protein adducts (Itabe et al., 1994) are all present in human lesions. Also, several (though not all (Fruebis et al., 1994)) lipid-soluble antioxidants slow the development of atherosclerosis in various hypercholesterolaemic animals (reviewed in Lynch and Frei, 1994). Low Density Lipoprotein LDL particles contain apo B embedded in the top layer of a spherical ball of lipid with both surface (cholesterol, phospholipid) and core (cholesterol ester, triglyceride) lipids. The oxidisability of various lipids is affected by the degree of lipid unsaturation, with
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polyunsaturated fatty acids, such as linoleate, being more vulnerable to oxidation than monounsaturated lipids (such as oleate) and cholesterol (Barclay et al., 1987; Barclay et al., 1990). Approximately 50% of LDL’s lipid contains polyunsaturated fatty acids, the majority of which are in form of linoleate esterified to cholesterol or the glycerol backbone of phospholipids (Stocker, 1994), making LDL an amenable substrate for peroxidation. Although the large proportion of polyunsaturated lipids in LDL make it a ready target for oxidation the lipoprotein also contains lipid-soluble redox-active compounds such as vitamin E, ubiquinol-10, and carotenoids (Table 15.1). Of these, vitamin E ( -tocopherol, -TOH) is, quantitatively (Esterbauer et al., 1987) and qualitatively the major lipid peroxidation chain-breaking antioxidant, as judged by careful investigations in homogeneous phase (Burton and Ingold, 1986). Thus it is not surprising that prevention of LDL lipid peroxidation (and prevention of atherosclerosis) has focused on the optimum level of vitamin E in LDL that is required to be protective against oxidative onslaughts (Jialal et al., 1995). Vitamin E and Ischaemic Heart Disease Epidemiological studies show an inverse relationship between ischaemic heart disease and plasma -TOH levels (Gey et al., 1991). It is less clear however, whether this
Table 15.1 Composition of human LDLa
Component
Weight %
mol/LDL
Protein Apolipoprotein B100
22±1.9
1
Lipids Free Fatty Acids
10–50
Phospholipids
700
Phosphatidylcholine Bisallylic
22.3±3.9
Hydrogensb
450 375
Cholesterol
9.6±0.7
600
Cholesterol Esters
42.2±3.8
1600
Cholesteryl linoleate
880
Cholesteryl arachidonate
95
Bisallylic Hydrogens
1165
Oxidation of Lipids and Lipoproteins
Triglycerides
5.9±2.7
395
180
Bisallylic Hydrogens
50
Total Bisallylic Hydrogens
1590
Antioxidants -Tocopherol
6–12
-Tocopherol
0.5
Ubiquinol-10c
0.5–0.8
Lycopene
0.2–0.7 0.1–0.4
-Carotene
a The values shown are derived from (Aviram et al., 1988; Esterbauer et al., 1992; Bowry and Stocker, 1993; Constantinescu et al., 1993; Reaven et al., 1993; Upston et al., 1996). b Bisallylic hydrogens refer to the polyunsaturated fatty acids in LDL (i.e., the most readily oxidisable lipid moieties of the lipoprotein particle). Linoleic and arachidonic acid contain 1 and 3 pairs of bisallylic hydrogens, respectively. c This range has been determined in our laboratory and is consistently higher than that reported by others (see eg., (Esterbauer et al., 1992)). Reasons for this have been discussed previously (Stocker, 1994).
relationship extends to dietary and/or supplemented vitamin E. For example, the Physician Health studies observed a reduced risk for CHD with supplemented (but not dietary) -TOH at ≥17 IU for men (Stampfer et al., 1993) and ≥60 IU per day for women (Rimm et al., 1993). In postmenopausal women however, the intake of 5–10 IU vitamin E per day from food (but not supplements) was inversely associated with a reduced risk of death from CHD (Kushi et al., 1996). In addition, short-term treatment with vitamin E appears to have no effect on the progression of CHD (Gillilan et al., 1977). Completed randomised trials (The Alpha-Tocopherol Beta Carotene Cancer Prevention Study Group 1994; Stephens et al., 1996) have provided inconclusive results (Gaziano, 1996).
IN VIVO LDL OXIDATION Where, how, and to what extent, LDL becomes oxidatively modified during atherogenesis remain largely unanswered questions. In addition to intrinsic properties of LDL (Table 15.1), factors that prolong the life/residence time of LDL may also be conducive to oxidation. For example, proteoglycans in the intima bind LDL and effectively “trap” the lipoprotein in the extracellular matrix. Lipoprotein retention in the
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intima and interaction with proteoglycans may be proatherogenic, e.g., pro-teoglycan-exposed LDL shows greater sensitivity than native LDL to in vitro oxidative modification by Cu2+ ions (Camejo et al., 1991; Hurt-Camejo et al., 1992). Putative Site of Oxidation It is generally upheld (Steinberg et al., 1989), though not proven, that lipoprotein/ LDL oxidation that contributes to atherosclerosis must occur in the arterial intima. In support, lipoprotein lipids in plasma are well protected from oxidation due to the robust antioxidant defenses, including small molecular weight antioxidants, protein sulphydryl groups, transition metal-binding proteins and enzymes (Stocker and Frei, 1991). It is noteworthy that LDL itself contains and in fact is the major transport vehicle for most lipid-soluble antioxidants of plasma (Table 15.1). Further, oxidised lipoproteins that may exist or form in plasma would be expected to be rapidly diluted by either hepatic clearance (Van Berkel et al., 1991) or accumulation in the arterial wall (Juul et al., 1996). Indeed, the concentration of oxidised lipids in plasma of healthy humans is extremely low, in the low nanomolar range, and there is no evidence that hydroperoxides are specifically associated with LDL (Bowry et al., 1992a). The situation is similar in patients with severe cardiovascular disease (Cleary et al., 1997), although plasma of these subjects shows signs of mild oxidative stress (though not damage), as indicated by a slightly elevated proportion of ubiquinone-10 to total plasma coenzyme Q (Cleary et al., 1997; Lagendijk et al., 1997). Using immunological techniques, oxidised LDL (Salonen et al., 1992a) and antibodies to “oxidised LDL” have been detected in circulation. In fact, Holvoet et al. (1995) suggested that circulating “oxidised LDL” may constitute a marker of unstable atherosclerotic disease. However, the specificity of anti-oxidised LDL antibodies for LDL and/or atherosclerosis remains questionable (Vaarala et al., 1993). A recent case-control study found no significant relation between autoantibodies against oxidised LDL and asymptomatic early atherosclerosis (Iribarren et al., 1997). Similarly, most studies find no association between serum anti-oxidised LDL antibodies and lipoprotein- or oxidation-related variables in circulation (Craig et al., 1995). Thus, while the precise relationship between immunologically detectable “oxidised LDL” and oxidative events in circulation remains inconclusive, the data do not support oxidative modification of LDL taking place in blood. Surprisingly, homogenates of advanced human plaque contain ascorbate, urate and -tocopherol ( -TOH) in quantities comparable to human plasma (Suarna et al., 1995) and sufficient to efficiently block
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LDL oxidation in vitro (Frei et al., 1988). Thus, at least some of the arguments generally put forward against the occurrence of oxidation of lipoproteins in circulation, also hold true for the intimal space. One way to overcome this conundrum is to speculate that LDL oxidation in the arterial wall occurs in “microenvironments” where oxidants are produced excessively and/or where the antioxidant shield is no longer intact. However, to date there is no direct evidence for such a microenvironment; specifically, biologically feasible conditions where LDL’s lipids become oxidised in the presence of -TOH plus ascorbate at the levels present in plaque homogenates, are not known. Extent of (Lipoprotein) Lipid Oxidation in Atherosclerotic Lesions Oxidised lipids are present in large quantities in atherosclerotic lesions (Glavind et al., 1952; Gilbert et al., 1969; Brooks et al., 1971; Harland et al., 1971; Carpenter et al., 1993). Several early studies noted in particular the presence of oxidised linoleate (Brooks et al., 1970; Harland et al., 1973; Teng and Smith, 1975). The latter is not surprising given that cholesterol linoleate (Ch18:2) represents the major oxidisable lipid in LDL (Esterbauer et al., 1992) and in addition to cholesterol, large quantities of cholesteryl esters (CE) are present in foam cells. As indicated earlier, many studies have demonstrated in vitro and in vivo proatherogenic activities of “oxidised LDL” (Berliner and Heinecke, 1996). The “oxidised LDL” used in most of these studies was generated in vitro, e.g., by prolonged exposure to nonphysiological form and concentration of cupric ion. Such treatment results in the rapid depletion of LDL’s -TOH, the formation and subsequent degradation of lipid hydroperoxides, and resulting oxidation of apo B (Esterbauer et al., 1992). Even the so-called minimallymodified LDL (MM-LDL) (Cushing et al., 1990) contains degradation products of phospholipid hydroperoxides (Navab et al., 1996) that are formed subsequent to depletion of LDL’s -TOH. While MM-LDL and “oxidised LDL” possess biological activities relevant to atherosclerosis, it remains largely unknown whether, and if so in what proportion, intimal LDL is oxidised to an extent comparable to MMLDL or even to in vitro Cu2+-oxidised LDL. Results from in vitro experiments carried out with “lesion LDL” (Ylä-Herttuala et al., 1989) have to be interpreted with caution, as the isolation procedure and/or the subsequent incubation likely cause oxidation of the lipoprotein beyond that present in vivo. Perhaps a more direct approach to this question is to ask how much -TOH is left in “lesion LDL” after isolation, as this reflects a minimal concentration of this antioxidant in intimal LDL. The ratio of -TOH to free cholesterol is consistently low in lesions, especially those rich in
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macrophage foam cells (Carpenter et al., 1995). This is suggestive that lesions are depleted of -TOH. However the excessive cholesterol accumulation occurring in lesions impacts on this ratio, giving misleading information on true -TOH levels. A more useful measure is to express -TOH levels relative to oxidisable lipid, particularly, Ch18:2. Using this approach, and when compared with plasma, -TOH levels are either not changed or, in fact elevated, in homogenates of advanced human lesions (Suarna et al., 1995) and apo B-containing low density fraction derived from them (Nui et al., in press). These findings are consistent with the presence of normal, if not elevated, levels of -TOH in plasma of patients suffering from severe atherosclerosis, compared with age-matched subjects (Cleary et al., 1997; Lagendijk et al., 1997). The above findings are consistent with most of the intimal lipid peroxidation occurring in the presence of -TOH. Alternatively, TOH may be successively depleted and subsequently replenished in response to oxidative stress. The distribution of regioisomers of primary oxidised lipids in atherosclerotic lesions may be used to delineate the two possibilities: in the presence of -TOH, a potent hydrogen donor, cis, trans lipid hydroperoxides accumulate preferentially, whereas in the absence of the vitamin, the thermodynamically more stable, trans, trans, isomers predominate (Kenar et al., 1996; Upston et al., 1996). Thus a ratio of trans, trans isomers greater than unity indicates lipid peroxidation occurring primarily in the absence of hydrogen donors. Preliminary findings (Upston, Terentis, Stocker unpublished) show however that cis, trans regioisomers of cholesteryl hydroxylinoleate are more abundant than trans, trans isomers in advanced human lesion, consistent with a majority (though not all) of the oxidised Ch18:2 being formed in the presence of -TOH. We conclude that the majority of oxidised lipids present in advanced human plaque are likely formed in the presence of -TOH, and that the majority of “lesion LDL”, while oxidised, may still contain vitamin E. Only a minority of “lesion LDL” may be oxidised to an extent beyond TOH depletion, representing a likely source/ site for the detected secondary lipid peroxidation adducts (Yl-Herttuala et al., 1989; Itabe et al., 1994). Putative In Vivo Oxidants Much research has concentrated on modelling LDL oxidation mediated by putative physiological oxidising species, including cells, in the presence of various antioxidants. In vitro, LDL lipid oxidation is initiated by a variety of oxidants including radical (i.e., one electron) and nonradical (i.e., nucleophilic) oxidants (Table 15.2). Several studies have attempted to determine the oxidants contributing to
