Chapter 1 GENERAL INTRODUCTION. 1.1 Lipoproteins and Lipid Metabolism

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1 GENERAL INTRODUCTION Contents 1.1 Lipoproteins and lipid metabolism 1.2 Atherosclerosis 1.3 HDL and atherosclerosis 1.4 ABCA-transporters 1.5 Lipid transfer proteins: CETP and PLTP 1.6 Macrophage RCT pathway: Key to study atherosclerosis regression? 1.7 Outline of the thesis 1.8 References 1.1 Lipoproteins and Lipid Metabolism Lipoproteins The major lipids in human plasma are cholesterol, cholesteryl esters (CE), triglycerides (TG) and phospholipids (PL). TG and CE molecules, which are insoluble in aqueous solutions, are carried in the core of spherical macromolecular complexes, called lipoproteins 1. Most lipoproteins share a common spheroid structure consisting of a neutral lipid core of hydrophobic CE and TG surrounded by a surface monolayer of PL, unesterified free cholesterol (FC) and apolipoproteins 1. Since lipoproteins constitute a heterogeneous population of particles, they are traditionally classified into six major classes according to their densities. Four of the major classes of lipoproteins very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), low density lipoprotein (LDL), and high density lipoprotein (HDL) are derived from the liver and are present in plasma from both fasted and non-fasted subjects. The other two major classes chylomicrons (CMs) and CM remnants are derived from the small intestine and are found in the plasma only after a fatty meal. The characteristics of the various human plasma lipoproteins are listed in Table 1 1. As shown, their relative contents of protein and lipid determine their hydrated density, size and electrophoretic mobility and hence, their classification. In more detail, CMs, VLDL, and LDL have apolipoprotein B (apob) as their primary protein, whilst apolipoprotein A-I (apoa-i) is the major protein constituent of HDL. CMs and VLDL are TG-rich lipoproteins, whilst LDL and HDL contain relatively high levels of CE and PL, respectively. Furthermore, the size of different lipoproteins is inversely correlated to their density, with VLDL being the largest lipoprotein and HDL the smallest one. The heterogeneity in lipoprotein size and composition induces changes in interaction with different tissues, thereby influencing the lipoprotein metabolism. 7

2 Table 1 Physical properties and composition of human plasma lipoproteins CMs VLDL LDL HDL Diameter (nm) Density (g/ml) < Mw (X 10 6 Da) Mobility a origin pre-β β α, pre-β Lipid composition b Triglyceride Free cholesterol Cholesterol ester Phospholipid Apolipoproteins A-I, A-II, A-IV - - A-I, A-II, A-IV B48 B100 B100 - C-I, C-II, C-III C-I, C-II, C-III - C-I, C-II, C-III E E - E a According to the electrophoretic mobility of plasma α- and β-globulins on agarose gel electrophoresis b The values given for composition are expressed as percentage of total weight Adapted from Ginsberg HN Lipid metabolism The liver plays an essential role in lipid metabolism. Lipoprotein metabolism can be divided into three distinct pathways, based on origin, function and fate of the lipid content of the particles involved 2-4. A condensed overview of metabolic routes is depicted below. (1) Exogenous lipid transport, describing the metabolic route of dietary lipids after their absorption from the intestine. In detail, dietary TG and CE are hydrolyzed in the intestine and subsequently assembled into CMs. These large TG-rich CMs are transported from the lymph to the blood circulation (Fig. 1). Nascent CMs consist mainly of TG (88%), but also PL, CE, FC, and apolipoproteins (e.g. apoa-i, apoa-ii, apoa-iv, apob-48, and apocs) (Table 1) 1. Upon entering the circulation, these CMs are processed by lipoprotein lipase (LPL), which hydrolyzes TG, thereby delivering liberated free fatty acids (FFA) to peripheral tissues such as adipose tissue (for storage into TG), skeletal muscle and heart (as energy source), and the liver (as storage or generation of lipoprotein particles) 5,6. Due to hydrolysis of the core lipids, CM particles shrink and the excess of surface material, i.e., PL, FC and apolipoproteins, are in part transferred to HDL particles 7. The CM remnants thus formed are rapidly taken up by the liver via a apoe-specific recognition site on hepatocytes, including the LDL receptor (LDLR), LDLr-related protein (LRP) 8, heparan sulphate proteoglycans (HSPG) 8, and possibly also scavenger receptor BI (SR-BI) 9. (2) Endogenous lipid transport, describing the distribution of lipids from the liver to peripheral tissues, in particular relevant during periods of fasting. In detail, the endogenous pathway begins with the production and secretion of TG-rich lipoproteins by the liver in the form of VLDL (Fig. 1). These lipids are either derived from incoming CM remnants, IDL, LDL, and HDL, or from de novo synthesis. TG-rich VLDL particles containing a single molecule of apob provide energy-rich material to the periphery during periods of fasting. Another important apolipoprotein on VLDL is apoe, which plays an role in the secretion 10,11 as well as in the metabolic fate of these VLDL particles, 8

3 General introduction the latter due to its interaction with specific receptors 12. Upon entering the blood circulation, VLDL is further enriched with apoe and apocs (e.g. apoc- I, apoc-ii, apoc-iii, and apoe) (Table 1) 1. Similar to CMs, TG-rich VLDL particles can undergo lipolysis catalyzed by LPL 5, leading to formation of VLDL remnants or IDL, which is partly cleared by the liver as mediated by apoe 13. The remainder is extensively processed by LPL and hepatic lipase (HL) to become cholesterol-rich LDL with apob100 as its sole apolipoprotein, which is recognized by the LDLR on the liver and peripheral tissues 13. Fig. 1. Schematic overview of pathways involved in lipoprotein metabolism. See text for explanation. Modified from Havel RJ et al 7. (3) Reverse cholesterol transport, reflecting transport of cholesterol from peripheral tissues to the liver. HDL is a relatively small lipoprotein, which carries approximately one-third of the cholesterol in human plasma and, is involved in the removal of excess cholesterol from cells 14. There are subclasses of HDL particles, including nascent discoidal HDL (pre-ß HDL) and spherical HDL (HDL 2 and HDL 3 ). One of the major functions of HDL is to transport cholesterol from peripheral tissues to the liver for elimination via the bile. This occurs by a pathway called reverse cholesterol transport (RCT), which involves the coordinate action of multiple cellular and plasma proteins (Fig. 2). 1. Nascent HDL particles, small particles lacking CE and containing apoa-i as their major apolipoprotein, are formed by the liver and the intestine 15. Cholesterol efflux from peripheral cells can occur by passive diffusion, or it may involve HDL or apoa-i receptors. For instance, ATP-binding cassette transporter A1 (ABCA1) may promote cholesterol efflux from peripheral cells to lipid-poor apoa-i. In addition, ABCG1 and SR-BI may promote cellular cholesterol efflux to mature 9