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lipoprotein oxidation in atherosclerotic lesions. There is now good evidence for the participation of myeloperoxidase and oxidants derived from this enzyme. Other oxidants, both enzymic and nonenzymic, have also been suggested to be involved. Myeloperoxidase Catalytically active myeloperoxidase (MPO) is present in human atherosclerotic lesions and co-localises with lipid-laden macrophages (Daugherty et al., 1994). The enzyme generates both tyrosyl radicals (a one electron oxidant) and hypochlorite (−OCl, a nucleophilic oxidant), both of which oxidise LDL (Hazell and Stocker, 1993;
Table 15.2 Radical (one electron) versus nonradical (nucleophilic) oxidants
Radical Oxidantsa
Nonradical Oxidants
Aqueous and lipophilic peroxyl radicals (ROO•)
Singlet oxygen (1O2)
Cu2+, Fe3+, Ham’s F-10 medium, autoxidation Hydroxyl (•OH) superoxide anion radicals (O2 −•) SIN-1 (donor of O2 −• and nitric oxideb), peroxynitriteb
Peroxynitrite (−OONO)b
Horse radish peroxidase/H2O2 Myeloperoxidase/H2O2/Cl−/tyrosine (tyrosyl radical)
Hypochlorite (−OCl)c
Soybean and human recombinant 15-lipoxygenase Human macrophagesd a
Radical oxidants or conditions giving rise to radical oxidants. The IUPAC recommended names for nitric oxide and peroxynitrite are nitrogen monoxide and hydrogen oxoperoxonitrate, respectively. Peroxynitrite is in equilibrium with its conjugated acid, peroxynitrous acid (HONOO). c In equilibrium with its conjugated acid, hypochlorous acid (HOCl). d When cultured in transition metal-containing medium. b
Savenkova et al., 1994). Also, a monoclonal antibody raised against −OCl-modified LDL and that reacts with −OCl-modified proteins (Malle et al., 1995), recognises epitopes in early and advanced human lesions; epitopes are present in endothelial and foam cells, as well as in an apoB-containing lipoprotein fraction (Hazell et al., 1996). Furthermore, 3-chlorotyrosine, formed when L-tyrosine reacts with
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−OCl
or MPO in the presence of hydrogen peroxide and chloride ions, is present in LDL isolated from human lesions (Hazen and Heinecke 1997). −OCl-modified LDL is potentially proatherogenic as it can cause transformation of macrophages into foam cells (Hazell and Stocker, 1993), and increase leukocyte adherence and migration into blood vessels (Liao et al., 1995). 15-Lipoxygenase Evidence for a role of 15-lipoxygenase in atherogenesis is supported by findings that its mRNA and the enzyme (Ylä-Herttuala et al., 1990) and specific lipid oxidation products (Kuhn et al., 1994; Folcik et al., 1995) are found in human atherosclerotic lesions. Also, 15-lipoxygenase can oxidise LDL in vitro (Belkner et al., 1993; Upston et al., 1996; Upston et al., 1997), and cells overexpressing 15-lipoxygenase intracellularly, induce extracellular LDL oxidation (Benz et al., 1995; Ezaki et al., 1995). This process, in addition to directly modifying LDL, is proposed to “seed” the lipoprotein with lipid hydroperoxides which subsequently become substrates for transition metal ion-induced oxidation (Rankin et al., 1991). Furthermore, expression of 15-lipoxygenase in rabbit iliac arteries results in appearance of oxidised lipidprotein adducts characteristic of oxidised LDL (Ylä-Herttuala et al., 1995), and a specific and non-antioxidant inhibitor of 15-lipoxygenase has been reported to lower lesion formation in rabbits fed a high fat, high cholesterol diet (Sendobry et al., 1997). However, it is unclear whether 15-lipoxygenase is causal or symptomatic of atherosclerosis as transgenic mice fed a high cholesterol diet show reduced atherosclerosis compared to controls (Shen et al., 1996) and this antiatherogenic effect may be due to 15-lipoxygenase promoting CE catabolism (Kühn and Chan, 1997). Non-enzymic lipid peroxidation That free radical processes contribute to intimal lipoprotein oxidation is suggested from the presence of non-enzymically formed arachidonate oxidation products such as isoprostanes (Morrow et al., 1992) or protein adducts with fragments of oxidised lipids (Itabe et al., 1994). Formation of non-enzymic isoprostanes cannot be blocked by inhibitors of cyclooxygenase and can take place in situ on membrane phospholipids (Morrow et al., 1992); in contrast, enzymically formed prostanoids are formed only after cleavage of arachidonate from phospholipids. Two different esterified isoprotanes are present in human atherosclerotic lesions and colocalise with foam cells adjacent to the necrotic core (Pratico et al., 1997).
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Transition metals Free, unchelated transition metals (principally iron and copper) are essentially absent in vivo due to the presence of storage and binding proteins in saturating levels. Despite this, a general theme has developed that chronic and degenerative diseases, including atherosclerosis, are caused in part by oxidative damage and that iron contributes to the production of reactive oxygen species. Thus, it has been suggested that the risk of CHD increases with increasing body iron stores. In support of this, a prospective epidemiologic study of heart disease in Finnish men found that the risk of heart attack increased with increasing levels of serum ferritin (Salonen et al., 1992b). However, the vast majority of the epidemiologic data, including results from prospective, cross-sectional, and case-control and autopsy studies (Miller and Hutchins, 1994), published since that initial study have failed to support the original hypothesis that high body iron stores increase the risk of CHD (reviewed in Sempos et al., 1996; Corti et al., 1997). Less epidemiological information is available for copper although, it is observed that low serum copper correlates negatively with hypercholesterolaemia (Ferns et al., 1997). However, Salonen et al., 1991 reported elevated serum copper, measured as caeruloplasmin concentration, as an independent risk factor for ischaemic heart disease and copper ions (see below) and copper-containing caeruloplasmin (Ehrenwald and Fox, 1996) are putative in vivo LDL oxidants. However, patients with Wilson’s disease, which have increased redoxactive copper and oxidation products in plasma (Ogihara et al., 1995), are known to develop neurological rather than cardiovascular complications. Evidence for an involvement of transition metal ions in in vivo LDL oxidation is largely indirect (Parums et al., 1990). A commonly cited work in support for transition metals in intimal LDL oxidation (Smith et al., 1992) used materials previously frozen, and thawing of biological samples artefactually converts redox-inactive into redoxavailable transition metals (Gutteridge et al., 1985). Also the work-up procedure used (Smith et al., 1992) releases transition metals from healthy arteries (Suarna, Dean, Stocker, unpublished), casting doubt on the specificity of the results observed for atherosclerotic vessels. Recent work from the group of Heinecke (Leeuwenburgh et al., 1997a), quantifying markers for protein oxidation in low density lipoprotein isolated from human atherosclerotic plaques, argues against an involvement of copper (or hydroxyl radical), while supporting a role for MPO. Despite its questionable in vivo involvement, Cu2+ has been and
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continues to be used extensively to model LDL oxidation in vitro (Esterbauer et al., 1989). The reasons for this stem from earlier studies showing cell-induced LDL oxidation only in Ham’s F10 medium, which contains both Cu2+ and Fe3+ (Steinbrecher et al., 1984). As noted earlier, oxidative modification of LDL induced by endothelial and smooth muscle cells, monocytes or macrophages requires the presence of transition metals in the media (Kritharides et al., 1995; Garner and Jessup,1996). Free or loosely-bound iron or copper have the potential to initiate lipid peroxidation by catalysing the breakdown of preformed hydroperoxides (Wilcox and Marnett, 1993) within lipoproteins such as LDL, and this process is enhanced in the presence of reducing agents. Large amounts of keto-derivatives of Ch 18:2 (Suarna et al., 1997) are present in advanced atherosclerotic lesions and this may be viewed as indirectly supporting an involvement of transition metals. However, 15lipoxygenase (Schewe et al., 1986) (see above) and hydroxyoctadecadienoic (Bronstein and Bull, 1997) and hydroxyeicosatetranoic acid dehydrogenases (Zhang et al., 1996) also metabolise lipid hydro(pero)xides to the corresponding ketoderivatives. Transition metals could however participate in the generation of non-enzymic prostanoids (see above), and/or the formation of malonyldialdehye- and hydroxynonenal-protein adducts detected by immunological techniques (Jürgens et al., 1993) and above. The latter could, however, also be derived from −OCl-dependent mechanisms (Hazen et al., 1997). Reactive nitrogen intermediates Reactive nitrogen intermediates are potential (free) metal-independent pathways for LDL oxidation. Peroxynitrite is formed when nitric oxide, generated by several cells present in the arterial wall, reacts with superoxide anion radical (Beckman et al., 1990). Peroxynitrite is a powerful oxidant for LDL (Darley-Usmar et al., 1992; Jessup et al., 1992), and also converts tyrosine to 3-nitrotyrosine (van der Vliet et al., 1995). The latter has been proposed as a marker for peroxynitrite in vivo (Crow and Ischiropoulos, 1996). Whether the level of chemically measured nitrotyrosine is elevated in human atheroslcerotic lesions is controversial (Evans et al., 1996; Leeuwenburgh et al., 1997b). Using a polyclonal antibody raised against nitrotyrosine, extensive protein nitration in atherosclerotic lesions has been reported (Beckman et al., 1994). Thus, reactive nitrogen species could contribute to protein and LDL modification in the intima. If so, a possible role for peroxynitrite (−OONO) remains to be established, as nitrating species are also formed by −OCl in the presence of nitrite (Eiserich et al., 1996), and there is good evidence for the presence of −OCl in lesions (see above).
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IN VITRO LDL LIPID OXIDATION The nature and concentration of potential oxidants, -TOH and other antioxidants associated with and surrounding LDL will all impact on the progression of atherosclerosis if, indeed, LDL lipid peroxidation initiates and promotes the disease. If, as indicated above, oxidised lipids and relatively normal levels of -TOH coexist in lesion lipoproteins, a mechanism whereby substantial lipid peroxidation can occur in the presence of -TOH would be required to explain such a phenomenon. Lipid Peroxidation: Chain-Breaking Action of Vitamin E in Homogeneous Phase The mechanism of non-enzymic, free radical-induced lipid peroxidation has been studied most extensively in homogeneous solution. The first step is abstraction of a bisallylic hydrogen from a vulnerable lipid (polyunsaturated fatty acid containing bisallylic hydrogen, LH) by the peroxidation initiating radical, with consequent formation of a carbon-centred lipid radical. Essentially immediate addition of oxygen occurs in aerated conditions to yield a lipid peroxyl radical (LOO•). This latter radical is the lipid peroxidation chaincarrying species and its reaction with other LH generates lipid hydroperoxides and other propagating LOO•. In the absence of chainbreaking antioxidants, large quantities of lipid hydroperoxides accumulate, i.e., peroxidation proceeds with a long chain length, defined as the number of peroxides formed per initiating radical. Lipid peroxidation eventually terminates via radical-radical termination processes (see Burton and Ingold, 1986 for review). Chain-breaking antioxidants interact with, and hence remove, chainpropagating LOO•. Phenols are good chain breakers, and -TOH is the most active lipid-soluble chain-breaking antioxidant in humans (Burton and Ingold, 1986). -TOH readily donates a hydrogen atom (Ingold and Howard, 1962), and the resulting -tocopheroxyl radical ( -TO•) is relatively inert and, in homogeneous solution, readily undergoes radical-radical termination reactions (Burton and Ingold, 1986). Thus, as long as -TOH is present, lipid peroxidation in homogeneous solution is inhibited strongly, with chain lengths of 1. Following consumption of -TOH, peroxidation proceeds at much higher rates, resulting in a clear “break” in rate of lipid peroxidation. “Lag time” was introduced to describe the length of time during which lipid peroxidation is inhibited strongly.
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MOLECULAR ACTION OF VITAMIN E IN LDL Similar to the situation in homogeneous solution, oxidation of LDL with high relative (to apo B) concentrations of Cu2+ions (Esterbauer et al., 1989), is characterised by an initial period of inhibited lipid peroxidation (during which -TOH is consumed), followed by more rapid lipid peroxidation after depletion of -TOH (Esterbauer et al., 1992; Esterbauer et al., 1987). Such similarities between homogeneous phase and (heterogeneous) LDL lipid peroxidation was interpreted as -TOH acting as a “conventional” antioxidant for LDL’s lipids, and terms such as “lag phase” were adapted for LDL oxidation (Esterbauer et al., 1992; Esterbauer et al., 1987). However, subsequent work, initially carried out with chemically well-defined (peroxyl) radical oxidants, showed a number of anomalies in the activities of -TOH during LDL oxidation (summarised in Table 15.3), demonstrating that the conventional mode of action of the vitamin cannot hold true for LDL. Tocopherol-Mediated Peroxidation: A Model of In Vitro LDL Lipid Peroxidation that Occurs in the Presence of Vitamin E Studies employing low fluxes of radicals or ratios of Cu2+ to LDL≤3 show that in the absence of endogenous ubiquinol-10 and other lowmolecular-weight antioxidants (see below), -TOH in LDL can be prooxidant and lipid peroxidation can proceed as a chain reaction of substantial length in the presence of the vitamin (Bowry et al., 1992b; Bowry and Stocker, 1993). This prooxidant activity of -TOH in LDL is observed with all radical oxidants listed in Table 15.2, including Cu2+ions. The overall process whereby -TOH facilitates LDL lipid peroxidation is termed tocopherol-mediated peroxidation (TMP) (Bowry and Stocker, 1993). The features (Table 15.3) and underlying principles of TMP have been described previously in detail (Bowry et al., 1992b; Bowry and Stocker, 1993; Ingold et al., 1993; Stocker, 1994; Waldeck and Stocker, 1996; Witting et al., 1997). In essence, TOH acts as both a phase-transfer and lipid peroxidation chaintransfer agent in LDL (Figure 15.1). The first activity is due to the fact that the vitamin is the most redox active component on LDL’s surface and thus can aid the transfer of an aqueous radical into the lipoprotein particle. The chain transfer activity of -TOH is due to the fact that -TO•, the most stable radical that can be formed in oxidising, tocopherol-containing LDL, cannot escape the lipoprotein particle within a time window within which it can abstract a hydrogen atom from LH. Thus, unless the
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radical (located on -TO•) becomes eliminated by either a second incoming radical (giving rise to radical-radical termination) or by “export” with the help of a co-antioxidant (see below), -TO• will initiate and carry a chain of lipid peroxidation in LDL (Figure 15.1). TMP may be seen as retarded lipid peroxidation when compared with LDL lipid peroxidation proceeding in the absence of -TOH, as the reaction of LH with -TO• (the rate-limiting reaction of TMP) is slow Table 15.3 Features of lipid peroxidation in -TOH-containing, isolated LDL induced by a steady flux of aqueous peroxyl radicals 1. Marked differences in the kinetics of lipid peroxidation depending on the radical flux to which LDL is exposed (Bowry and Stocker, 1993; Witting et al., 1995; Neuzil et al., 1997a). 2. Rapid initial consumption of ubiquinol-10 followed by that of -TOH (Stocker et al., 1991). 3. Lipid peroxidation following ubiquinol-10 depletion proceeding in a chain reaction despite the presence of -TOH (Sato et al., 1990; Stocker et al., 1991). 4. Maximal rate of lipid peroxidation in post-ubiquinol-10 period independent of the rates of radical generation and lipid peroxidation initiation (Bowry et al., 1992b; Bowry and Stocker, 1993). 5. Inversed deuterium kinetic isotope effect for lipid peroxidation in postubiquinol-10 period (Witting et al., 1995). 6. Decreased rate of lipid peroxidation concomitant with depletion of TOH (Bowry and Stocker, 1993; Neuzil et al., 1997a).
-
7. Increase in the initiation and rate of lipid peroxidation by -TOH enrichment (Bowry et al., 1992b; Bowry and Stocker, 1993; Witting et al., 1995; Thomas et al., 1996a; Neuzil et al., 1997a). 8. Decrease or prevention of initiation and lipid peroxidation by depletion (Bowry and Stocker, 1993; Neuzil et al., 1997a).
-TOH
9. Overall pro- or anti-oxidant effect of -TOH depending on the radical flux to which LDL is exposed (Bowry and Stocker, 1993; Kontush et al., 1996; Neuzil et al., 1997a). 10. Strong suppression of lipid peroxidation by ubiquinol-10 or small phenolic and quinolic antioxidants kinetically inferior to -TOH (Mohr et al., 1992; Bowry and Stocker, 1993; Bowry et al., 1995; Thomas et al., 1996a). 11. Prevention of lipid peroxidation in oxidising LDL by ascorbate but not urate (“urate paradox”) (Bowry and Stocker, 1993; Bowry et al., 1995). Feature 3 is seen exclusively in LDL oxidising under low radical fluxes.
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Features 5–7, 10 are particularly apparent in LDL oxidising under low radical fluxes.
compared to that of LH with LOO• (the rate-limiting reaction of uninhibited peroxidation). However, given enough time, -TO• can peroxidise a majority of LDL’s lipids (Waldeck and Stocker, 1996). Oxidation of LDL lipids with TMP features have been reported by several other groups (Sato et al., 1990; Maiorino et al., 1993; Iwatsuki et al., 1995; Kontush et al., 1996; Gotoh et al., 1996). Importantly, TMP is a general feature for radical-induced oxidation of lipid emulsions, including isolated HDL (Bowry et al., 1992a), VLDL (Mohr and Stocker, 1994), lipoproteins in plasma (Neuzil et al., 1997a), and even aggregated lipoproteins, such as LDL-proteoglycan complexes (Morris and Stocker,
Figure 15.1 Tocopherol-mediated peroxidation (TMP) of LDL lipids and its inhibition. LDL lipid peroxidation initiated by one-electron oxidants can give rise to -TO•, which in turn initiates and propagates TMP of LDL lipids yielding lipid
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hydroperoxides (LOOH) and regenerating -TO• (reactions 3–5). LDL’s -TOH participates in the inhibition of lipid peroxidation in either of two ways: Under conditions of high radical flux, a second radical likely enters LDL and intercepts -TO• (reaction 2) before it can initiate TMP. This type of lipid peroxidation inhibition is associated with consumption of -TOH. Alternatively, -TO• is intercepted by a coantioxidant (XH) giving rise to a co-antioxidantderived radical (X•, reaction 6). Here, antioxidation is achieved by subsequent export of X• from LDL (reaction 7) and decay of X• to non-radical products (NRP, reaction 8). In this case, inhibition of LDL lipid peroxidation is associated with depletion of the co-antioxidant(s) rather than -TOH. Reaction 1 and reactions 3–5 refer to the phase- and chain-transfer activities of LDL’s -TOH, respectively.
unpublished). However, TMP can be relevant only for oxidising conditions that give rise to -TO•. Thus, nucleophilic oxidants, such as −OCl, oxidatively modify LDL independent of TMP, largely via direct reaction with apo B (Hazell and Stocker, 1993) that does not appear to be affected by -TOH (Hazell and Stocker, 1997). Inhibition of tocopherol-mediated peroxidation by coantioxidants: How vitamin E together with other antioxidants efficiently protects lipoprotein lipids against radicalinduced peroxidation The above illustrates that -TOH has the capacity to render lipoproteins more reactive towards one-electron radical oxidants (Table 15.2). Whether this results in an overall pro- or antioxidant activity depends on the fate of -TO•. The vitamin acts as an antioxidant as long as compounds are present that reduce -TO• and effectively eliminate the radical character from the lipoprotein by formation of relatively harmless aqueous radicals (Bowry et al., 1995). In fact, in the presence of such compounds (referred to as co-antioxidants) lipid peroxidation is effectively abated, particularly if -TOH also acts as the phase transfer agent, i.e., when radical reactions involving lipids and hence lipid hydroperoxide formation, are not involved. Inhibition of TMP by -TOH plus co-antioxidants represents a unifying model of defence against radical-induced lipoprotein lipid peroxidation, independent of the nature of the oxidant involved. It can also be considered a superior defence over the conventional inhibition of lipid peroxidation in lipoproteins by chain-breaking antioxidants: the
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latter depends on scavenging of LOO• which, compared to -TO•, is less accessible to aqueous (co-)antioxidants and more effective in propagating lipid peroxidation. A number of natural and synthetic co-antioxidants have been identified (Bowry et al., 1995; Witting et al., 1996). Natural coantioxidants include ubiquinol-10 (Stocker et al., 1991), ascorbate (Bowry and Stocker, 1993), billirubin (Neuzil and Stocker, 1994) and the tryptophan metabolite, 3-hydroxyanthranillic acid (Thomas et al., 1996b). Among these, ubiquinol-10 and ascorbate represent the first line of defence of lipophilic and aqueous coantioxidants, respectively (Frei, 1991; Stocker and Frei, 1991; Witting et al., 1997). Synthetic co-antioxidants include a number of hydroquinones and diphenols (Bowry et al., 1995; Witting et al., 1996). Among them, tocopheryl hydroquinone is the most active compound known to date. In addition to reducing -TO• in oxidising LDL, this hydroquinone maintains ubiquinol-10 in its reduced, co-antioxidant active form, and also directly scavenges incoming radicals (Neuzil et al., 1997b). Interestingly, -tocopheryl hydroquinone may be considered a physiological co-antioxidant, as nucleophilic oxidants like HOCl and peroxynitrite effectively convert -TOH to -tocopheryl quinone (Jessup et al., 1992; Hazell and Stocker, 1993), which itself may become reduced in vivo to the hydroquinone (Kohar et al., 1995). Tocopheryl hydroquinone is not found in native lipoproteins, though would be expected to be associated with them if derived from -TOH. Tocopherol-Mediated Peroxidation: A Possible Contribution to Intimal Lipid Peroxidation? As indicated earlier, advanced human atherosclerotic plaque contains large quantities of oxidised lipids, normal concentrations of -TOH, and the majority of the oxidised lipids appear to have formed in the presence of the vitamin. Principally, TMP could explain formation of these oxidised lipids if ascorbate, or other coantioxidants were absent or inaccessible, and the rate with which intimal lipoproteins encounter radical oxidants is sufficiently low to allow -TO• to initiate/propagate lipid peroxidation (Figure 15.1). Co-antioxidants in atherosclerotic lesions Only small amounts of co-antioxidants are required to afford LDL substantial protection against TMP (Bowry et al., 1995), and advanced lesions contain relatively large amounts of ascorbate (Suarna et al., 1995). Thus, it does not appear that the natural shield of coantioxidants is generally overwhelmed, unless compartmentalisation of co-antioxidants and lipid peroxidation is envisaged. For example, it is conceivable that the negatively charged ascorbate becomes unavailable
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to -TO• within oxidising intimal lipoproteins aggregated by proteoglycans containing negatively charged glycosaminoglycans. Such compartmentalisation has however not been demonstrated experimentally (see above). Equally however, there is no evidence at present that lipoprotein-associated co-antioxidants (for which a putative microenvironment is not relevant) are present in lesions. Indeed, of the two relevant lipid-soluble compounds, ubiquinol-10 and -tocopheryl hydroquinone, only the corresponding oxidised, co-antioxidant-inactive quinones are present in advance plaques (Suarna et al., 1995). Therefore, a lack of available co-antioxidants remains a plausible explanation for the occurrence of intimal lipid peroxidation in the presence of -TOH. Radical flux in the intima In addition to the availability of effective co-antioxidants, the rate with which intimal lipoproteins encounter radical oxidant/s is an important, confounding parameter (Figure 15.1). In the absence of co-antioxidants, high rates of radical encounter by LDL or other lipoproteins will result in rapid consumption of -TOH. This is associated with initial protection from peroxidation of the lipoprotein’s lipids and an apparent “lag phase”. Under lower fluxes of the same radical(s), -TOH in the same lipoprotein will be consumed more slowly, and such slow consumption of the vitamin is accompanied by significant lipid peroxidation, so that a (clear) “lag phase” is no longer observed (Bowry and Stocker, 1993). Thus, the length of “lag phase” in LDL undergoing oxidation, frequently used as a parameter to describe LDL “oxidisability”, reflects the outcome of a particular oxidising condition; it has no known bearing on the outcome of the same LDL exposed to different oxidising conditions. With other words, to assess the potential relevance of an in vitro test on in vivo “LDL oxidisability” both the oxidant and the oxidant: LDL ratio (or radical flux) must be considered (Stocker, 1994). With regards to the most commonly used in vitro “LDL oxidisability” test (Esterbauer et al., 1989), there is no convincing evidence that copper ions are biologically relevant (see above) and the ratio of copper to LDL used is extremely unphysiological. The frequency of radical encounter is determined by the rate of radical production, the efficacy with which aqueous antioxidants that surround lipoproteins scavenge initial radicals, and the concentration of lipoproteins in the intima. Judged by the amounts of apo B per protein, LDL is ≈ half as concentrated in atherosclerotic plaque than plasma (Pepin et al., 1991), while the concentration of aqueous antioxidants in extracellular fluids is somewhat lower than that in plasma (Dabbagh and Frei, 1995). Unfortunately, information on the rate of radical
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formation in the arterial wall is presently not available. However, an upper limit may be estimated from the maximal rate of superoxide anion generation of maximally stimulated neutrophils, and assuming that 0.5% of this becomes converted to hydroperoxyl radical, which can initiate LDL lipid peroxidation (Bedwell et al., 1989). The obtained value (1×10−8mol L−1 min−1) is significantly less than 2×10−7mol L−1 min−1, i.e., the rate of oxidation of LDL in the commonly used in vitro Cu2+/LDL “oxidisability” test (Esterbauer et al., 1989), as judged by TOH consumption. Overall, it thus appears feasible that intimal lipoproteins encounter conditions where TMP is relevant, and that a deficiency of effective coantioxidants rather than -TOH alone is responsible for the observed intimal lipid peroxidation (Thomas et al., 1995). Indeed, preliminary results suggest that supplementation with synthetic and natural coantioxidants inhibits intimal lipid peroxidation in hypercholesterolaemic animals used as animal models of atherosclerosis (unpublished). Future studies will be needed to verify this and to test whether, and if so to what extent, inhibition (or promotion) of intimal lipid peroxidation decreases (enhances) the progression of atherosclerosis.