4 HDL 15,18. A portion of lipid-free apoa-i undergoes glomerular filtration in the kidney and tubular re-absorption through cubilin. 2. Subsequent activity of lecithin:cholesterol acyltransferase (LCAT) leads to formation of large, CE-rich HDL 18,19, thus producing spherical HDL particles, converting HDL 3 to larger HDL 2. The latter mature HDL particles can then be taken up by the liver by 3 pathways: (1) as part of a holo-hdl uptake mechanism, probably involving proteoglycans (PG), apoe, and possibly other factors; (2) via hepatic SR-BImediated selective uptake of CE and FC 18,20 ; and (3) by cholesterol ester transfer protein (CETP)-mediated transfer to TG-rich lipoproteins (TRLs) 21, with subsequent uptake of TRL remnants in the liver, involving LDLR, PG, or LRP. Importantly, CETP is not expressed in rodents which may, in part, explain the high HDL levels found in these animals. There is also an exchange of surface PL between the different lipoproteins mediated by phospholipid transfer protein (PLTP) 22. Both in mice and humans, the lipolysis of TG in HDL is efficiently catalyzed by hepatic lipase (HL), whereas the lipolysis of PL in HDL is catalyzed by endothelial lipase (EL), thus assisting in the remodeling of mature HDL to smaller HDL particles. 3. Upon delivery of HDL-CE to the liver, the CE s are hydrolyzed and reused for lipoprotein assembly. Alternatively, cholesterol is secreted into the bile either as neutral sterols or bile salt via ABCG5/8 (halftransporters that work together as heterodimers) and ABCB11 (BSEP)-mediated pathways 23,24. Kidney Intestine Liver ABCG5/G8 ABCB11 Bile Fig. 2. Schematic illustration of reverse cholesterol transport. See text for explanation. Thus, in order for the cycle of RCT to persist, new acceptors of cellular cholesterol (ie, apoa-i, pre-β-hdl) must be continuously synthesized or regenerated in reactions that are catalyzed by lipid transfer proteins (i.e., 10

5 General introduction CETP, PLTP, HL and EL), acting in conjunction with ABC-transporters (i.e., ABCA1, ABCG1, ABCG5/G8, and ABCB11) to enhance cellular cholesterol efflux. 1.2 Atherosclerosis Overview Cardiovascular diseases (CVD), such as ischemic heart diseases (incl. acute myocardial infarction) and cerebrovascular diseases (incl. stroke), are currently the leading cause of death and illness in developed countries, and is expected to become the pre-eminent health problem worldwide. According to World Health Organization (WHO) estimates, in 2003, 16.7 million people around the globe died of CVD each year. This is over 29% of all deaths globally 25. The major contributor to the growing burden of CVD is atherosclerosis, a progressive disease characterized by a focal accumulation of lipids and fibrous elements within the large arteries 26,27. Atherosclerotic lesions can cause flow limiting stenosis leading to lack of oxygen and nutrition supply in the tissues located distally from the plaque. However, the most severe clinical events follow the rupture of the lesion, which exposes the pro-thrombotic material in the plaque to the blood and causes sudden thrombotic occlusion of the artery. In the heart, atherosclerosis can lead to myocardial infarction and heart failure, whereas in the brain, it can cause ischemic stroke and in peripheral tissues, it can result in renal impairment, hypertension, aneurysms and critical limb ischemia 26. Epidemiological studies have identified numerous environmental and genetic risk factors which are associated with an increase risk of atherosclerosis development. For instance, atherosclerosis has been related to hyperlipidemia with increased VLDL/LDL-cholesterol and TG levels, often accompanied by low HDL-cholesterol levels 28,29. In addition, several other factors such as hypertension, diabetes mellitus, obesity, male sex, smoking, age, family history, physical inactivity and infections are associated with an increase risk of atherosclerosis development Pathogenesis of atherosclerosis The initiation of atherosclerosis has been debated for many years and several hypotheses have been proposed. One of the earliest, the responseto-injury hypothesis stated that endothelial injury leads to an inflammatory response as part of a healing process in the arterial wall 30. Subsequently, the response-to-oxidation hypothesis has evolved to focus on specific proinflammatory oxidized phospholipids that result from the oxidation of LDL phospholipids containing arachidonic acid and that are recognized by the innate immune system in animals and humans, proposing that lipoprotein oxidation is the important link in atherosclerosis 31. In 1995, it was suggested that retention of lipoproteins is the initiating step which leads to oxidation, inflammation and endothelial dysfunction, consistent with previous hypotheses 31. This so-called response-to-retention hypothesis was based on pioneering work in the 70s and 80s showing that lipoproteins can interact with the arterial wall