ANTIOXIDANT CAPACITY OF HDL Whereas elevated plasma LDL levels remain a strong risk factor for atherosclerosis there is an inverse relationship between HDL and coronary heart disease (Stampfer et al., 1991). Although the exact mechanism underlying this protective action remains unknown it is commonly attributed to HDL’s ability to mediate reverse cholesterol transport from extrahepatic tissues to the liver for clearance. For example, HDL promotes cholesterol efflux from, and lowers intracellular cholesterol, in foam cells (Miyazaki et al., 1992; Fielding and Fielding, 1995). However reverse cholesterol transport does not fully explain HDL’s protective role in coronary heart disease. For example, hypertriglyceridaemic patients have relatively high levels of pre- HDL, the preferred cholesterol acceptor. Also, lecithin: cholesterol acyltransferase (LCAT) levels correlate negatively with HDL cholesterol, although LCAT drives cholesterol esterification in HDL thereby allowing cholesterol efflux from cells to the lipoprotein down a concentration gradient (Barter and Rye, 1996). As the most abundant lipoprotein in tissue fluids, HDL may perform a variety of additional functions that, in isolation or in total, may contribute to the lipoprotein’s protective effects. For example, HDL inhibits cytokine-induced expression of adhesion molecules (Cockerill et al., 1995) responsible for monocyte adherence to the endothelium, an
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early event in atherosclerosis. Also, HDL modulates a number of proatherogenic effects of oxidised LDL; it inhibits LDL oxidation induced by Cu2+ (Parthasarathy et al., 1990; Mackness et al., 1993) or human aortic cells (Navab et al., 1991) and blocks monocyte adhesion to endothelium induced by “oxidised LDL” (Maier et al., 1994). Thus the antioxidant capacity of HDL may be attributed to direct inhibition of LDL oxidation and/or curtailing certain bioactivities of oxidised LDL. Role of Associated Proteins in HDL’s Antioxidant Activities A number of proteins associated with HDL have been postulated to modulate the effects of oxidised LDL, including paraoxonase, platelet activating factor (PAF) acetylhydrolase, LCAT and apolipoprotein AI (apo AI). Indeed, the loss of apo AI, paraoxonase, and PAF acetylhydrolase from HDL isolated following an acute phase response correlates with a loss of HDL’s protective effect against LDL oxidation (Van Lenten et al., 1995). Paraoxonase Paraoxonase is an arylesterase whose biological substrate/s is unknown, however it catabolises organophosphates. Its activity in human serum is associated with a subset of HDL. Significantly lower levels of paraoxonase activity have been reported in patients with familial hypercholesterolaemia and insulin-dependent diabetes, diseases associated with atherosclerosis (Mackness et al., 1991a). However, a putative association between low paraoxonase activity and increased coronary artery disease (Serrato and Marian, 1995) has not been confirmed (Garin et al., 1997; Ruiz et al., 1995). HDL and purified paraoxonase decrease the level of lipid peroxides in LDL oxidation induced by Cu2+ ions when measured indirectly (Mackness et al., 1991b) and the degree of protection offered by HDL is related to its paraoxonase activity although this was not a strong association (R=0.47; p<0.06) (Mackness et al., 1993). These protective activities have been attributed to hydrolysis of oxidised phospholipids containing short acyl chains (Watson et al., 1995a). Thus, paraoxonase could play a role in preventing oxidative and proatherogenic changes to LDL in vivo, if these grossly oxidised lipids are important. Platelet activating factor acetylhydrolase The inflammatory actions of PAF in vivo are controlled by PAF acetylhydrolase, an enzyme associated with both LDL and HDL in
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plasma. In addition to its endogenous substrate, PAF acetylhydrolase acts on short chain acyl moieties in phospholipids that arise due to oxidation and subsequent fragmentation of the fatty acids (Stremler et al., 1991). PAF acetylhydrolase inhibits the bioactivity of oxidised LDL in vitro by facilitating hydrolysis, and hence removal, of active oxidised phospholipids in LDL (Watson et al., 1995b). The enzyme itself is sensitive to oxidation (Ambrosio et al., 1994; Dentan et al., 1994). Thus, to mediate the above effects, PAF acetylhydrolase may have to migrate between HDL and LDL, and/or substrates generated during oxidation of lipoproteins may have to “move” to the active enzyme. Lecithin: cholesterol acyl transferase LCAT esterifies cholesterol to fatty acids derived from lecithin and is crucial in reverse cholesterol transport as it lowers HDL’s cholesterol content and thereby maintains a gradient for this lipid from cells to extra-cellular acceptors. Although expression of human LCAT in mice elevates HDL cholesterol, it may also enhance reverse cholesterol transport by increasing the heterogeneity of HDL and stimulating apolipoprotein A formation. In addition, the level of proatherogenic VLDL and LDL is reduced in plasma of LCAT transgenic mice (Vaisman et al., 1995; Mehlum et al., 1995). Similar effects are observed in transgenic rabbits which, unlike rodents, also produce cholesteryl ester transfer protein (CETP) (Hoeg et al., 1996). Thus LCAT may be regarded as antiatherogenic although individuals who are deficient in LCAT generally do not present with premature atherosclerosis (see Kuivenhoven et al., 1997 for review). In addition to the above activities, there is some evidence that HDLassociated LCAT may protect against iron-induced oxidation of LDL (Klimov et al., 1989). LCAT has intrinsic phospholipase A2 activity, and hence may lower the content of oxidised phospholipids in oxidised lipoproteins, similarly to PAF acetylhydrolase. In addition, LCAT appears to act on both oxysterols (Szedlacsek et al., 1995) and oxidised fatty acids of oxidised phospholipids (Nagata et al., 1996). This gives rise to oxidised cholesteryl esters associated with HDL that can be taken up “selectively” by liver cells and hence provides a potential clearance route for potentially proatherogenic, oxidised lipids. Apolipoproteins AI and AII Apo A in HDL also exhibits activities that may be antiatherogenic. For example, im-munoisolated apo AI-containing HDL contains both caeruloplasmin and transferrin which can decrease transition metalinduced LDL oxidation in vitro (Kunitake et al., 1992). In addition,
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HDL also directly reduces lipid hydroperoxides, formed in the lipoprotein upon exposure to aqueous peroxyl radical-induced oxidation, to the corresponding hydroxides (Sattler et al., 1995). Such reduction of lipid hydroperoxides occurs concomitantly with the oxidation of specific methionine residues of apo AI and apo AII to methionine sulfoxides (Garner et al., 1998). As lipid hydroxides no longer breakdown to secondary radicals and other reactive moieties, this could represent yet another antioxidant activity of HDL which may contribute to its overall antiatherogenic effects. Potential Role of HDL in the Detoxification of Oxidised Lipoprotein Lipids Exchange of lipoprotein core lipids between different lipoprotein classes is mediated by CETP (Tall, 1993), however whether CETP activity is pro- or antiatherogenic in vivo is unresolved. CETP may be proatherogenic as its concentration and/or activity is increased in many human dyslipidaemias associated with atherosclerosis and species with low plasma CETP activity are generally resistant to diet-induced atherosclerosis (Tall, 1986). In addition, CETP transgenic mice with hypertriglyceridaemia have low plasma HDL cholesterol, diminished HDL particle size and an increased fractional catabolic rate of apo AI, all of which are symptomatic of human atherosclerosis (Hayek et al., 1993). Other studies show however that in probucol-induced reversal of atherosclerosis in animals, CETP is also increased (McPherson et al., 1991). Mediating the transfer of CE from HDL to LDL, CETP may be seen as antiatherogenic (by contributing to reverse cholesterol transport) or proatherogenic (by generating atherogenic lipoproteins). Whether CETP promotes cholesterol and oxidised or unoxidised CE efflux from arterial foam cells is not known. Both macrophages and smooth muscle cells in the vasculature have the capacity to synthesise CETP, so that the protein is likely present in the intima. In addition, CETP can mediate the transfer of oxidised CE from LDL to HDL (Christison et al., 1995), and oxidised CE in HDL are selectively taken up at a faster rate than unoxidised CE by hepatocytes in vitro (Sattler and Stocker, 1993; Christison et al., 1996) and in vivo (Fluiter et al., 1996). In advanced lesions, oxidised lipids are present in apoAI- and apoB-containing high- and low-density fractions, respectively, so that transfer of these potentially proatherogenic, oxidised lipids between different particles is likely to occur. The direction of this transfer will depend on the concentration of both oxidised lipids in the different particles and the relative pool size of donor/acceptor particles. Of the lipoprotein pool, HDL is quantitatively the major lipoprotein and HDL particles have a relatively high turnover rate. These properties coupled
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16 Plasma Lipoprotein Disorders and Endothelial Function Mark R.Adams and David S.Celermajer * Department of Cardiology, Royal Prince Alfred Hospital, Missenden Road, Camperdown, New South Wales 2050, Australia
The endothelium is a monolayer of cells between the vascular lumen and smooth muscle. It has become apparent over the last two decades that the endothelium plays a role in a wide array of homeostatic functions within healthy vessels. Hypercholesterolaemia is associated with multiple abnormalities of endothelial function, including disturbances in the normal control of vascular tone, platelet adhesion, coagulation and leukocyte adhesion to the endothelium. Several techniques have been developed to allow the measurement of endothelium-dependent vasodilator function, and have demonstrated that endothelial dysfunction may be present from an early age in the presence of hypercholesterolaemia. Endothelial abnormalities seem to relate especially to oxidised LDL and endothelial dysfunction plays an important role in the process of atherogenesis. Other lipoproteins may also play crucial roles in this process; in particular HDL cholesterol has important protective actions at the endothelial level. The process of endothelial dysfunction is potentially reversible and such reversibility may represent an early step in the regression of atherosclerotic changes. In addition, improvements in endothelial function are associated with a survival benefit and with a reduction of cardiovascular events. Although further investigation is required, the use of cholesterol lowering agents and novel compounds such as antioxidants or NO donors may provide clinical benefit in terms of improving arterial physiology, decreasing ischaemic
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episodes and preventing mortality associated with atherosclerotic vascular disease. KEYWORDS: Lipids, Endothelial physiology, Atherosclerosis, Nitric Oxide.