6 According to the most accepted response-to-injury hypothesis, atherogenesis is initiated by injury to the endothelium, which is a thin monocellular layer that covers the inner surface of the blood vessels, separating the circulating blood from the tissues 30,37. Injury to the endothelium, induced by various possible risk factors like consuming a highsaturated-fat diet, smoking, hypertension, hyperglycemia, obesity, or insulin resistance, induces changes in the permeability of the arterial wall, expression of several adhesion molecules in the endothelial cells [e.g., intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, P-selectin and E-selectin], and the production of cytokines as well as growth factors These changes in endothelial function result in an increased migration of monocytes and T-lymphocytes from the circulation into the intima of the arterial wall. On migration through the endothelial layer, monocytes differentiate into macrophages, scavenge the modified LDL from the vessel wall, and start producing several growth stimulating and chemoattractive molecules. In addition to macrophages, these chemokines are also produced by the injured endothelium, its adherent leukocytes, and possibly also by the underlying smooth muscle cells. The most important ones include monocyte chemotactic protein-1 (MCP-1) together with its receptor [chemokine receptor 2 (CCR2)], granulocyte/macrophage colonystimulating factor (GM-CSF), nuclear factor- B (NF- B), interleukin-1 (IL- 1 ), tumour necrosis factor alpha (TNF-α), interferon γ (IFN-γ) 42,43. By excessive storage of cholesterol as CE, macrophages develop into foam cells, which are the first sign of atherosclerosis. The earliest visible atherosclerotic lesion, consisting of accumulated lipid within macrophages in the intima of the arterial wall is called the fatty streak 44,45. The macrophage can release its cholesterol through efflux, which involve several pathways as described in detail in section 1.3. With time, smooth muscle cells can also take up lipids, thereby contributing to foam cell formation. In addition, smooth muscle cells can synthesize large amounts of collagen, elastin, and proteoglycans, and the fatty streak evolves into an intermediate fibrofatty lesion, which consists of several layers of foam cells and smooth muscle cells with T-lymphocytes, surrounded by a relatively poorly developed matrix of connective tissue. Further increase of the lesion severity subsequently results in the formation of an advanced fibrous lesion, which are characterized by a fibrous cap covering a core of extracellular lipid and necrotic debris, together with macrophages, T-lymphocytes, smooth muscle cells and sometimes calcification 46. Macrophages play a key role in the thrombotic complications of atherosclerosis by producing matrix metalloproteinases (MMPs) that can degrade extracellular matrix that lends strength to the plaque's fibrous cap. When the plaque ruptures as a consequence, it permits the blood to contact another macrophage product, the potent pro-coagulant protein tissue factor. Eventually the macrophages congregate in a central core in the typical atherosclerotic plaque. Macrophages can die in this location, some by apoptosis, hence producing the so-called 'necrotic core' of the atherosclerotic lesion. A uniformly thick fibrous cap provides stability to the atherosclerotic plaques, whereas a thin and non-uniform fibrous cap leads to an instable plaque. The ruptured lesions are prone to hemorrhage and thrombosis, an event which has been related to the majority of clinical manifestations: in the coronary circulation, 12

7 General introduction unstable angina pectoris, or acute myocardial infarction 47. A schematic overview is depicted in Fig. 3. Initiation Fibrous cap formation and thinning Migration Fig. 3. Pathogenesis of atherogenesis. The development of atherosclerosis from the endothelium injury to the fibrous cap formation and thinning. See text for explanation. Modified from Peter L Properties of HDL in Atherogenesis The strong inverse relationship between HDL levels and atherosclerosis has been known for more than 30 years 48. Surprisingly, no effective therapy specifically directed at HDL has yet been developed, which reflects uncertainty about the multiple mechanisms underlying the protective effect of HDL against the development of atherosclerosis Almost every step in the pathogenesis of atherosclerosis has been reported to be favorably influenced by HDL 52-70, as summarized in Table Cholesterol efflux, RCT and atherogenesis The central role that HDL plays in RCT is pivotal to cholesterol homeostasis in the whole organism, and is believed to constitute the principal mechanism by which HDL exerts an atheroprotective effect on the vasculature. The key pathways of RCT are depicted in section The specific process involving cholesterol efflux from macrophage foam cells in the artery wall and transport to the liver for excretion into bile has been termed macrophage RCT 71,72. The principal molecules involved in cholesterol efflux from macrophage foam cells are ABCA1 and ABCG1. Genetic knock-down studies suggest that ABCA1 and ABCG1 together account for about 60 70% of the net cholesterol efflux to HDL or serum from cholesterol-loaded liver X receptor (LXR)-activated macrophages 71. In addition to ABCA1 and 13

8 ABCG1, macrophage SR-BI can promote cholesterol efflux to mature HDL 15. Whether macrophage SR-BI contributes in a meaningful way to macrophage cholesterol efflux and RCT in vivo has not been resolved. In SR-BI deficiency, cholesterol uptake in the liver is disturbed, because SR-BI can also promote selective uptake of HDL cholesterol by cells 18. The role of SR- BI in promoting net removal of cholesterol mass from macrophages has to be further clarified. Table 2 Anti-atherogenic activities of HDL particles Activity Documented protective effects 1. Facilitation of RCT Efflux of cholesterol from foam cells in artery wall Anti-inflammatory Inhibition of the synthesis of platelet-activating factor 53 Inhibition of leukocyte adhesion to the arterial wall via attenuation of the expression of VCAM-1 and other cytokine-induced cell adhesion molecules 54,55 Inhibition of expression of MCP-1 56,57 3. Improved endothelial function Stimulation of endothelial NO synthase activity 58,59 Enhanced endothelium-dependent vasodilation 60 Prevention of endothelial cell apoptosis 61,62 Stimulation of prostacyclin synthesis Anti-oxidative Protection of LDL from oxidation 64 via apo AI-mediated anti-oxidative actions 65 via paraoxonase-mediated anti-oxidative actions Anti-thrombotic 6. Anti-infectious Protection of erythrocytes against the generation of procoagulant activity 67 Stimulation of prostacyclin synthesis 56 Inhibition of thrombin-induced endothelial tissue factor expression 68 Reduction of the pyrogenic activity of bacterial lipopolysaccharide 69 Lysis of Trypanosoma Brucei 70 Experiments with transgenic animals suggest that disruption of one or more steps in RCT results in accelerated atherosclerosis, whereas overexpression of pivotal proteins in RCT, such as apoa-i, LCAT, and SR- BI, exerts atheroprotective effects. 15,18,19,138 This information supports the concept that cholesterol efflux and RCT are key anti-atherogenic properties of HDL. However, the important lesson from experimental approaches is that disruption of RCT and resulting atherosclerosis may occur in the presence of either decreased or increased HDL cholesterol levels, depending on which step of RCT is dysfunctional. For instance, decreased HDL levels associated with increased accumulation of cholesterol in peripheral tissues and/or atherosclerosis were observed in humans and animals with apoa-i or ABCA1-deficiency, 18,87,88 in which the initial steps of RCT are inhibited. By contrast, increased HDL concentrations combined with enhanced arteriosclerosis were presented in animals with dysfunctional SR-BI, in which the later step of RCT is impaired 18. Therefore, it is not the increase or decrease of plasma HDL cholesterol concentration per se, but rather the concentrations of various HDL subclasses, the cellular mobilization and transport of lipids, and the kinetics of HDL metabolism that 14

9 General introduction critically determine the atherosclerotic risk. Diagnostic measures allowing more accurate insight into the efficacy of cholesterol flow along RCT pathways still await development Other mechanisms HDL may impart anti-atherogenic effects through its function as an autonomous protective factor for the endothelium 73. Endothelial dysfunction characterized by decreased bioavailability of nitric oxide (NO), a potent vasodilator, and increased affinity of the endothelial surface for leukocytes is often encountered in the early stages of atherosclerosis. In advanced plaques, denudation of the endothelium as a consequence of increased apoptotic cell death can be observed. HDL-induced activation of endothelial nitric oxide synthase (enos), NO release, and vasorelaxatory effects were documented 58,59. Furthermore, HDL attenuates expression of VCAM-1, ICAM-1, and E-selectin, as well as cytokines such as IL-8 that promote leukocyte extravasation 60,74,75. Endothelial apoptosis was prevented in the presence of HDL, and this effect was associated with inhibition of typical apoptosis pathways such as the activation of caspases 61,62,76,77. In addition, HDL activates protein kinase Akt, a ubiquitous mediator of antiapoptotic signaling 76. Thus, HDL can suppress expression of cytokine-induced endothelial cell adhesion molecules and block the migration of macrophages into the subendothelial space of blood vessels. In addition, the anti-atherogenic mechanism of HDL in inhibiting LDL lipid oxidation, especially via the 12-lipoxygenase-induced pathway, is believed to be crucial for preventing the scavenger receptor-mediated uptake of modified LDL particles into macrophage cells located within the subendothelial intima 79. The oxidation of phospholipids is largely generated by the lipoxygenase and myeloperoxidase pathways in the formation of cellderived reactive oxygen species (ROS) 80. These ROS oxidise lipoprotein phospholipids containing arachidonic acid, which in turn, makes the particles pro-inflammatory. The ability of HDL to inhibit oxidation and inflammation is by way of the high levels of antioxidants inherent to this lipoprotein, such as apoa-i 65, and the enzymes; paraoxonase (PON)-1 66,81 and platelet activating factor acetyl-hydrolase (PAF-AH) 82. These prevent oxidation reactions and catalyse the breakdown of oxidised phospholipids on the LDL particle. 1.4 ABCA-transporters ATP-binding cassette (ABC) transporters are membrane proteins that are widely distributed in prokaryotes and eukaryotes, and most of them use ATP to generate the energy needed to transport substrates (i.e., drugs, toxins, peptides, lipid derivatives, and so on) across membranes 83. To date, 51 members of ABC transporter family have been identified in mice, which, based on structural similarities, have been divided into seven subfamilies, designated ABC A-G 83. Hitherto more than 48 human ABC protein genes have been identified and sequenced. Evidence has accumulated during the past years to suggest that a subgroup of 13 structurally related full-size transporters, referred to as ABCA transporters, mediates the transport of a variety of physiologic lipid compounds. This subfamily can be divided into 15

10 two subgroups, based on a phylogenetic analysis 84. One subgroup comprises five proteins (ABCA5-6 and ABCA8-10), the genes encoding these transporters are clustered on chromosome 17q24 in humans. The other subgroup consists of seven proteins (ABCA1-4, A7, A12-13), encoded by genes in different chromosomes. The emerging importance of ABCA transporters in human disease is reflected by the fact that as yet four members of this protein family (ABCA1, ABCA3, ABCA4, ABCA12) have been causatively linked to completely unrelated groups of monogenetic disorders 85,86. The following part will mainly focus on the best explored transporter, ABCA1, with an emphasis on its role in cellular lipid transport and atherosclerosis. Next, in a brief summary of current knowledge, other promising candidates of the ABCA subfamily for human diseases will also be discussed ABCA1 ABCA1 has been originally identified as an engulfment receptor on macrophages, and it has recently been shown to play an essential role in the handling of cellular lipids. Indeed by promoting the efflux of excessive cellular cholesterol to apo acceptors such as apoa-i, ABCA1 controls the formation of HDL and thus the whole process of RCT. Although the primary action of ABCA1 may be to act as a PL translocase, different views have emerged on how ABCA1 mediates cholesterol efflux 17 : (a) ABCA1 forms assembly of a FC/PL/apoA-I complex, and thus promotes PL and FC efflux from a membrane domain in a single step. (b) ABCA1 acts in a two-step process, first locating in a border region between liquid and cholesterol-rich liquid ordered domains (rafts), then promoting PL and FC efflux to apoa-i. (c) Combining the two earlier models, the third model suggests that ABCA1 first promotes PL efflux to apoa-i to form PL/apoA-I complex, which then remove cholesterol from rafts (Fig. 4). Fig. 4. Different models for ABCA1-mediated cholesterol efflux. See text for explanation. Adapted from Tall AR et al 17. ABCA1 is ubiquitously expressed, with highest expression levels in placenta, fetal tissues, lung, adrenal glands, brain, and liver. At the cellular level, tissue macrophages as well as macrophage-like cell lines of mouse or 16