INTRODUCTION Atherosclerosis first develops in early childhood, with subendothelial accumulation of lipid in the aorta. Macroscopic changes such as fatty streaks and early atherosclerotic lesions have been documented in the coronary arteries of teenagers and young adults (Stary, 1989). The development of such lesions has been related to the presence of vascular risk factors, and in particular the presence of hyperlipidaemia, especially LDL and VLDL subfractions (PDAY investigators, 1990). Recently, investigators have shown that endothelial dysfunction may develop in children with *Corresponding Author. Department of Cardiology, Royal Prince Alfred Hospital, Missenden Rd, Camperdown, 2050, Sydney, Australia
vascular risk factors, before the appearance of macroscopic changes (Celermajer, 1992; Sorensen, 1994). In experimental animals, hypercholesterolaemia perturbs a number of endothelial functions in a dose-dependent and potentially reversible manner. In total, these findings suggest that endothelial dysfunction is a key early event in the development of atherosclerosis and that dyslipidaemias have an important role in the pathogenesis of abnormal endothelial physiology. The possible mechanisms by which atherogenic lipids interfere with normal endothelial function will be discussed in this chapter. Therapeutic manipulation of plasma lipids has been shown in large clinical trials to reduce morbidity and mortality from cardiovascular events and to reduce the incidence of both painful and silent myocardial ischaemia (Scandinavian Simvastatin Survival Study Group, 1994; Shepherd et al., 1995). Despite these clinical improvements, however, there is often very little regression of atherosclerosis plaque burden in the coronary arteries (Brown and Maher, 1994; Blankenhorn et al., 1993; Brown, 1990). One of the reasons for this clinical improvement may be a restoration of normal endothelial function, with decreased vasoconstriction and increased dilatation, which has been shown to accompany cholesterol lowering (Treasure et al., 1996; Anderson et al., 1996). Strategies for the potential reversal of endothelial dysfunction will therefore also be addressed.
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ENDOTHELIAL FUNCTION/DYSFUNCTION Over the last two decades it has become apparent that the vascular endothelium is not simply an inert cellular barrier, but is a dynamic organ which performs a wide array of homeostatic functions (Celermajer, 1997) (Figure 16.1). As the endothelium forms a monolayer of cells between the vascular lumen and smooth muscle, the endothelium is in a position to regulate smooth muscle tone and growth within the vessel wall, to sense haemodynamic changes within the vessel and to influence events within the vessel lumen, such as platelet aggregation, thrombosis, vessel permeability and leukocyte adhesion (Rubanyi, 1993). Regulation of Vascular Tone One of the most important roles the endothelium plays is in the control of vascular smooth muscle tone. The endothelium regulates tone via transduction of blood borne signals, modification of circulating factors, such as angiotensin and adenosine, and by the direct production and secretion of various vasoactive substances. The most important of these endothelial products that have so far been described are the potent vasodilators prostacyclin and endothelium-derived relaxing factor (EDRF), or nitric oxide (NO), as well as the vasoconstricting agents endothelin-1 and thromboxane. Nitric Oxide The existence of an endothelium-derived relaxing factor (EDRF) was first demonstrated by Furchgott and Zadwadzki (1980), after they observed that aortic rings denuded of endothelium paradoxically constricted in the presence of acetylcholine. Since this time it has been demonstrated that the activity of EDRF is mediated by nitric oxide (NO) or a closely related nitrosothiol compound (Palmer, 1987). Nitric oxide is formed within endothelial cells by the enzyme nitric oxide synthase (NOS) in response to a number of both pharmacologic and physical stimuli (Palmer et al., 1988). L-arginine is the physiologic substrate for nitric oxide synthase in a reaction which is stereo-specific; the products of this reaction are nitric oxide and L-citrulline. Nitric oxide synthase can be blocked by a number of competitive inhibitors, such as NG-monomethyl-L-arginine (L-NMMA), which has facilitated the in vitro and in vivo research of nitric oxide synthase. Nitric oxide has an extremely short half-life, and is secreted continuously in the resting state by healthy endothelium (Hartmann et al., 1987; Vallance et al., 1989). For this reason NO is important in
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maintaining arterial relaxation in both systemic and pulmonary circulations (Stamler et al., 1994). The endothelial release of NO is further stimulated by physical factors such as shear stress in response to blood flow (Bassenge and Heusch, 1990) and in response to a variety of other circulating agents such as acetylcholine, bradykinin, thrombin and serotonin, which act through specific endothelial cell receptors (Mehta, 1995). The action of NO on vascular smooth muscle cells is mediated by an increase in the intracellular activity of guanylate cyclase, with a resultant increase in the levels of cyclic guanosine monophosphate (cGMP) (Arnold et al., 1977). This in turn results in a reduction in intracellular calcium and therefore smooth muscle relaxation (Fösterman et al., 1986).
Figure 16.1 Functions of the normal endothelium. The endothelium secretes factors into the vessel lumen influencing coagulation (eg., prostacyclin, tissue plasminogen activator) and platelet adhesion and aggregation (eg., prostacyclin, thromboxane, nitric oxide). Cell adhesion molecules expressed on the luminal endothelial cell surface (such as intercellular adhesion molecule-1 and vascular cell adhesion molecule1) regulate leukocyte adhesion. Other factors secreted abluminally (such as nitric oxide, endothelin and prostacyclin) regulate vessel tone and growth, and in the endocardium may also influence myocardial contractility.
Apart from its important role in the maintenance of vascular relaxation, NO has been shown to have many other important actions within the vasculature. These include inhibition of platelet aggregation
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and adhesion (de Graaf et al., 1992; Yao et al., 1992), inhibition of leukocyte adhesion (De Caterina et al., 1995; Adams et al., 1997), the modulation of vascular smooth muscle cell growth and the modulation of endothelin production (Zeiher, 1996). The range of these important functions has led investigators to propose that NO is an important antiatherosclerotic agent (Cooke and Tsao, 1994). Arachidonic acid metabolites Endothelial cells contain the enzyme cyclooxygenase which produces prostacyclin from arachidonic acid (Moncada et al., 1977). Prostacyclin is a powerful vasodilator in both systemic and pulmonary circulations and is produced by endothelial cells in response to a variety of stimuli including acetylcholine, histamine, platelet-derived growth factor and bradykinin (Moncada et al., 1977). Prostacyclin causes an increase in cyclic adenosine monophosphate (cAMP) within smooth muscle cells leading to relaxation, and in addition prostacyclin has antiplatelet and antithrombotic activity (Dinerman and Mehta, 1990). Another important product of arachidonic acid produced by the endothelium is thromboxane A2, which is a powerful vasoconstrictor and promoter of thrombosis and platelet aggregation (Rubanyi, 1993). Endothelins In 1985, Rubanyi and Vanhoutte described a 21 amino acid peptide which was secreted by the endothelium in response to hypoxia, and which caused powerful vasoconstriction. This substance has since been purified, sequenced (Yanagisawa et al., 1988) and extensively studied. There are at least three isoforms of endothelin, however, only one of these isoforms, endothelin-1 (ET-1), has been isolated from human endothelial cells (Masaki, 1989). As well as hypoxia, ET-1 is released from endothelial cells in response to a variety of other stimuli, such as adrenaline, and like NO, it has a short half-life (Anggard et al., 1989). Small concentrations of ET-1 are present in healthy adults, and it may play a role in the maintenance of vascular resistance (Davenport et al., 1990), however, although increased levels have been observed in systemic hypertension and coronary artery disease, its pathophysiological role in these conditions is uncertain (Cody, 1992). Endothelium-derived hyperpolarising factor The endothelium also produces a factor which causes hyperpolarisation of smooth muscle cells, independent of nitric oxide and cGMP (Feletou and Vanhoutte, 1988). This endothelial factor increases potassium ion conductance thereby causing hyperpolarisation and relaxation of
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smooth muscle cells. The exact identity of this factor and its significance has not been determined (Cohen and Vanhoutte, 1995). Regulation of Thrombosis and Haemostasis The endothelium plays an important role in the maintenance of blood fluidity by influencing both platelet aggregation and thrombosis as well as fibrinolysis. Both prostacyclin and NO released from endothelial cells are potent inhibitors of platelet aggregation and may act in a synergistic manner (de Graaf et al., 1992; MacDonald et al., 1988). Similarly there appears to be a synergistic effect between NO, prostaglandin E2, and tissue-plasminogen activator to disaggregate platelets (Stamler et al., 1989). These important interactions between platelets and endothelial cells not only regulate platelet aggregation but also effect vascular tone and the coagulation system (Moncada et al., 1977). In addition the endothelium has a number of other actions which have a direct effect on the coagulation system. The endothelium inactivates thrombin (Machovic, 1986) and enhances protein C formation by a thrombin-thrombomodulin interaction (Esmon, 1987). Endothelial cells also secrete the powerful thrombolytic agent tissuetype plasminogen activator in response to procoagulant stimuli such as stasis, noradrenaline and thrombin (Hekman and Loskutoff, 1987). The endothelium also plays a role in haemostasis by secreting procoagulant factors such as collagen and fibronectin towards the basal lamina, where they are only exposed to circulating blood if endothelial integrity is disrupted (Dinerman and Mehta, 1990). Regulation of Leukocyte Adhesion In normal, healthy blood vessels, leukocytes circulate freely within the vascular space, although they may quickly adhere to the endothelium and migrate into the subendothelial space in the presence of inflammation. The adhesion of leukocytes, in particular monocytes, to the endothelium is thought to be an important early step in atherogenesis (Ross, 1993). The endothelium plays a crucial role in controlling leukocyte adhesion, chiefly by modulation of cell adhesion molecules expressed on the surface of endothelial cells (Jang et al., 1994). Molecules such as intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E selectin are expressed in response to a variety of stimuli including hypercholesterolaemia (Li et al., 1993) and cytokines such as interleukin-1 . Other factors, such as endothelium-derived NO and antioxidants have been shown to suppress the expression of some of these cell adhesion molecules through the common pathway of inhibition of nuclear factor-kB (NF-kB) (De Caterina et al., 1995;
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Weber et al., 1994). Endothelial Dysfunction As endothelial cells perform a wide range of regulatory functions, it is clear that endothelial injury, leading to dysfunction, could result in a series of cellular and molecular interactions involving endothelial cells, platelets, leukocytes and smooth muscle cells, potentially leading to atherogenesis. The concept of endothelial damage leading to the initiation of atherogenesis is at the heart of the “response to injury hypothesis” of atherosclerosis proposed by Ross and Glomset (1976). Abnormal endothelial function may lead to increased adhesion of monocytes to the endothelium and their transmigration into the subendothelial space. Monocytes may then take up oxidised LDL via both oxidised LDL receptors and scavenger receptors to form foam cells within the vessel wall (Goldstein et al., 1983), leading to the development of fatty streaks and atherosclerosis. Other important consequences of endothelial dysfunction include platelet aggregation and adhesion, with the release of various growth factors and the migration, proliferation and differentiation of smooth muscle cells (Ross, 1993). Although the exact mechanism leading to the initiation of this process is unclear, a number of factors associated with endothelial injury have been identified, in particular the adverse effects of oxidised LDL (Steinberg, 1991). Other causes of endothelial dysfunction include physical factors such as shear stress, chemicals such as homocysteine and oxygen-derived free radicals. An important sign of endothelial dysfunction is a reduction in the bioavailability of nitric oxide in the vessel wall. In the setting of hypercholesterolaemia, it is unclear whether this loss of NO activity is due to a decrease in the endothelial production of NO, and/or due to increased degradation by factors such as superoxide radicals. This reduction in NO activity is thought to be a key factor in atherogenesis, as NO itself has important antiatherosclerotic actions (Cooke and Tsao, 1994). The reduction in local NO availability has been documented in many groups of human subjects at risk of atherosclerosis, including young children with hypercholesterolaemia (Celermajer et al., 1992), passive and active cigarette smokers (Celermajer et al., 1993; Celermajer et al., 1996), diabetic subjects (Clarkson et al., 1996) and hypertensives (Yokokowa et al., 1991). As well as the important contribution of endothelial dysfunction to atherogenesis, endothelial dysfunction may play a role in the development of ischaemic syndromes. Impaired endothelial vasodilator function at the site of coronary artery stenoses may lead to vasoconstriction in response to physiologic stimuli such as exercise and increased blood flow (Gordon et al., 1989; Nabel et al., 1990). There is
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evidence that this endothelial dysfunction may be reversible by reversing the causative agent (Anderson et al., 1995; Treasure et al., 1995) or by the use of a variety of other therapies, ranging from antioxidant vitamins to hormonal therapy (Levine et al., 1996; Lieberman et al., 1994).