11 General introduction human origin are consistently expressing high levels of ABCA1. Despite the ubiquitous expression of ABCA1, the accumulation of cholesterol in both human and mouse models of ABCA1 dysfunction occurs principally in macrophages. Mutations in the human ABCA1 gene are the underlying molecular defect in Tangier disease (TD) 87. Homozygous TD is a rare disorder associated with extremely low levels of HDL cholesterol (less than 5% of normal) and apoa-i (less than 1% of normal). The hallmark pathology is CE accumulation in tissue macrophages, leading to accumulation of foam cells in various organs, such as the liver and spleen. Importantly, fibroblasts from Tangier subjects show a dramatic reduction in cholesterol and phospholipid efflux to apoa-i, indicating a defect in the initial step of RCT 88. Moreover, a systematic survey of TD patients suggests that homozygotes have an approximately four- to six-fold increased risk of atherosclerotic cardiovascular disease (CVD), compared with age-matched controls. Obligate heterozygotes for the TD mutation have decreased HDL cholesterol (about 50% of normal), normal LDL cholesterol, and an apparent increase in risk for atherosclerosis 88. ABCA1 knockout (ABCA1 / ) mice have a phenotype similar to that of TD patients. Upon cross-breeding of ABCA1 / mice with hypercholesterolemic mouse models (ABCA1 / /apoe / or ABCA1 / /LDLr / double knockout mice), the accumulation of foam cells in peripheral tissues was especially pronounced 89. However, neither of these mouse models developed atherosclerotic lesions. By using the bone marrow transplantation technique, a chimeric mouse model with selective ABCA1 deficiency in hematopoietic cells did develop atherosclerosis upon feeding with a high-cholesterol Western-type diet 90. The progression of foam cell formation and the atheroma development are consistently significantly influenced by the ABCA1 expression on macrophages, independent of the levels of circulating HDL 90. It has to be noted that the global amount of lipid efflux from macrophages provides only a minimal contribution to circulating HDL levels. In fact, the major cholesterol contribution to HDL generation presumably comes from ABCA1 on the basolateral membrane of hepatocytes 91. In addition to its role in cellular cholesterol efflux, ABCA1 in macrophages may exert another protective function in inflammation, as evidenced by in vitro studies which showed that ABCA1-deficient macrophages have an increased response to chemotactic factors 92, in line with our previous studies which identified ABCA1 as a leukocyte factor that controls the recruitment of inflammatory cells into tissues in LDLr / mice 90. Furthermore, it has been reported that the lack of ABCA1 causes a significant reduction of apoe protein level in the brain of ABCA1 / mice 93. ApoE isoforms strongly affect Alzheimer disease (AD) pathology and risk. These results suggest that ABCA1 may play a role in the pathogenesis of parenchymal and cerebrovascular amyloid pathology and thus may be considered a therapeutic target in AD, in addition to its well-known contribution to CVD Other ABCA-transporters In addition to ABCA1, three other members of the ABCA subfamily (ABCA3, ABCA4, and ABCA12) have been causatively linked to human diseases. In detail, ABCA3 encodes a lamellar granule membrane protein, which is essential for alveolar surfactant lipid transport and secretion in alveolar lung 17

12 cells 94, and neonatal surfactant deficiency is triggered by mutations in the ABCA3 gene; ABCA4 is expressed exclusively in photoreceptors of the eye for the transport of retinol 95, and degenerative retinopathies are triggered by mutations in the ABCA4 gene; ABCA12 harbors missense mutations in autosomal-recessive congenital ichthyosis 96. The biological function of the remaining 9 ABCA-transporters currently awaits clarification. In particular, ABCA2, ABCA5 and ABCA7 represent promising candidate genes for hereditary diseases in various physiological systems. (1) ABCA2 has been identified as a highly expressed gene in oligodendrocytes in the mammalian brain and associated with lysosomes. ABCA2 shares high homology with ABCA1 (50%). Analysis of the putative ABCA2 promoter sequence revealed potential binding sites for transcription factors that are involved in the differentiation of myeloid and neural cells 97. (2) ABCA5 is highly expressed in oligodendrocytes and astrocytes of the brain, alveolar type II cells of the lung, and Leyding cells of the testis. Furthermore, ABCA5 plays an important role in the endolysosomal system, and inactivation of ABCA5 gene in mice results in exophthalmos and collapse of the thyroid gland as well as a dilated heart, with fatal heart failure 98. (3) ABCA7 mrna was detected predominantly in myelolymphatic tissues with highest expression in peripheral leukocytes, thymus, spleen, and bone marrow. Among the known members of the ABCA subfamily in humans, ABCA7 protein shows the highest homology known to ABCA1 (54%) and ABCA4 (49%) 99. However, differences between ABCA1 and ABCA7, especially in tissue distribution profile and transcriptional regulatory mechanism, implicate that ABCA7 may have a more specific role than mimicking ABCA1. The physiological function of ABCA7 is still essentially unknown. During in vitro differentiation of human monocytes into macrophages, gene expression analysis described that ABCA2, ABCA5, and ABCA7 are all cholesterol-responsive genes 100. In particular, expression of ABCA5 and ABCA7 is up-regulated upon incubation of monocyte-derived macrophages with acetylated LDL and down-regulated upon induction of cholesterol efflux by HDL 3, indicating ABCA5 or ABCA7 may play a role in macrophage cholesterol homeostasis. However, the in vivo effects of macrophage ABCA5 and ABCA7 expression are currently unknown. 1.5 Lipid Transfer Proteins: CETP and PLTP CETP and PLTP are both remodeling enzymes that transfer lipids between lipoproteins. CETP was originally designated Lipid Transfer Protein-I (LTP-I) and PLTP was designated LTP-II upon isolation and purification of these two proteins from human plasma 101. In humans, the CETP gene is located on chromosome 16 (16q21), while the PLTP gene is located on chromosome 20 (20q12 q13.11). When human CETP and PLTP were subsequently cloned, sequence analysis revealed a 21.7 % amino acid identity between the two proteins 102. In addition to humans, homologous CETP and PLTP genes have been found in other mammalian species. 18

13 General introduction PLTP activity has been detected in all species studied thus far. In contrast, substantial CETP activity is found only in man, rabbit, chicken and trout 103. Importantly, CETP is not present in the mouse, the most commonly used experimental animal model for atherosclerosis 104. In addition, CETP and PLTP belong to the same gene family: the lipid transfer/lipopolysaccharide (LPS) binding protein family 105. The other family members include bactericidal/permeability-increasing protein (BPI) and LPS-binding protein (LBP), which mediate anti-bacterial and pro-inflammatory activities via binding to LPS on the outer membrane of Gram-negative bacteria 105,106 Based on the available computational model of the closely related BPI 107, the crystal structure of CETP has been recently constructed 108. As shown in Fig. 5, CETP has an elongated 'boomerang' shape with dimensions of 135Å X 30Å X 35Å and a fold homologous to that of BPI 107. The fold consists of two similar domains connected by a linker, residues in CETP. The structure of CETP can be divided into four structural units: one barrel at each end of the protein (barrels N and C), a central β-sheet between the two barrels and a C-terminal extension that is not present in BPI. Each barrel contains a highly twisted β-sheet and two helices (A and B in barrel N, A' and B' in barrel C), with helices B and B' being longer than A and A'. These three structural units in CETP overlay well with the homologous units in BPI. The fourth unit, Glu465 Ser476 at the C terminus of CETP, forms a distorted amphipathic helix, helix X, which unwinds slightly at the end. Fig. 5. Overall structure of CETP. (a) Ribbon diagram of N-terminal (green) and C-terminal (yellow) domains, with linker in red. Whenever possible, N-terminal side is shown on left throughout the figures. CE1 (magenta) and CE2 (cyan) are shown as space fills and phospholipid as black bonds. N-glycosylation sites are shown as blue bonds, with 341 and 396 labeled. '5' marks the observed N terminus. Helices A, B, A', B' and X are labeled. Helix X belongs to the C-terminal domain but interacts with residues of the N-terminal domain. (b) The view after a 90 rotation. The four structural units shown are barrel N (green), central β-sheet (orange), barrel C (yellow) and helix X (cyan). The Ω1 flap is in gray. N-glycosylation sites 88 and 240 are labeled. Adapted from Qiu X et al 108. The amino terminal domains in CETP and PLTP are highly homologous whereas the C-terminal domains vary in hydrophobicity and hence, providing functional specificity 109. The possibility that CETP and PLTP might 19

14 act as independent risk factors in the development of atherosclerosis has raised the question of whether they are protective against or detrimental to the disease progression CETP The glycoprotein CETP is secreted by the liver and circulates in plasma principally bound to HDL 110. As described in Fig. 6, CETP equilibrates CE across various lipoprotein particles. VLDL is normally TG rich and HDL is CE rich. CETP filled with CE (step 1) binds VLDL and releases the bound phospholipid (phosphatidylcholine, PC). As VLDL is TG rich, one or two TGs can enter the tunnel and deposit an equal amount of CE into VLDL. The TGbound CETP departs from VLDL carrying two phospholipids from the surface, leaving the VLDL particle with a higher CE content (step 2). It then engages HDL and releases the bound phospholipid (step 3). As HDL is CE rich, one or two new CEs can enter the tunnel and an equal amount of bound TG is deposited into HDL. The CE-filled CETP dissociates from HDL carrying two phospholipids from the surface (step 4) and hence completes a full cycle of heteroexchange, which results in a lower CE content in HDL. The nonspecific nature of the tunnel suggests similar binding affinities for CE and TG. Homoexchange occurs when a lipid of the same kind as that bound is loaded into the tunnel, which results in no net change in lipoprotein lipid content. Both types of neutral-lipid exchange require the discharge and reloading of phospholipid, forming a basis for CETP-mediated phospholipid exchange that can also occur independently of TG or CE exchange. Fig. 6. Proposed mechanism for CETP-mediated HDL remodeling. Details are described in the text. Modified from Qiu X et al 108. Epidemiological and experimental evidence has shown that CETP may play an important role in the development of atherosclerosis 111. However, there is continued controversy regarding the function of CETP in the development of atherosclerosis. Rabbits are highly susceptible to diet-induced atherosclerosis and display naturally high levels of CETP 112. When rabbits were injected with antisense oligonucleotides against CETP to inhibit expression, HDL cholesterol levels were elevated and atherosclerosis was substantially reduced 113. Small CETP inhibitors have recently been shown to 20

15 General introduction not only increase HDL cholesterol levels, but also to affect the size distribution of HDL subpopulations and the apolipoprotein and enzyme composition in rabbits Human subjects with CETP deficiency and high levels of HDL cholesterol (>60 mg/dl) showed reduced risk of CAD, whereas CETP-deficient subjects whose HDL levels were moderately increased (40 60 mg/dl) presented a higher risk of developing CAD 117. Inhibitors of CETP have now been tested in human subjects and shown to increase the concentration of HDL cholesterol while decreasing that of LDL cholesterol and apob 118. Mice are naturally CETP deficient but studies with transgenic animals expressing human CETP have proven to be of help to further unravel the role of CETP in HDL metabolism. Studies in CETP transgenic mice have provided mixed results. When the human CETP gene is introduced in mice, a dose-related reduction in HDL-C levels and a small increase in VLDL and LDL cholesterol occur 119. However, the effects of CETP expression in this species can be neutral, proatherogenic, or antiatherogenic, depending upon the metabolic context These results would predict that, in mice that have robust pathways for uptake of apobcontaining lipoproteins, the expression of CETP, although it reduces HDL cholesterol levels, might be expected to be anti-atherogenic, consistent with published studies Conversely, in mice that have markedly defective clearance of apob-containing lipoproteins, expression of CETP might be expected to promote atherogenesis, also consistent with published studies These studies confirm the lesson learned from animal studies that the role of CETP in lipoprotein metabolism and in the development of atherosclerosis may reflect the interaction of this protein with several factors 128. What implications might these studies have for the role of CETP (and its inhibition) on RCT and atherosclerosis in humans? One hypothesis is that in normolipidemic healthy humans, hepatic SR-BI is expressed at low levels and the CETP pathway is a critical pathway for the hepatic clearance of HDL-derived CE 129. However, in the setting of impaired clearance of apobcontaining lipoproteins (including not only familial hypercholesterolemia but also other genetic and environmental factors that reduce hepatic apoblipoprotein uptake), the CETP pathway may instead be more proatherogenic by transferring HDL-CE to apob-containing lipoproteins, which are then inefficiently cleared. It would follow from this hypothesis that CETP inhibition in persons with highly effective apob-lipoprotein clearance (such as patients on high-dose statins) might not be protective (or even proatherogenic), whereas in those with defective clearance it might be antiatherogenic. Collectively, although CETP inhibition is consistently associated with decreased atherosclerosis in rabbits, studies in humans and CETP transgenic mice show that CETP expression can be either pro- or anti-atherogenic and more research regarding the role of CETP in atherosclerosis is warranted, before CETP inhibition can be used as a therapeutic approach to treat CVD in humans PLTP PLTP transfers phospholipids but not neutral lipids between plasma lipoproteins 130. PLTP-mediated processes are physiologically vital in the transfer of surface remnants from lipolyzed TG-rich lipoproteins to nascent 21