CLINICAL ASSESSMENT OF ENDOTHELIAL DYSFUNCTION A number of techniques have been developed during the last decade to examine endothelial function in vivo (Celermajer, 1997). These techniques have variously examined endothelial function in conduit arteries and resistance vessels, in both the coronary and the peripheral circulations. One common feature that most of these techniques share is that they rely on detecting the degree to which the endothelium is able to produce nitric oxide, in response to a number of physical and pharmacologic stimuli. Other measures of endothelial damage and/or dysfunction that have been assessed have ranged from the examination of serum markers of endothelial damage to the measurement of nitric oxide metabolites in urine. Coronary Artery Endothelial Function The in vivo assessment of coronary artery endothelial function was described by Ludmer and colleagues in 1986. With this technique, coronary artery diameter was measured using quantitative angiography, and then the change in diameter was measured in response to the intracoronary infusions of acetylcholine. Subsequent investigation has demonstrated that the vasodilatory response to acetylcholine is dependent on functional endothelium and that this endotheliumdependent dilatation is dependent mainly on endothelial production of NO (Hodgson and Marshall, 1989; Lefroy et al., 1993). Where there is endothelial dysfunction, the infusion of acetylcholine results in paradoxical vasoconstriction, due to the direct vasoconstricting effect of acetylcholine on smooth muscle cells. The endothelium-dependent response to acetylcholine is then contrasted with the vasodilatory response to an exogenous nitrate such as nitroglycerin, which releases NO within smooth muscle cells and therefore causes an endotheliumindependent dilatation. Investigators measuring coronary artery endothelial function have also utilised other pharmacologic agents to induce endothelium-dependent dilatation, such as substance P or serotonin, and have also observed endothelium-dependent dilatation in response to physiologic stimuli such as increased blood flow (Nabel et al., 1990), exercise (Gordon et al., 1989) and even mental stress
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(Yeung et al., 1991). The coronary microcirculation endothelial function can also be assessed invasively, often at the same time as the conduit arterial function is being assessed. Once again intracoronary acetylcholine infusions or other stimuli are used to provoke resistance vessel endothelium-dependent dilatation. Rather than assessing changes in vessel diameter, changes in intracoronary flow velocity are measured using Doppler catheters or flow wires (Drexler and Zeiher, 1991). The investigation of coronary artery physiology using these techniques has confirmed the pivotal role of the endothelium in maintaining vascular control and the way it may be disturbed in the presence of disease. As well as endothelial dysfunction being present in atherosclerotic arteries, endothelial dysfunction was documented in angiographically normal arteries where disease existed elsewhere (Zeiher et al., 1991) and that endothelial dysfunction predated the development of atherosclerosis (Drexler and Zeiher, 1991). Thus the measurement of endothelial function has allowed the relationship between cardiovascular risk factors and early atherosclerosis to be explored, and to assess various interventions aimed at the reversal of these early changes. Despite these important advances, the above technique has been limited in studying the very early changes of atherosclerosis because of its invasive nature. Although the technique carries no appreciable risk over that of coronary angiography (Zeiher et al., 1989), it is not feasible to study children or even asymptomatic adults. As the coronary circulation can only be adequately imaged using invasive catheter-based techniques, at the present time, noninvasive methods have examined arteries in the peripheral circulation. Peripheral Arterial Endothelial Function A method of assessing the endothelial function of forearm microcirculation was described by Panza et al. in 1990. This method utilises an intra-arterial infusion of acetylcholine into the brachial artery as a stimulus for endothelium-dependent dilatation of the forearm microvasculature. Changes in blood flow are then measured using venous strain gauge plethysmography. This technique has provided investigators with valuable information regarding microvascular endothelial dysfunction in the presence of a variety of risk factors, such as hypercholesterolaemia (Calver et al., 1992; Creager et al., 1990) and has been utilised as an endpoint in studies of reversing endothelial dysfunction (Creager et al., 1992). However, although this technique is less invasive than coronary artery studies, it still requires placement of an intra-arterial catheter and complex infusion protocols, along with the need to control a range of external factors to ensure adequate reproducibility (Creager et al., 1990).
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A technique for measuring peripheral artery flow mediated dilatation has recently been described (Celermajer et al., 1992), which is completely non-invasive and utilises high-resolution external vascular ultrasound to measure changes in brachial or femoral artery diameter in response to hyperaemia, and in response to the administration of sublingual nitroglycerin. The normal vasodilator response of the artery to increased flow is dependent on the endothelial release of NO, and can be blocked by the infusion of L-NMMA (Joannides et al., 1995). Furthermore, by comparing this response to that elicited by endothelium-independent stimulus of nitroglycerin, conclusions can be drawn regarding endothelial function. This technique is particularly useful as it has been shown to correlate well with endothelial function measured in the coronary circulation, and with the degree of coronary atherosclerosis seen on angiography (Anderson et al., 1995; Neunteufl et al., 1997). Of particular interest however, is that this non-invasive technique provides insights into the very early stages of atherosclerosis, demonstrating that even as early as the age of four years endothelial dysfunction may have developed in those at risk (Celermajer et al., 1993). In addition, the serial study of young asymptomatic adults is possible, allowing the evaluation of potential strategies to reverse the early stages of atherosclerosis.
HYPERLIPIDAEMIA AND ENDOTHELIAL DYSFUNCTION Numerous in vitro studies as well as experimental animal models of atherosclerosis and human in vivo studies have demonstrated a link between dyslipidaemias and endothelial dysfunction (Freiman et al., 1986; Andrews et al., 1987; Creager et al., 1990). Although the mechanism whereby dyslipidaemias lead to atherogenesis has not been fully elucidated, they have been implicated in abnormalities of most of the important functions that endothelial cells perform, including regulation of vascular tone, endothelial adhesiveness and thrombosis. Loss of Endothelium-Dependent Dilatation Hypercholesterolaemia Since the demonstration of the existence of an endothelium-derived relaxing factor, numerous investigators have found that there is a marked decrease in its activity in the presence of hypercholesterolaemia. In the hypercholesterolaemic rabbit model of atherosclerosis, ex vivo and in vivo endothelium-dependent dilatation in response to acetylcholine is impaired early in the atherogenic process
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(Jayakody et al., 1987; Chappell et al., 1987; Cooke et al., 1991). In fact, endothelium-dependent dilatation may be impaired within hours of exposure to increased levels of cholesterol. Similar findings have been noted in the large vessels of cholesterol fed primates, in response to acetylcholine and thrombin (Freiman et al., 1986), as well as in the coronary microcirculation in response to acetylcholine and bradykinin (Sellke et al., 1990). Hypercholesterolaemia has also been shown to affect endotheliumdependent dilatation of conduit vessels in man. Vita and colleagues (1990) have studied the response of human coronary arteries to acetylcholine. The investigators found that endothelium-dependent dilatation was reduced not only in the presence of atherosclerosis (Ludmer et al., 1986), but also that endothelium-dependent dilatation could be markedly abnormal even in angiographically smooth coronary arteries, and furthermore that the degree of this impairment was related to the presence of coronary risk factors. In particular the relationship between total cholesterol and the acetylcholine response was quite strong, with an r value of −0.58. Similar results have been found by other groups investigating coronary physiology; Zeiher et al. (1991) for example found a progressive loss of normal coronary responses to acetylcholine with increasing progression of atherosclerosis from angiographically normal arteries in subjects with hypercholesterolaemia to arteries with advanced atherosclerosis. This progressive decline in endothelium-dependent dilatation is not limited to large coronary arteries, but has also been demonstrated in the microcirculation (Drexler et al., 1991). Impairment of endothelium-dependent dilatation has also been demonstrated in the peripheral circulation in hypercholesterolaemia. This has important clinical research implications, as it facilitates the assessment of endothelial function and assessment of interventions aimed at reversing this dysfunction in otherwise healthy subjects. Using forearm plethysmography techniques, Creager et al. demonstrated in 1990 that endothelium-dependent responses to methacholine were abnormal in the microcirculation of adults with hypercholesterolaemia (mean total cholesterol 7.1 mmol/l) compared to normal controls (mean total cholesterol 4.6mmol/l) (Figure 16.2). In contrast, Gilligan and coworkers (1994) found that although the microvascular endotheliumdependent response to acetylcholine was markedly impaired in the presence of hypercholesterolaemia, the response to bradykinin was preserved. Whether this reflects a selective impairment of NO release in response to some stimuli, but normal responses via other pathways, requires further evaluation. The development of a technique to measure large vessel endothelium-dependent dilatation non-invasively has given insights into how hypercholesterolaemia might affect endothelial function at the
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very early stages of atherosclerosis. The first manuscript describing of this technique (Celermajer et al., 1992) reported a marked loss of flowmediated dilatation of the femoral artery of children with familial hypercholesterolaemia, compared to normocholesterolaemic controls. This impairment of endothelial function is related to the level of total cholesterol (Sorensen et al., 1994) and interacts with other risk factors such as cigarette smoking, male gender and family history of premature arterial disease (Celermajer et al., 1994).
Figure 16.2 Dose response curves of changes in forearm blood flow, measured by plethysmography in response to methacholine (Creager et al., 1990), show that endothelium-dependent dilatation is reduced in hypercholesterolaemic subjects, compared to controls. Adapted from Creager et al. 1990. Copyright 1990, The American Society for Clinical Investigation, Inc.
The mechanism whereby hypercholesterolaemia causes impairment of endotheliumdependent dilatation has not been fully elucidated, however it is clear that there is a decrease in the overall bioavailability of NO in hypercholesterolaemia. Studies in experimental animal models of atherosclerosis suggest that there is a significant decrease in the availability of nitric oxide in the vessel wall after cholesterol feeding (Cohen et al., 1988; Böger et al., 1995) as well as in hypercholesterolaemic humans (Casino et al., 1993). It is not clear,
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however, whether this is due to a decrease in production by endothelial cells and/or due to increased NO breakdown. Decreased endothelial NO production has been documented by Lefer et al., (1993) in in vitro studies of hypercholesterolaemic rabbits. Several investigators (Gryglewski et al., 1986) have also found indirect evidence for decreased endothelial nitric oxide production, showing decreased urinary excretion of N in the setting of hypercholesterolaemia. Several investigators, however, have also demonstrated an excess of superoxide anion production in the vessel wall in response to hypercholesterolaemia (Ohara et al., 1993), which may be due to both native and modified LDL (Pritchard et al., 1995). An excess of superoxide production would result in the breakdown of nitric oxide to inactive and potentially toxic compounds such as peroxynitrite (Gryglewski et al., 1986). Garcia and coworkers (1995) recently studied the effects of superoxide dismutase infusion in an in vivo study in humans and found that the removal of superoxide anions did not restore NO activity, suggesting that the endothelial defect in hypercholesterolaemia may be due to reduced NO production. A number of factors could be involved in reduced endothelial NO production. Hypercholesterolaemia has been linked to increased levels of asymmetrical dimethylarginine (ADMA), a competitive antagonist of nitric oxide synthase, which could lead to decreased production of nitric oxide by substrate competition (Bode-Böger et al., 1996; Vallance et al., 1992). Furthermore, oxidised LDL may impair nitric oxide synthase production through activation of protein kinase C (Ohara et al., 1995) as well as by interfering with endothelial cell signal transduction (Liao and Clark, 1995). Oxidised LDL can also lead to reduced transcription of mRNA for nitric oxide synthase, as well as direct destabilisation of mRNA after transcription (Liao et al., 1995). As well as reducing the availability of NO, a number of other events occur in hypercholesterolaemia which may alter vascular physiology. Oxidised LDL in particular has been shown to induce mRNA expression and release of endothelin from endothelial cells (Boulanger et al., 1992), although it is possible that this may relate to the reduced inhibitory action of NO (Boulanger and Lüscher, 1990). Hypercholesterolaemia has also been associated with changes in secretion and metabolism of prostaglandins (Holland et al., 1988). Native LDL and Oxidised LDL There is evidence that native LDL has far less direct effects on the normal regulatory functions of the endothelium than oxidatively modified LDL (Steinberg et al., 1996). Native LDL, and in particular small, dense LDL particles, which are more susceptible to oxidation, may be metabolised by endothelial cells and monocytes within the
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vessel wall, resulting in the consumption of antioxidant defences. Once the normal antioxidants, such as -tocopherol, are consumed, the LDL particles are subject to a variety of modifications. Reactive hydroxyfatty acids may be formed from polyunsaturated phospholipids and there is production of lysophosphatidylcholine, which seems to play an essential role in the disruption of normal endothelial function (Kugiyama et al., 1990). Apolipoprotien-B may also undergo covalent modification leading to fragmentation, and preventing normal binding to the native LDL receptor (Steinbrecher, 1987). The resulting oxidised LDL particle is highly negatively charged and has a number of toxic effects on the vessel wall, which lead (inter alia) to reduced NO activity. Evidence from experimental animal studies suggest that oxidised LDL is much more potent in causing loss of endothelium-dependent dilatation than native LDL (Simon et al., 1990; Cox and Cohen, 1996). In vivo studies in humans have related the level of oxidised LDL, the susceptibility of a subject’s LDL to oxidation, and autoantibody titres to oxidised LDL to the degree of endothelial dysfunction in both the large vessels (Anderson et al., 1996) and in the microcirculation of the coronary arteries (Raitakari et al., 1997). Furthermore, recent work by Heitzer and colleagues (1996) suggests that oxidised LDL and cigarette smoking may act synergistically to impair endothelial function, and that the oxidation of LDL may be potentiated by external factors such as cigarette smoking. Therefore oxidised LDL has a wide range of deleterious effects on normal endothelial function. There is also evidence, however, that native LDL also has important effects on the vascular endothelium, especially in the early stages of endothelial dysfunction. In particular native LDL has been shown in vitro to increase superoxide anion production by nitric oxide synthase, and to alter endothelial prostacyclin activity, endocytic activity and permeability (Pritchard et al., 1995). Lipoprotein (a) Patients with familial hypercholesterolaemia and increased levels of LDL have been shown to have impaired endothelium-dependent dilatation. In these subjects the level of lipoprotein (a) is an independent predictor of the degree of endothelial dysfunction in the femoral artery (Sorensen et al., 1994), and in the coronary circulation (Schächinger et al., 1997). In vitro studies have also shown that oxidised lipoprotein (a) is more potent than oxidised LDL in impairing endothelium-dependent dilatation (Galle et al., 1995).
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High-density lipoprotein In addition to the beneficial effects that HDL cholesterol has on reverse cholesterol transport and thrombosis, increased HDL levels may attenuate the negative effects of hypercholesterolaemia on endothelium-dependent dilatation. In vitro studies of rabbit aorta preparations have yielded inconclusive results, with HDL failing to consistently help in retaining endothelium-dependent responses to acetylcholine (Takahashi et al., 1990; Vedernikov et al., 1988). In vivo coronary artery studies by Kuhn and coworkers (1991) and Zeiher et al. (1994), however, have found an amelioration of abnormal vasoconstriction in the presence of elevated HDL levels. Triglycerides Reduced endothelium-dependent dilatation has been described in a number of conditions associated with hypertriglyceridaemia, such as the obesity/insulin resistance syndrome (Steinberg et al., 1996) and in non-insulin dependent diabetes mellitus (McVeigh et al., 1992), although this may relate to elevated LDL levels (Clarkson et al., 1996). Chowienczyk and colleagues (1997) have examined peripheral artery small vessel function in subjects with isolated hypertriglyceridaemia associated with lipoprotein lipase dysfunction, and found no relationship between triglyceride levels and the degree of endothelial dysfunction. Effects on Endothelial Adhesion As leukocyte (and in particular monocyte) adhesion to the endothelium is an important early event in atherogenesis, investigators have been interested in how cholesterol may alter endothelial function to enhance this process. Various experimental models of atherosclerosis have demonstrated that monocytes adhere to endothelial cells after cholesterol feeding, and that these changes occur prior to the development of macroscopic lesions (Joris et al., 1983; Faggiotto et al., 1984). This increase in monocyte adhesion to endothelial cells has been associated with a reduction in the bioavailability of NO (Lefer and Ma, 1993) and oxidised LDL has been associated with an increase in monocyte binding to endothelial cells in humans (Mata et al., 1996). These changes in monocyte adhesion may be, at least in part, explained by an up-regulation in endothelial expression of various adhesion molecules. In 1991, Cybulsky and Gimbrone demonstrated increased expression of endothelial cell adhesion molecules in rabbits after cholesterol feeding. Since then, investigators have shown that oxidised LDL
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induces the endothelial expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) (Khan et al., 1995), and that certain constituents of LDL, such as lysophosphatidylcholine, may be instrumental in this action (Kume et al., 1992). In contrast to this, other investigators have found the HDL may inhibit the endothelial expression of ICAM-1, VCAM-1 and Eselectin (Cockerill et al., 1995). Recent findings suggest that these changes in monocyte adhesion may be mediated at the level of cell adhesion molecule gene transcription by the up-regulation or activation of the nuclear factor-kB within endothelial cells (Ahmad et al., 1995). The up-regulation of nuclear factor-kB appears to be sensitive to redox state (Marui et al., 1993) and to be inhibited by NO (De Caterina et al., 1995; Adams et al., 1997). Other Endothelial Changes Cell growth and division The initiating events that lead to endothelial cell damage may involve direct toxic effects of oxidised LDL. There is evidence that oxidised LDL may trigger apoptosis of endothelial cells via reactive oxygen species and the enhancement of CPP32-like protease activity (Dimmeler et al., 1997). Also oxidised LDL disrupts normal regulatory functions of the endothelium which are important in both stimulating and inhibiting vascular growth, via the secretion of a variety of cytokines and NO (Ross, 1993). This, together with the loss of endothelial regulation of the inflammatory process which can lead to the up-regulation of growth promoting genes, may play an important role in intimal thickening, involving the proliferation of smooth muscle cells and monocytes (Simari et al., 1996). Thrombosis Oxidised LDL can lead to the loss of normal endothelial anticoagulant mechanisms. The loss of NO activity may result in increased platelet aggregation and adhesion (Radomski et al., 1987). Oxidised LDL can initiate a number of other procoagulant effects within the endothelium, including up-regulation of plasminogen activator inhibitor mRNA and down-regulation of mRNA for tissue plasminogen activator and thrombomodulin (Arnman et al., 1994).
REVERSAL OF ENDOTHELIAL DYSFUNCTION As endothelial dysfunction may be both an important initiating event in
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atherogenesis as well as a causative factor in the development of acute coronary syndromes in established coronary artery disease, it has been postulated that the correction of endothelial dysfunction may be associated with an improvement in mortality and morbidity from atherosclerosis, and even the prevention of its progression (Heistad and Armstrong, 1994). Strategies that have been trialled for endothelial protection include cholesterol lowering, the reversal of other vascular risk factors as well as a number of novel approaches, such as antioxidants (Celermajer, 1997). Lipid Lowering Cholesterol lowering, by dietary, pharmacologic or other means, has been associated with an improvement in cardiovasular morbidity and mortality in a number of clinical trials (Blankenhorn et al., 1993; Watts et al., 1992; MAAS Investigators 1994). However, these beneficial effects are accompanied by only a minimal regression of plaque and the survival benefits are seen long before there is evidence of structural plaque regression (Brown et al., 1990). For these reasons, several investigators have proposed that the benefits from cholesterol lowering may relate not to large reductions in plaque burden, but rather to changes in plaque composition leading to plaque stabilisation with an increase in fibrosis and a decrease in the plaque’s soft lipid core (Kinlay et al., 1996). In addition, lipid lowering may lead to a decrease in inflammatory cells which produce metalloproteinases which degrade the plaque’s fibrous cap, leaving the plaque more vulnerable to fissuring and rupture (Libby, 1995). An alternative, or indeed complementary explanation for the above observations is that lipid lowering may lead to an improvement in endothelial function. Experimental animal models of atherosclerosis have shown an improvement in endothelium-dependent dilatation after withdrawal of the cholesterol rich diet or institution of cholesterol lowering therapy (Harrison et al., 1987). In 1993, Leung and colleagues studied coronary artery endothelial function in hypercholesterolaemic patients before and after six months cholesterol lowering with dietary modification and cholestyramine. In this study total cholesterol was reduced by 28.7% with a corresponding improvement in endotheliumdependent dilatation of 27.9%, with a change from net coronary constriction to dilatation. Similarly endothelial function in the coronary microcirculation of hypercholesterolaemic subjects can be improved by cholesterol lowering using the HMG CoA reductase inhibitor Pravastatin (Egashira et al., 1994). Other investigators have confirmed that therapy with the HMG CoA reductase inhibitor Lovastatin improves large vessel coronary artery endothelium-dependent dilatation in adults with hypercholesterolaemia (Treasure et al., 1995a; Andersen
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et al., 1995). In the study by Treasure and colleagues, in hypercholesterolaemic patients undergoing coronary angioplasty, cholesterol lowering therapy for six months reduced total cholesterol by 31% and significantly improved endothelium-dependent dilatation (Figure 16.3). Similar results have been found by investigators studying peripheral arterial endothelial function with HMG CoA reductase inhibitors such as Atorvastatin (Simons et al., 1998). Other studies of peripheral arterial function have shown that the improvements in endothelial function after cholesterol lowering occur within a relatively short time. Endothelial dysfunction may develop after only two weeks of increased cholesterol
Figure 16.3 Treasure et al. (1995) examined coronary arterial responses to serial infusions of acetylcholine in two groups of subjects, approximately 6 months after randomisation to either Lovastatin or placebo. Responses are expressed as the percentage of change in diameter from the base-line value in all coronary artery segments. Endothelium-dependent dilatation was significantly better in the Lovastatin group compared to placebo (p=0.013). Adapted from Treasure et al. 1995. Copyright 1995, The Massachusetts Medical Society.
levels and improve towards normal within twelve weeks when cholesterol levels are reduced (Stroes et al., 1995). Vogel et al. (1996) found an improvement in brachial artery flow-mediated dilatation after
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only two weeks of treatment with a HMG CoA reductase inhibitor, although when the cholesterol lowering agent was withdrawn, there was a rapid reappearance of endothelial dysfunction as cholesterol levels rose. Similar results have been found in the microcirculation of the forearm, with an improvement occurring within four weeks, which continued to improve up to three months (O’Driscoll et al., 1997). Recently it has been shown that rapid lowering of LDL cholesterol using apheresis may lead to an improvement in endothelium-dependent dilatation within two hours (Tamai et al., 1997), suggesting that LDL may directly impair endothelial function and that the clinical benefits from LDL cholesterol reduction may start as soon as cholesterol is reduced. Whether or not these changes are sustainable past six months treatment duration requires further prospective evaluation. Other Agents Apart from lowering of serum cholesterol, other approaches have been taken in an attempt to reverse the endothelial dysfunction associated with hypercholesterolaemia. As oxidised LDL plays an important role in atherogenesis, and a number of lipid soluble antioxidants are important in preventing LDL oxidation, investigators have proposed the use of antioxidant therapy either alone or as an adjunct to cholesterol lowering. In animal models of atherosclerosis, antioxidant supplementation reduces monocyte adhesion to endothelium exposed to oxidised LDL (Lehr et al., 1994). In humans, Andersen et al. (1995) found that adding treatment with the antioxidant Probucol lead to an improvement in coronary artery endothelium-dependent dilatation over and above that seen with cholesterol lowering alone. In addition, in patients taking lipid lowering therapy, their degree of endothelial dysfunction may relate to the susceptibility of their LDL to oxidation (Anderson et al., 1996). As there is decreased bioavailability of NO in hypercholesterolaemia, supplementation with L-arginine, NO’s physiologic substrate, has been investigated both in hypercholesterolaemic animal models and humans. Several investigators have found that in the hypercholesterolaemic rabbit model, oral supplementation with L-arginine not only improves endothelium-dependent dilatation, and reduces platelet aggregation and monocyte adhesion to endothelial cells, but also markedly decreases atherosclerosis (Cooke et al., 1992; Böger et al., 1995; Tsao et al., 1994). In hypercholesterolaemic humans, acute intravenous L-arginine may improve peripheral microvascular endothelial function (Creager et al., 1992). Clarkson et al., (1996) have recently shown that four weeks therapy with oral L-arginine may improve endothelial function in hypercholesterolaemic young adults. Whether L-arginine or other NO
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donors are able to produce sustained improvements (i.e., beyond four weeks) in the endothelial dysfunction associated with hypercholesterolaemia requires further investigation.
CONCLUSIONS Hypercholesterolaemia is associated with multiple abnormalities of endothelial function, including disturbances in the normal control of vascular tone, platelet adhesion, coagulation and leukocyte adhesion to the endothelium. These abnormalities seem to relate especially to oxidised LDL and play an important role in the process of atherogenesis. Other lipoproteins may also play crucial roles in this process; in particular HDL cholesterol has important protective actions at the endothelial level. The process of endothelial dysfunction is potentially reversible and such reversibility may represent an early step in the regression of atherosclerotic changes. In addition, improvements in endothelial function are associated with a survival benefit and with a reduction of cardiovascular events. Although further investigation is required, the use of cholesterol lowering agents and novel compounds such as antioxidants or NO donors may provide clinical benefit in terms of improving arterial physiology, decreasing ischaemic episodes and preventing mortality associated with atherosclerotic vascular disease. References Adams, M.R., Jessup, W., Hailstones, D. and Celermajer, D.S. (1997). L-arginine reduces human monocyte adhesion to vascular endothelium and endothelial expression of cell adhesion molecules. Circulation , 95 , 662–668. Ahmad, M., Marui, N., Alexander, R.W. and Medford, R.M. (1995). Cell type-specific transactivation of of the VCAM-1 promoter through an NF-kappa B enhancer motif. J.Biol. Chem ., 270 , 8976– 8983. Anderson, T.J., Meredith, IT., Yeung, A.C., Frei, B., Selwyn, A.P. and Ganz, P. (1995a). The effect of cholesterol-lowering and antioxidant therapy on endothelium-dependent coronary vasomotion. N.Engl. J.Med. , 332 , 488–493. Anderson, T.J., Uehata, A., Gerhard, M.D., Meredith, I.T., Knab, S., Delagrang, D. et al. (1995b). Close relation of endothelial function in the human coronary and peripheral circulations. J.Am. Coll. Cardiol. , 26 , 1235–1241. Anderson, T.J., Meredith, I.T., Charbonneau F., Yeung, A.C., Frei, B., Selwyn, A.P. et al. (1996). Endothelium-dependent coronary vasomotion relates to the susceptibility of LDL to oxidation in humans. Circulation , 93 , 1647–1650.
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SUBJECT INDEX Abetalipoproteinaemia and MTP defects 149 –151 Adhesion molecules and endothelial dysfunction 329, 336 –337 and HDL receptors 118, 125 inhibition by HDL 73, 76 Alzheimer’s disease and apoE 214 –219 pathological features 215 ApoAI anti-oxidant activity 315 ligand for HDL receptors 120 –122 mutations 26 ApoAII anti-oxidant activity 315 ligand for HDL receptors 120 ApoB100 familial defective 14 –16 ApoB/E receptor (see LDL receptor) ApoCII familial deficiency 18 ApoE 202 –219 and Alzheimer’s disease 214 –219 gene and sites of synthesis 202 –203 role in remnant metabolism 207 –209 structure and function 205 –207 ApoE polymorphisms and Alzheimer’s disease 216 –219 and type III hyperlipoproteinaemia 209 –214 impact on lipid metabolism 203 –205 Arterial endothelium sites of plaque initiation 56 Arterial intima cholesterol accumulation and lipid core formation 58 –59 entry, efflux and retention of LDL 56 –58 Atherogenesis 73 –74 and chylomicron metabolism 88 –92 Atherosclerosis carotid and femoral artery 46
Subject index
462
Cerebro-tendinous xanthomatosis 16 Cholesterol efflux 72 metabolism xvi –xvii risk factor for CAD 35 –37 Cholesterol ester storage disease 24 Cholesteryl ester transfer protein 96 deficiency 172 –174 mechanism of action and function 168 –169 molecular structure 168 Cholesteryl ester transfer protein and atherosclerosis 188 animal studies 171 –172 human studies 172 –175 predictions from in vitro studies 169 –171 Chylomicrons xiii assembly 146 –148 atherogenicity 79 origin and secretion 86 –88 Chylomicron metabolism in dyslipidaemic states 99 –101 in normolipidaemic subjects with CAD 101 monitoring metabolism in vivo 101 –103 therapeutic opportunities 103 Chylomicron remnants xiii and apoE 207 –209 and inflammation 95 –97 and vascular smooth muscle cells 97 –99 and vasodilatation 99 atherogenicity 79, 85 –103 metabolism by macrophages 95 –97 uptake by LDL receptor 92 Coronary artery disease risk factors 34 Dyslipidaemia 10 –27 prevalence 10 mechanisms 10 –12 Endothelial function 325 –340 and leukocyte adhesion 329, 336 –337 and thrombosis and haemostasis 329 and vascular tone 326 –328 Endothelial dysfunction 329 –330 and hyperlipidaemia 332 –337 clinical assessment 330 –332 reversal 337 –340 Endothelial (post-heparin) lipases 158 –173 and chronic human diseases 165 –166
Subject index
463
diagnosis of lipase defects 163 modulation by hormones 164 –165 non-enzymatic actions 160 regulation of plasma lipoprotein 160 –161 physiological actions 161 structure 158 synthesis and processing 158 –160 Extracellular matrix trapping of intimal LDL 57 Fish eye disease 47, 189 HDL xv and endothelial dysfunction 336 and family studies 43 –44 and intervention studies 78 –79 and population studies 41–43, 76 –78 and cholesterol efflux 72, 74 –75 animal models 73, 77 antioxidant properties 72, 75–76, 313 –316 catabolism 70 deficiency 76 elevation 76 formation 69 –70 functions 71 –73 inhibition of endothelial adhesion molecules 73, 76 ligand for HDL receptors 120 –122 mechanisms of inhibition of atherosclerosis 73 –76 protection against CAD 5, 41–44, 66 –79 regulation of apolipoprotein-specific HDL 71 structure 67 HDL cholesterol and protection against CAD 77 HDL receptors 109 –125 and HDL receptors 118, 125 HB1 and HB2 117 –119 identification, cloning and properties 109 –118 nature of ligands 119 –122 signalling 122 –125 SR-BI 113 –117 HDL subpopulations 67 –69 protection against CAD 42, 77 –78 Hepatic lipase 71 deficiency 163 Hypercholesterolaemia familial 12, 38–39, 100 primary 12 –17 homozygous familial 12 –14
Subject index
464
heterozygous familial 14, 39 polygenic 16 Hyperlipidaemia familial combined 20, 76, 100 –100 primary mixed 20 Hyperlipoproteinaemia hyper lipoproteinaemia, familial 16 hyperlipoprotein(a)aemia 17 type I 162 type III 100, 209 –214 type IV and V 100 –101 Hypertriglyceridaemia familial 19–20, 40–41, 76 familial hepatic lipase deficiency 19 familial lipoprotein lipase deficiency 17 –18 primary 17 –20 Hypolipoproteinaemia abetalipoproteinaemia 24 familial hypobetalipoproteinaemia 25 hypo lipoproteinaemia 26 –27 primary 24 –27 secondary 27 Tangier disease 26 –27 IDL xv atherogenicity 4 Lecithin: cholesterol acyltransferase 70, 181 –182 anti-oxidant activity 314 –315 gene mutations 187 –192 LCAT knock-out 183 overexpression of human LCAT 182 –183 Lecithin: cholesterol acyltransferase deficiency 44, 76, 180 –193 and atherosclerosis 186 –187 familial deficiency 183 –186 fish-eye disease 186 LDL xv and endothelial dysfunction 335 –336 and family studies 38 –39 and population studies 37 –38 atherogenicity 4–5, 53 –63 entry, efflux and retention by arterial intima 56 –58 intimal metabolism 58 –59 proatherogenic modification 59–60, 300 risk factor for CAD 36, 38 tocopherol-mediated peroxidation 308 –313 LDL receptor deficiency 90 –92
Subject index
465
gene 133 –137 mutations 12 –14 structure and function 130 –133 LDL receptor regulation 130 –142 by cholesterol 137 –140 by non-steroidal compounds 140 –142 LDL subpopulations and phenotypes 54 –56 atherogenicity 38, 56, 60 –63 Lipid transport xiii –6 endogenous 2 –3 exogenous xvii –1 Lipoprotein lipase and atherosclerosis 163 caloric distribution 161 –162 genetic deficiency 162 pharmacological alterations 166 physiological regulation 162 regulation of lipolytic actions 160 Lp(a) 8, 44 and endothelial dysfunction 336 and family studies 45 –46 and population studies 44 –45 atherogenicity 5, 44 –46 clinical correlates 17, 46 genetics 45 –46 Macrophages in atherogenesis 58 metabolism of chylomicron remnants 95 –97 Metabolic syndrome 211 Microsomal triglyceride transfer protein 188 defects 149 –151 lipoprotein assembly 151 –155 Oxidized LDL and endothelial dysfunction 335 –336 in vitro oxidation 307 –308 in vivo oxidation 301 –307 proatherogenic modification 59–60, 300 Paraoxonase 314 Phospholipid transfer protein 109, 226 –295 and atherosclerosis 293 –295 and HDL conversion 290 –292 animal models 293 distribution and regulation 292 functions 288 –292 structure and gene organisation 226 –288
Subject index
466
Phytosterolaemia 16 Plasma lipoproteins xiii –xvi atherogenicity 79 –5 disorders, epidemiology 33 –46 oxidation 299 –316 Platelet activating factor acetylhydrolase 314 Population studies Framingham 36 GRIPS 44 Helsinki Heart Study 44, 79 MRFIT 36 Lipid Research Clinics Coronary Primary Prevention Trial 44, 79 Physicians Health Study 40, 42, 44 PROCAM 44 Stanford Five-City Project 44 Postprandial lipoproteins arterial delivery and retention 93 –95 Proteoglycans trapping of intimal LDL 57 Reverse cholesterol transport 30–79, 85 Secondary hyperlipidaemias 55 and alcohol 23 and chronic renal failure 22 and coffee 23 and chlorinated hydrocarbons 23 and diabetes mellitus 21 and exogenous sex-hormones 21 and gout 21 and growth hormone 21 and hypothyroidism 21 and immunosuppresants 23 and nephrotic syndrome 22 and obesity 21 and obstructive liver disease 22 and other drugs 24 and pregnancy 21 and progressive partial lipodystrophy 21 and storage disorders 22 and thiazides and -blockers 23 SR-BI 113 –117 Tangier disease 47 Transgenic animal models and CETP 171 –172 and LCAT 182 –183 and PLTP 293
Subject index
467
antiatherogenic models 73 Triglyceride and endothelial dysfunction 336 and family studies 40 –41 and population studies 39 –40 metabolism xvii Triglyceride-rich lipoproteins correlation to HDL cholesterol 40 risk factor for CAD 40 Vitamin E and ischaemic heart disease 300 –301 tocopherol-mediated peroxidation in LDL 308 –313 VLDL xiv assembly 146 –148 atherogenicity 79 –4