16 HDL particles and in the generation of pre-β-hdl, the initial acceptor of excess peripheral cell cholesterol. Also, during the formation of mature HDL particles, PLTP is involved to mediate lipid transfer between HDL particles, converting HDL 3 to larger HDL 2. According to a tentative model 131, PLTP interacts with the surface of HDL and induces an increase in the surface pressure either by penetrating the surface or by increasing the amount of surface lipid by net lipid transfer. ApoA-I molecules are displaced from the particles because of the increased pressure. This leads to formation of particles that are unstable because of partially exposed hydrophobic cores. Two unstable particles interact through their hydrophobic core surfaces and fuse to a thermodynamically stable particle. The fusion product may then participate in additional rounds of fusion with other fusion products or original particles (Fig. 7). Human PLTP transgenic mice have been generated and show a complicated phenotype 132. Transgenic mice that express moderate levels (~30 % increase) of human PLTP do not exhibit marked changes in lipoprotein metabolism, unless these mice are crossed into a human apoa-i background, and only after that, increases in α-hdl and preβ-hdl are observed 132. Transgenic mice expressing high, stable PLTP levels ( fold increase in activity) display a 30 40% decrease in plasma HDL-C and a concomitant rise in preβ-hdl formation, as compared to wild type controls 133. On the other hand, PLTP-deficient (PLTP / ) mice show a substantial reduction in plasma HDL levels and, after consuming a high-fat diet, an accumulation of VLDL 134. Surprisingly, crosses of PLTP / mice into apob transgenic, apoe-/-, or LDLr-/- backgrounds resulted in diminished atherosclerosis in all three of these standard mouse atherosclerosis models 132. In part this was related to the reduction of levels of apobcontaining lipoproteins seen in apob transgenic and apoe-/- mice. However, an anti-atherogenic effect of PLTP deficiency was also seen in LDLr-/- mice, despite a lack of reduction in apob lipoprotein levels. A possible clue to understanding this unexpected observation was the finding that PLTP could facilitate the in vitro transfer of vitamin E from Toll-like receptor into HDL 135. An analysis of vitamin E revealed a build-up of levels in VLDL and LDL of PLTP / mice, associated with a reduction in susceptibility of apob lipoproteins to Cu-mediated oxidation in vitro. Moreover, there was a reduction in antibodies to oxidized LDL in plasma. Thus, in addition to reducing levels of apob lipoproteins, PLTP deficiency resulted in an increase in their content of vitamin E and resistance to oxidation, which is atheroprotective. In humans, immunohistochemistry studies revealed that PLTP protein is highly expressed by macrophages within atherosclerotic lesions, suggesting a potential role for this protein in lipid-loaded macrophages 136. Recently, plasma PLTP activity was reported to be related to CVD in humans 137. Patients within the highest quintile of PLTP activity displayed a 1.9-fold increased risk for CVD compared to patients within the lowest quintile. The authors from this study concluded that PLTP activity is an independent predictive value for CVD. However, human genetic PLTP deficiency has not been described thus far, and the effects of PLTP polymorphisms on lipoprotein metabolism have as yet not been clearly delineated. 22

17 General introduction HDL 3 HDL 2 Fig. 7. Proposed model for PLTP-induced fusion of HDL particles. PLTP modulates the size and composition of HDL in a process called HDL conversion. Details are described in the text. Modified from Lusa S et al Macrophage RCT Pathway: Key to Study Atherosclerosis Regression? Looking back at animal and clinical studies published since the 1920s, the notion of rapid regression and stabilization of atherosclerosis in humans has evolved from a fanciful goal to one that might be achievable pharmacologically, even for advanced plaques. Possible mechanisms responsible for lesion shrinkage include decreased retention of apoblipoproteins within the arterial wall, efflux of cholesterol and other toxic lipids from plaques, emigration of foam cells out of the arterial wall, and influx of healthy phagocytes that remove necrotic debris and other components of the plaque. Successful regression of atherosclerosis generally requires robust measures to improve plasma lipoprotein profiles. Examples of such measures include extensive lowering of plasma concentrations of atherogenic apob-lipoproteins and enhancement of reverse lipid transport from atheromata into the liver, either alone or in combination. Unfortunately, the clinical agents currently available cause less dramatic changes in plasma lipoprotein levels, and, thereby, fail to stop most cardiovascular events. Hence, there is a clear need for testing of new agents expected to facilitate atherosclerosis regression. Additional mechanistic insights will allow further progress. Importantly, regression of atherosclerosis might be expected to be accompanied by a loss of CE mass from foam cells. The concept that promotion of macrophage RCT could prevent progression or even induce regression of atherosclerosis is thus remarkably attractive. Data in animals suggest that atherosclerosis regression can be achieved through HDLbased interventions such as apoa-i overexpression, which has been shown to promote macrophage RCT 138. In humans, as noted earlier, infusion of a single dose of pro-apoa-i increased fecal sterol excretion 139, and a weekly 23

18 infusion of recombinant apoa-i Milano /phospholipid complexes for 5 weeks appeared to induce regression of coronary atherosclerosis in a small study 140. All these findings suggest that therapies designed to promote macrophage RCT might a possibility worth exploiting. One approach is to increase the concentration of acceptors by intravenous infusion of apoa-i (wild-type or Milano) 141 or peptides based on the apoa-i structure 142. A second approach is to turn on the macrophage RCT pathway. In this regard, the most conceptually attractive is LXR agonism, as this upregulates both ABCA1 and ABCG1 expression, promotes macrophage cholesterol efflux in vitro 143, increases macrophage RCT in vivo 144, and reduces atherosclerosis in mice 145. Although some LXR agonists have resulted in hepatic steatosis and increased plasma TG and LDL cholesterol in animal models 146,147, there is still hope that this approach will be tested in humans and may prove ultimately safe and effective. In addition, there have been reports that synthetic agonists of PPAR-α, PPAR-γ, and possibly PPAR-ß/-σ may promote macrophage cholesterol efflux , and thus, existing drugs (fibrates, thiazolidinediones) and new compounds under development in this area may be another way to promote macrophage RCT. Whether inhibition of CETP 151 or promotion of LCAT activity 18,19 or hepatic SR-BI expression 18 will be viable therapeutic approaches to increase macrophage RCT and retard or regress atherosclerosis has yet to be determined. As we learn more about the molecular regulation of macrophage cholesterol efflux and RCT, there will undoubtedly be additional targets for the development of new therapies that potentially may afford the best opportunity to regress atherosclerosis. 1.7 Thesis outline The first part of the thesis focuses on the role of ABC-transporters in lipoprotein metabolism and atherosclerosis. The process of hepatic cholesterol uptake from serum coupled to intracellular processing and biliary excretion plays a pivotal role in cholesterol homeostasis of the body. Importantly, the liver consists of several different cell types with specific localizations and functions. Therefore, in Chapter 2 the mrna expression patterns of ABC-transporters and their cellular localization were systematically investigated to identify novel members involved in specific functions relevant for lipid homeostasis in the liver. Cholesterol homeostasis in macrophages is of prime importance, as dysregulation of the balance of cholesterol influx and cholesterol efflux will lead to excessive cholesterol accumulation. ABCA1 is one of the key ABC-transporters that facilitate cholesterol efflux from macrophages. Previously, we have shown that inactivation of macrophage ABCA1 induces atherosclerosis in LDLr / mice 90. In Chapter 3, we created a chimeric mouse model with selective ABCA1 overexpression in hematopoietic cells, including macrophages, by using the bone marrow transplantation technique. The therapeutic effect of macrophage ABCA1 overexpression on atherosclerotic lesion development was investigated in LDLr / mice. In addition to atherosclerosis progression, atherosclerosis regression is another important clinical goal, since most patients enter the clinic with established atherosclerosis. The concept that 24

19 General introduction promotion of cholesterol efflux from macrophages by up-regulating ABCA1 could prevent the progression or even induce the regression of atherosclerosis is thus remarkably attractive. In Chapter 4, the possible effect of macrophage ABCA1 overexpression on atherosclerosis regression was investigated in LDLr / mice with different stages of established lesions. Based on our previous findings, ABCA5 is one of the putative novel candidates involved in macrophage cholesterol homeostasis. In Chapter 5, bone marrow from ABCA5 knockout mice was transplanted to LDLr / mice, and the effects of ABCA5 deficiency on macrophage cholesterol homeostasis and atherosclerotic lesion development was evaluated. The second part of the thesis mainly focuses on the role of lipid transfer proteins (CETP and PLTP) in lipoprotein metabolism, atherosclerosis, and inflammatory diseases. By using the bone marrow transplantation technique, we created chimeric LDLr / mouse models with selective PLTP deficiency or human CETP overexpression in hematopoietic cells and thus macrophages. The role of macrophage-derived PLTP and CETP in lipoprotein metabolism and atherosclerotic lesion development was investigated in Chapter 6 and 7, respectively. Of note, macrophage foam cells are primarily restricted to atherosclerosis, whereas activated macrophages are a common feature of many inflammatory diseases. In Chapter 8, leukocyte CETP expression in acute coronary syndromes was studied in both humans and CETP transgenic mice. Furthermore, LPS is one of the most potent endotoxins that, through the activation of cellular immunity, induce a cytokine-mediated systemic inflammatory response in the host 152. In Chapter 9 we investigated whether CETP is important for a general host defense mechanism against systemic inflammation. The effect of CETP in the resistance to a sublethal dose of LPS was evaluated in a murine model. The results obtained from our experiments and the implications of these studies for future research are described in Chapter References 1. Ginsberg HN. Lipoprotein physiology. Endocrinol Metab Clin North Am. 1998; 27: Mahley RW, Hussain MM. Chylomicron and chylomicron remnant metabolism. Curr. Opin. Lipidol. 1991; 2: Mahley RW, Innerarity TL. Lipoprotein receptors and cholesterol homeostasis. Biochim. Biophys. Acta. 1983; 737: Havel RJ. Biology of cholesterol, lipoproteins and atherosclerosis. Clin Exp Hypertens.1989; 11: Fielding CJ, Renston JP, Fielding PE. Metabolism of cholesterol-enriched chylomicrons. Catabolism of triglyceride by lipoprotein lipase of perfused heart and adipose tissues. J Lipid Res. 1978; 19: Zechner R. The tissue-specific expression of lipoprotein lipase: implications for energy and lipoprotein metabolism. Curr Opin Lipidol. 1997; 8: Havel RJ, Kane JP. Introduction: Structure and metabolism of plasma lipoproteins. In: Sriver CR, Beaudet AL, Sly WS, Valle D, eds. The metabolic basis of inherited disease. 6th Ed. New York: McGraw-Hill. 1989; Mahley RW, Ji ZS. Remnant lipoprotein metabolism: key pathways involving cell-surface heparan sulfate proteoglycans and apolipoprotein E. J Lipid Res. 1999; 40: Out R, Kruijt JK, Rensen PC, Hildebrand RB, de Vos P, Van Eck M, Van Berkel TJ. Scavenger receptor BI plays a role in facilitating chylomicron metabolism. J Biol Chem. 2004; 279: