The role of abca1 in atherosclerosis: lessons from in vitro and in vivo models - Thesis

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The role of abca1 in atherosclerosis: lessons from in vitro and in vivo models

Singaraja, R.R.

Publication date

2003

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Final published version

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Citation for published version (APA):

Singaraja, R. R. (2003). The role of abca1 in atherosclerosis: lessons from in vitro and in vivo

models.

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Lay-out:: Medische Fotografie en Illustratie, AMC Printing:: Buijten & Schipperheijn, Amsterdam

lessonss from in vitro and in vivo models

ACADEMISCHH PROEFSCHRIFT

terr verkrijging van de graad van doctor aann de Universiteit van Amsterdam opp gezag van de Rector Magnificus

prof.mr,, P.F. van der Heijden

tenn overstaan van een door het college voor promoties ingesteldee commissie, in het openbaar te verdedigen

inn de Aula der universiteit

opp woensdag 17 september 2003, te 14.00 uur

door r

Roshnii Rebecca Singaraja

Promotores: : Overigee leden: Faculteitt Geneeskunde Prof.dr.. J.J.P. Kastelein Prof.dr.. M.R. Hayden Prof.dr.. H. Pannekoek Prof.dr.. P.H. Reitsma Prof.dr.. H.R. Buller Dr.. A.K. Groen Dr.. J.A. Kuivenhoven

Chapterr 1: Introduction 7

PARTT I: Insights from an abcal animal model

Chapterr 2: Human ABCA1 BAC transgenic mice show increased HDL-C and ApoA-l dependant 33 effluxx stimulated by an internal promoter containing LXREs in intron 1.

Roshnii R. Singaraja, Virginie Bocher, Erick R. James, Susanne M. Clee, Lin-Hua Zhang, Blairr R. Leavitt, Bing Tan, Angela Brooks-Wilson, Anita Kwok, Nagat Bissada, Yu-zhou Yang,, Guoqing Liu, Sherrie R. Tafuri, Catherine Fievet, Cheryl L. Wellington, Bart Staelsandd Michael R. Hayden. J Biol Chem. 2001;276:33969-33979.

Chapterr 3: Alternate transcripts expressed in liver and macrophages in response to diet imply 63 tissuee specific regulation of human ABCA1

Roshnii R. Singaraja, Erick R. James, Jennifer Crim, Alu Chatterjee and Michael R. Hayden.. Manuscript in preparation

Chapterr 4: Increased activity of ABCA1 protects against atherosclerosis 85 Roshnii R. Singaraja, Catherine Fievet, Graciela Castro, Erick R. James, Nathalie Hennuyer,

Susannee M. Clee, Nagat Bissada, Jonathan C. Choy, Jean-Charles Fruchart, Bruce M. McManus,, Bart Staels and Michael R. Hayden. J Clin Invest. 2002; 110:3542.

Chapterr 5: Macrophage-specific ABCA1 overexpression inhibits atherosclerotic lesion development 103 Mirandaa Van Eck, Roshni R. Singaraja, Erick R. James, Michael R. Hayden, and Theo

J.C.. Van Berkel. Manuscript in preparation

Chapterr 6: Alterations of plasma lipids in mice via adenoviral mediated hepatic Overexpression

off human ABCA1 117 Cheryll L. Wellington, Liam R. Bunham, Steven Zhou, Roshni R. Singaraja, Henk Visscher,

Allisonn Gelfer, Colin Ross, Erick R. James, Guoquing Liu, Mary T. Huber, Yu-Zhou Yang,, Robin J. Parks, Albert Groen, Jamila Fruchart-Najib, and Michael R. Hayden. JJ Lipid Res. [epub ahead of printing]

Chapterr 7: Efflux and Atherosclerosis: The clinical and biochemical impact of variations in the 139 ABCA11 gene

Roshnii R. Singaraja, Liam R. Brunham, Henk Visscher, John Kastelein, and Michael R. Hayden.. In press, Artenoscler Thromb Vase Biol

Chapterr 8: The range of phenotypes in TD and FHA depends on severity of defects in ABCA1 163 Roshnii R. Singaraja, Henk Visscher, Erick R. James, Veronique Rigot, Yannick Hamon,

Giovannaa Chimini, and Michael R. Hayden. Manuscript in preparation

Chapterr 9: Identification and functional analysis of a naturally occurring E89K mutation in the 187 ABCA11 gene of the WHAM chicken.

Alann D. Attie, Yannick Hamon, Angela R. Brooks-Wilson, Mark P. Gray-Keller, Marcia L.. MacDonald, Veronique Rigot, Angie Tebon, Lin-Hua Zhang, Jacob D. Mulligan, Roshnii R. Singaraja, J. J. Bitgood, M. E. Cook, John J. Kastelein, Giovanna Chimini, andd Michael R. Hayden. J Lipid Res. 2 02;43:1610-1617.

Chapterr 10: Truncation mutations in ABCA1 suppress normal upregulation of full-length ABCA1 201 byy 9-cis-retinoic acid and 22-R-hydroxycholesterol.

Cheryll L. Wellington, Yu-Zhou Yang, Steven Zhou, Susanne M. Clee, Bing Tan, Kenichi Hirano,, Karin Zwarts, Anita Kwok, Alison Gelfer, Michel Marcil, Scott Newman, Kirsten Roomp,, Roshni Singaraja, Jennifer Collins, Lin-Hua Zhang, Albert K. Groen, Kees Hovingh,, Alison Browniie, Sherrie Tafuri, Jacques Genest Jr, John J. Kastelein, and Michaell R. Hayden. J Lipid Res. 2002;43:1939-1949.

Chapterr 11: Summary and conclusions 221

Chapter r

I I

Introduction n

Atherosclerosis s

Cardiovascularr diseases are the major cause of death in adults in most developed and many developingg countries, and are now the commonest cause of death worldwide (1-3). In 1990, heartt disease and cerebrovascular disease were the first and second leading causes of death accountingg for 6.3 and 4.4 million deaths worldwide. These disorders also lead to substantial morbidityy and disability and are a main source of the rising cost of health care. Worldwide, t h e r e f o r e ,, the need for effective strategies t o prevent CHD has never been greater. Hypercholesterolemia,, hypertension, low HDL-cholesterol, elevated levels of circulating triglyceridee and lipoprotein(a), cigarette smoking, obesity, diabetes mellitus and physical inactivity aree factors that powerfully influence risk of atherosclerosis (4).

Atherosclerosiss is a progressive disease characterized by the accumulation of lipids and fibrous elementss in the large arteries. It is estimated that by approximately the age of 50, 3 0 % of the intimall surface of the coronary arteries is covered by atherosclerotic lesions (5). The arterial endotheliumm is permeable to proteins within the plasma (6). Substances may move through thee endothelial layer either by transcytosis or through gap junctions between endothelial cells (4).. Physical forces such as shear stress affect the endothelial cells in vessel junctions and curvatures.. These areas are preferential sites for lesion formation and show increased permeability too macromolecules such as LDL (7). However, sheer stress alone is insufficient to induce lesion formation.. High plasma concentrations of pro-atherogenic lipoproteins such as LDL cholesterol levelss exceeding 2 mmol/L (80mg/dL) at sites of increased stress is a major contributor (8). A primaryy initiating event in atherosclerosis is the accumulation of LDL in the sub endothelial matrixx by passive diffusion through EC junctions and its interaction with proteoglycans (9). Thee rate of entry of lipoproteins into the vessel wall has been shown to be greater than the rate att which they leave (8). Although native LDL is not taken up rapidly enough by macrophages too generate foam cells, trapped LDL undergoes modifications through oxidation, lipolysis, proteolysiss and aggregation that contribute to foam cell formation as well as inflammation (10,, 11). Minimally oxidized LDL stimulates the overlying ECs to produce a number of pro-inflammatoryy molecules (12) inducing the entry of leukocytes into the arterial wall (13). Growth factorss secreted bu the endothelial cells such as macro^ha^e-colon" stimulatincs factor stimulate thee growth, differentiation and proliferation of monocytes and macrophages (4). Oxidized LDL bindss to macrophage scavenger receptors (14) and is rapidly taken up by macrophages leading too foam cell formation. With time, the foam cells die, contributing their lipid contents to the necroticc core of the developing lesion (15, 16). This causes the migration from the medial layer off smooth muscles cells that secrete fibrous elements (17, 18). Occlusive fibrous plaques developp and enlarge in size, because of the migration of new mononuclear cells into the shoulderss of the plaques (Figure 1).

Endotheliall Dysfunction Fatty-Streakk Formation

Advanced.. Complicated Lesion

Macrophagee accumulation Formation of Fibrous-cap formation Necroticc core

Smooth-musclee Foam-cell T-cell migrationn formation activation

ndd Adherence off and entry

off leukocytes

Unstablee Ruptured Plaque

Plaguee rupture Thinning of fibrous cap Hemorrhagee from plague

Figuree 1. The development of atherosclerosis

(A)) Schematic of a section of an arterial wall. Components of the plasma such as macrophages or lipoproteins mayy cross the endothelial layer and enter the mtima. Lipoproteins may bind to proteoglycans of the extracellular matrixx and become trapped within the intima. (B) Lipoproteins accumulated within the intima may be taken up byy macrophages, resulting in foam cells. Accumulations of foam cells constitute a fatty streak lesion (C) Layers off foam cells form isolated extracellular lipid pools as the foam cells die. Convergence of lipid pools results in a focall lipid core followed by the formation of a fibrous cap through smooth muscle cell migration into the intima andd proteoglycan deposition. (D) Formation of a defect in the lesion surface leading to plague rupture and resultingg in a hematoma or thrombosis formation. Adapted from Ross (115).

Whilee the growing fibrous plaques may occlude the artery, rupture of the plaques also lead to clinicall consequences. During plaque rupture, lipids from the core of the lesions are released andd exposed. This may stimulate pathways involved in wound healing, and may generate inflammationn or thrombosis (19, 20). Plaque ruptures often occur on the edges of lesions wheree foam cell content is high (4). Additionally, thrombi on the luminal surface of the lesion cann break off, leading to occlusion of smaller vessels in the heart resulting in Ml, or in the brain resultingg in stroke. Restricted blood flow to coronary arteries, or transient intermittent coronary obstructionn due to thrombus formation may result in angina (18).

Itt has been estimated that over half of patients with premature CAD have a genetic lipoprotein disorderr (21).

Tangierr Disease and FHA patients and phenotypes

Inn 1961 a new disorder of cholesterol and lipoprotein metabolism was described in t w o siblingss from Tangier Island, a sandbar on Chesapeake Bay (22). The clinical features of these siblingss were hypocholesterolemid, enldiyed tonsils of unusual appearance, hepatosplenomegaly, lymphadenopathy,, and nearly complete lack of plasma high density lipoprotein, with foam cellss observed in the tonsils and lymph nodes that contained large amounts of cholesterol esterss (23). The disease was named Tangier Disease reflecting the origin of the first described patients.. Since then, about 60 families worldwide have been described as having Tangier Disease,, with homozygotes having marked deficiency of HDL-C and ApoA-l (both <10mg/df), decreasedd LDL-C (about 4 0 % of normal), and mild hypertriglyceridemia ( 1 6 2 % of normal) (24).. Homozygotes also develop cholesterol ester deposition in their tonsils (orange tonsils), liver,, spleen, gastrointestinal tract, lymph nodes, bone marrow and Schwann cells, the latter leadingg to peripheral neuropathies.

Numerouss epidemiological and clinical studies have demonstrated that an inverse and independentt association between HDL-C and coronary heart disease is present (25). More than 4 0 %% of patients with myocardial infarction have low HDL-C as a cardiovascular risk factor (26). Overall,, cardiovascular disease was observed in 2 0 % of those with TD compared with 5% of controlss (p<0.05), and in those between 35 and 65 years of age, CVD occurred in 4 4 % of thosee with TD, compared with 6.5% in control males and 3.2% in control females (24). Metabolicc studies on TO patients revealed that HDL and its precursors are rapidly catabolized (27)) and in contrast to normal mononuclear phagocytes (MNP), MNP from those with TD degradee internalized HDL in unusual lysosomes, indicating a defect in cellular lipid metabolism (28,, 29). HDL mediated cholesterol efflux and intracellular lipid trafficking and turnover are alsoo abnormal in fibroblasts form TD patients (30-32).

Thee TD locus was originally mapped to chromosome 9q31 (33). Subsequently, the defect underlyingg TD was determined to be in the ABCA1 gene, a member of the ATP binding cassette transporterr superfamily (34-36). In addition, it was determined that familial hypoalpha lipoproteinemiaa (FHA), a previously separate disorder characterized by low levels of HDL-C, mappedd to the same locus and was caused by heterozygous mutations in ABCA1 (34) indicating thatt TD and FHA were allelic.

Thee ABCA1 gene

Proteinss are classified as ABC transporters based on the presence of ATP binding domains, also k n o w nn as nucleotide binding folds (NBFs). These ATP binding domains contain three characteristicc conserved regions, the Walker A and B domains, which are separated by approximatelyy 90-110 amino acids, and a signature (C) motif, located just upstream of the Walkerr B site (Figure 2) (37, 38). In addition, ABC transporters consist of one or t w o sets of

Figuree 2. Topographical model of the ABCA1 protein.

Thee ABCA1 protein is 2261 ammo acids long, and consists of two halves, each containing a set of six transmembranee domains, one large extracellular loop, and a nucleotide binding fold consisting of the Walker A andd B sequences and the Walker C motifs that make up the ATP binding cassette. The transmembrane domains aree shown and the residues that are predicted to be in the transmembrane domain are identified. The residues constitutingg the Walker domains are also shown.

transmembranee domains, each usually comprised of six membrane spanning u-helices and providingg substrate specificity (39).

ABCC genes are organized in t w o ways. Some are half transporters and consist of one NBF and onee transmembrane domain region. These half transporters form either homo or heterodimers inn order to function. Other ABC genes are full transporters consisting of t w o NBF's and t w o transmembranee domain regions (37). The mammalian ABC genes are divided into seven subfamilies,, ABCA to ABCG, based on similarity in gene structure, order of the domains, and sequencee homology in the NBF and TM domains.

Geneticc variations in the ABC genes are the cause of several disorders. For example, mutations inn ABCA4 result in Stargardt disease, retinitis pigmentosum 19, cone-rod dystrophy, and age-relatedd macular degeneration (40, 41), and mutations in ABCC7 (CFTR) result in cystic fibrosis (42).. Mutations in both ABCB1 (MDR1) and ABCC1 (MRP1) are associated with cancer (43-47),, and mutations in ABCG5 and G8 cause sitosterolemia (48) (Table 1).

Tablee 1. Mutations in ABC transporters are the underlying cause of several different diseases.

Genee Disease

A R C A 11 T a n q i ^ r rV.r'.lr_{>> Frirriüi-V H y f v ^ i p h . l " p " p r ^ t r i p r r r}

;l 3 <J.J 3 5 ;

ABCA44 Stargardt disease, Retinitis pigmentosum 19, cone-rod dystrophy, age-related macular degenerationn (40, 41)

ABCB44 Progressive familial intrahepatic cholestasis type 3 (PFIC) (49) ABCB77 X-lmked sideroblastic anemia and cerebellar ataxia (50, 51) ABCB111 Progressive familial intrahepatic cholestasis type 2 (52-57) ABCC66 Pseudoxanthoma elasticum (58-61)

ABCC88 Persistent hyperinsulmemic hypoglycemia of infancy (PHHI) (62-66) ABCD11 X-linked adrenoleukodystrophy (67)

ABCC22 Dubin-Johnson syndrome (52-57) ABCC77 Cystic fibrosis (42)

ABCG5andd ABCG8 Sitosterolemia (48)

ABCA11 gene regulation

Thee ABCA1 gene shows complex regulation. ABCA1 protein levels and ApoA-l mediated lipid effluxx have been shown to be up regulated by cAMP stimulation, putatively mediated through increasedd phosphorylation of ABCA1 (68). This upregulation has been described in rodent monocytee derived cell lines J774 and RAW264, but not in human kidney CaCo-2, human liver HepG2,, human monocyte derived THP-1 nor in human fibroblasts from skin (69). LXR/RXR agonistss also increase ABCA1 protein and ApoA-l mediated lipid efflux. A functional LXR element wass described in Exonl of the ABCA1 gene (70), and three other functional LXR elements have beenn described in intron 1 of the ABCA1 gene (71). LXRs are nuclear hormone receptors that are activatedd by oxysterols and control the regulation of several genes in the sterol metabolic pathways (72).. In addition, the ABCA1 promoter has been shown to bind the zinc finger gene ZNF202, a transcriptionall repressor of several genes involved in lipid metabolism (73). The ABCA1 promoter alsoo contains several E-box elements. One E-box element at position -140 is conserved and downregulatess ABCA1 expression (74, 75). In addition, functional 5p1/3 and oncostatm m-responsivee elements have also been described in the ABCA1 promoter (74) (Figure 3).

Threee groups of transcription start sites have been described in the ABCA1 gene. The first start sitee occurs 40bp downstream of a TATA box, and putative binding sites for AP1, NFKB, Sp1 and SRFBPP are also found in this region (76, 77). This transcript was identified in placenta. The second startt site occurs 82bp downstream of the first start site, and a functional LXR/RXR site is found slightlyy upstream of this start site (70, 78). This transcript was identified in the human monocytic celll line THP-1 and the human liver cell line, HepG2. The third group of transcripts occurs in intronn 1 of the ABCA1 gene. Three different transcriptional start sites have been identified in this region,, each forming a novel exon 1 that splices into the previously identified exon 2 of ABCA1 (71,, 79). These transcripts occur downstream of TATA and CAAT sequences and contain functional LXR/RXRR elements located upstream. These transcripts were identified in human liver, and in liver

Oxysterolss Lipids retinoidss retinoids

II I

LXR/RXRR PPAR/RXR (nott functionally characterized)

DR44 DR4 DR4 DR1 DR1 -71744 -7656 -4686 -2495 -1706

Figuree 3. Regulatory elements in the ABCA1 promoter and intron 1.

AA schematic diagram of both the most upstream and the intronl promoter of ABCA1. ABCA1 is upregulated throughh the LXR/RXR elements at position -63 of the most upstream promoter and -4686, -7656, and -71 74 of thee intronl promoter. In addition, ABCA1 is upregulated by cAMP, and the GC boxes at-91 and-157,andthe E-boxx motif at position -140 have been shown to be essential for ABC1 upregulation. ABCA1 is down regulated byy the ZNF202 transcription factor. Also shown are the two potential PPAR elements discovered in the intronl off ABCA1 that have not been functionally characterized.

fromm mice expressing the human ABCA1 gene. In addition to these transcripts, one other transcript containingg the most 5' transcription start site, but lacking part of exon 3 and all of exon 4 was detectedd in human endothelial, smooth muscle and fibroblast cells (80).

ABCA11 protein distribution and localization

Thee ABCA1 protein is expressed in cells of the myeloid lineage and has been detected in humanss in activated monocytes, macrophages and in foam cells, and in the mouse it has been detectedd in both peritoneal and bone marrow derived macrophages (81-8S). In addition, parenchymall cells such as those in the liver and adrenals also express high levels of ABCA1 (84).. ABCA1 RNA and protein are highly expressed in the testes, kidneys, lungs and spleen (85).. It is also highly up regulated in the uterus and in developing placentas where it has been foundd in both the decidual and labyrinthine layers (81, 84, 86).

Intracellular^,, ABCA1 is localized mainly at the plasma membrane and elegant time lapse fluorescent microscopicc studies also showed that ABCA1 fused to GFP showed intracellular trafficking between thee endoplasmic reticulum, endosomes, lysosomes and the plasma membrane (87, 88).

Severall groups have performed recent studies in order to determine the topology of the ABCA1 protein.. Using flag tags and antibodies, and also deciphering the glycosylation status of ABCA1, thee most current topological analysis of ABCA1 determined that ABCA1 consists of t w o large extracellularr loops, one between the first and second transmembrane domains, and the other followingg the intracellular NBF1 domain (89, 90).

ABCA11 is heavily glycosylated (72) and it also is phosphorylated (68,91). Mutation of both glycosylationn and phosphorylation sites in ABCA1 resulted in a reduction in ApoA-l mediated lipidd efflux. In addition, the ABCA1 protein contains other functional domains. ABCA1 contains aa PEST sequence that affects its stability and enhances its cleavage by calpains (92) and thereforee controls cell surface concentration and lipid efflux activity of ABCA1. It also contains

Oxysterolss Oxysterols cAMPP retinoids ZNF202

: :

<=&= =

=HD=# #

NF-kappaa B GnT-motif GC Box E-Box GC-E -6444 -225 -157 -140 -9

Classicall Vacuum Flippase pumpp cleaner

Figuree 4. Models of possible mechanisms of action of ABC transporters.

Modelss for substrate transport by ATP binding cassette transporters. In the classical pump model, substrates in thee cytosol are pumped across the plasma membrane and released into the aqueous environment in the extracellularr surface. In the vacuum cleaner model, substrates partition into the lipid layer, and interact with the proteinn which then pumps the substrate into the extracellular environment. In the flippase model, substrates partitionn into the lipid bilayer, and are translocated to the outer layer, where they repartition again into the extracellularr environment Adapted from Sharom, F.J (94)

aa c-terminal PDZ recognition peptide (93), and binds to the PDZ domain containing protein, 32-syntrophin.. Syntrophins interact with microtubule proteins and are thought to target secretory vesicless to plasma membrane domains (93). Profile scans performed on ABCA1 also identified severall other transport and vesicle binding domains (unpublished observation).

Althoughh the fact that ABCA1 transports lipids across plasma membranes has firmly been established,, the determination of the exact mechanism by which this occurs still remains elusive.. Three possible models have been suggested for the transport of substrates across plasmaa membranes by ABC transporters. The first is a classical pump model by which molecules inn the cytosolic aqueous compartment are pumped directly across the plasma membrane and intoo the aqueous environment in the extracellular compartment. In this model, substrates move throughh a transport channel within the protein, and do not contact the lipid phase. In the second,, a vacuum cleaner model, substrates are partitioned into the lipid bilayer and come into contactt with the transporter. The substrates are then pumped across the plasma membrane intoo the exxraceiiuiar environment. The third is a flippase model during which substrates partition intoo the inner leaflet of the lipid bilayer and interact with the transporter and then are flipped acrosss the plasma membrane into the outer leaflet. The substrate is then repartitioned into the extracellularr environment (94) (Figure 4),

Reversee cholesterol transport p a t h w a y

Inn 1965, the first observation that cholesterol can be removed from cells in vitro by exposing t h e mm t o serum was made (95). The concept of reverse cholesterol transport (RCT) was subsequentlyy formulated (96) as a multi-step process by which there is net movement of

cholesteroll from peripheral tissues such as macrophages back to the liver via the plasma c o m p a r t m e n t .. The concept of RCT provided a theoretical framework for understanding cholesteroll homeostasis. Every day, approximately 9 mg of cholesterol per kg of body weight thatt is synthesized by peripheral tissues have to be moved to the liver for effective catabolism (97).. Disruption of the RCT pathway may favor the deposition of cholesterol in the arterial wall, andd thereby contribute to the development of atherosclerosis. HDL acts as the acceptor and transportt particle for effluxed cholesterol and as such occupies a central position in RCT. The RCT pathwayy consists of five steps: (1) uptake of cholesterol from cells by specific acceptors (cholesterol efflux)) (2) esterification of cholesterol within HDL by lecithimcholesterol acyltransferase (LCAT) (3)) transfer of cholesterol to the ApoB containing lipoproteins (cholesterol transfer) (4) remodeling off HDL (5) uptake of HDL cholesterol by the liver (cholesterol uptake) (98) (Figure 5). ABCA1 is essentiall for the first step in RCT, the efflux of lipids across plasma membranes.

Macrophagee Liver

Figuree 5. ABCA1 and the reverse cholesterol transport pathway.

AA schematic model of the reverse cholesterol transport pathway, showing the transport of cholesterol from peripherall cells such as macrophages, to the liver for excretion as bile. Five steps are involved in the reverse cholesteroll transport pathway. (1) uptake of cholesterol from cells by specific acceptors (cholesterol efflux). Nascentt pre-(! HDL, secreted by the intestine or liver, is a potent acceptor of effluxed cholesterol from peripheral tissues.. (2) esterification of cholesterol within HDL by lecithinxholesterol acyltransferase (LCAT). When free cholesteroll is estenfied, it is moved to the centre of the HDL particle, and nascent HDL particles are converted intoo HDL, particles. (3) transfer of cholesterol to the ApoB containing lipoproteins (cholesterol transfer). HDL, particless are converted into large HDL_ particles by acquiring phospholipids and apolipoproteins that are released duringg lipolysis of triglycerides in chylomicrons or VLDL by LPL. (4) Remodeling of HDL. Cholesteryl esters from HDLL are transferred into VLDL, IDL or LDL by CETP. These particles are subsequently taken up by the liver (5) uptakee of HDL cholesterol by the liver (cholesterol uptake). Cholesterol esters in the HDL particles are taken up byy the liver through the scavenger receptor class B1 (SR-B1). SR-B1 performs the uptake of cholesterol ester withoutt internalization or degradation of the HDL particle. ABCA1 is essential for the first step in RCT, the efflux off lipids across plasma membranes.

ABCA11 and lipid efflux

Cholesteroll efflux involving HDL can occur through three distinct pathways. First, the efflux of cholesteroll from cells to HDL can be mediated through the scavenger receptor B1 (SRB1), whichh is a HDI receptor th.it mediate: the uptake uf diulesteiol estets into cells such as hepatocytess (99,100). Second, free cholesterol can desorb from the plasma membrane and incorporatee into HDL particles. This occurs through passive diffusion (101). These t w o processes aree bidirectional. Third, active unidirectional efflux of cholesterol and phospholipids from cells too ApoA-l, theapolipoproteinof HDL is mediated by ABCA1 (34,102). Although ABCA1 promotes thee efflux of both cholesterol and phospholipids, if these are transported together or if their transportt is independent of each other has been until recently unclear. Three models have beenn suggested for the mechanism of efflux due to ABCA1 (103). In the first model, ABCA1 translocatess both cholesterol and phospholipids to the plasma membrane outer leaflet in one step.. ApoA-l can then act as an acceptor for these lipids promoting joined efflux. In the second model,, ABCA1 translocates phospholipids and cholesterol by a two-step process, first translocating phospholipidss that are taken up by ApoA-l, which leads to the formation of lipid-poor particles thatt then take up cholesterol from membrane microdomains as the second step. The third suggestedd model is a intermediate of the first t w o models, where cholesterol and phospholipids effluxess are normally coupled since they are in close membrane microdomain proximity, but cann be dissociated if the microdomains are far apart (103). Several recent studies suggest that thee second or third models are most accurate.

Fieldingg and colleagues found a dissociation between phospholipid efflux and cholesterol effluxx to ApoA-l in the presence of vanadate and okadaic acid, both of which inhibited free cholesteroll efflux but not phospholipid efflux (104). Their data suggested that ABCA1 dependent phospholipidd efflux preceded free cholesterol effiux to ApoA-l, and showed that the phospholipid-ApoA-ll complex was a much better acceptor of free cholesterol efflux than just ApoA-l alone (104).. Wang and colleagues also set out to determine if phospholipid and cholesterol efflux aree coupled, and came to the conclusion that the binding of ApoA-l to ABCA1 causes the formationn of ApoA-l-phospholipid complexes that subsequently promote free cholesterol efflux (105).. They did this through a series of elegant experiments where when cyclodextrin was addedd in order to deplete cells of cholesterol, ABCA1 mediated cholesterol efflux was abolished, butt phospholipid efflux to ApoA-l still occurred. Also, conditioned media from cyclodextrin treatedd ABCA1 containing cells had the ability to elicit cholesterol efflux from cells not expressing ABCA1.. Yamauchi and colleagues compared ApoA-l mediated cholesterol and phosphocholine effluxx from several fibroblast cell lines (106). They found that all but one cell line were ApoA-l dependentt phosphocholine efflux competent. However, about half of the cell lines that were phosphocholinee efflux competent did not promote ApoA-l mediated cholesterol efflux. All the celll lines were able to promote diffusion-mediated cholesterol efflux, and all the cell lines

exceptt for the one that showed no efflux at alt contained detectable levels of ABCA1. Finally, Sunn and colleagues found that when fibroblasts are co-transfected with ABCA1 and Stearoyl-CoAA Desaturase 1 or 2 (SCD1 or SCD2), ABCA1 mediated cholesterol efflux but not phosphocholinee efflux is inhibited. This inhibition is caused by a decrease in membrane ordered regionss indicated by a decrease in Triton-X 100 resistant domains in the presence of SCD 1 and 22 (107). Taken together, these data show that ABCA1 mediated phosphocholine and free cholesteroll efflux are separable events, and that ABCA1 is required for the generation of ApoA-l-phospholipidd complexes (Figure 6). Once generated, these complexes are able to take up cholesteroll efficiently, even in the absence of ABCA1.

Animall models of ABCA1

Sincee the discovery of ABCA1 as the gene mutated in TD and FHA, several animal models involvingg ABCA1 have been generated. Three of these models describe phenotypic consequences off the loss of functional ABCA1, t w o of which are knock-out mouse models and the other, a naturallyy occurring chicken with loss of function mutations in ABCA1 ( 8 1 , 108, 109, 110). The knock-outt mouse models were generated on t w o distinct strains, and show differences in the extentt and severity of lipid accumulation in various tissues, presumably because of the strain differences.. However, both models show absence of HDL in homozygotes. In addition, females

Figuree 6. Model of cholesterol and phospholipid efflux by ABCA1.

Althoughh three possible models for cholesterol and choline efflux have been proposed (103), several lines of evidencee point to this model as the most representative. In this model, ABCA1 translocates phospholipids and cholesteroll by a two-step process, first translocating phospholipids at the plasma membrane that are taken up byy lipid poor ApoA-i, which leads to the formation of nascent lipid-poor HDL particles that are potent cholesterol acceptorss and that take up free cholesterol from membrane microdomains as the second step

off both models displayed difficulties in breeding, with markedly reduced litter sizes and number off pregnancies. In contrast, the distribution of accumulated lipids differed markedly between thee t w o models, with one showing accumulation in the testes, thymus, liver and placenta (81) andd the other showing accumulation in the lung (108). The reasons for these differences remainn largely unproven, although it is likely that differences in the background strains of the animalss could affect phenotypes, caused by the presence of modifier genes.

Thee phenotype of the WHAM chicken showed similarities with the knock-out mouse models, thee most significant of which was an almost complete lack of circulating HDL. In addition, the WHAMM chicken showed accumulation of lipids in the liver and small intestine (110). Threee mouse models over expressing ABCA1 have also been generated. The generation and characterizationn of one of these mouse models is described in detail in this thesis. Two other mousee models over expressing ABCA1 were generated (111-114). The earliest was a model generatedd in the FVB background, and used BAC transgenic technology for its generation. Onee BAC containing a full length ABCA1 gene containing up to 70 kb of upstream sequences, andd another containing a truncated ABCA1 gene that did not contain the ABCA1 promoter, Exonll and part of intron 1 were used to generate transgenic mice. These BAC mice both showedd no changes in plasma HDL-C levels. The full length BAC mice showed increased cellularr cholesterol efflux, and ABCA1 was expressed in macrophages. The truncated BAC showedd the presence of human ABCA1 transcripts only in the liver. No further characterization off these mice has been reported.

Anotherr ABCA1 transgenic mouse model was generated using cDNA technology, and in these micee the ABCA1 cDNA was driven by the ApoE promoter with its macrophage and hepatic controll elements (112). The copy numbers is these mice were 30 and 40, and protein over expressionn was detected in the liver (4 and 9 fold) and in macrophages (3 and 6 fold) (113). ABCA11 expression was not detected in the brain, adrenals, heart, small intestine, spleen, lung orr kidney. Increased ApoA-l mediated cholesterol efflux was observed f r o m peritoneal macrophagess isolated from these mice. The transgenic mice showed elevated plasma TC, PL, FC,, CE and HDL-C compared with controls. Levels of ApoA-l and ApoB were also increased. Thee degradation of HDL was delayed in these mice, and was the cause of the increase in plasmaa HDL-C that was observed.

Whenn these cDNA transgenic mice were fed an atherogenic diet, they developed an anti-atherogenicc profile, showing 63% decrease in plasma cholesterol, 6 7 % decrease in free cholesterol,, 6 3 % decrease in cholesteryl ester, 53% decrease in non-HDL cholesterol, and a 6 4 %% decrease in ApoB. In addition, these mice showed a 280% increase in HDL-C levels, 220% increasee in ApoA-l levels, and a 280% increase in ApoE levels (113). These mice also showed aa 65% decrease in atherosclerotic lesion area. However, when these mice were crossed to the ApoE-/-- mouse model, a 200 - 260% increase in aortic lesion area was observed. In addition,

whenn these mice were crossed to the LDLr-/- mice, there was an increase in TC and non-HDL-CC with no changes observed in the levels of plasma HDL-C (114).

Rationalee for this thesis

Thee discovery of ABCA1 as a gene critical for the maintenance of plasma lipid homeostasis, andd its role in the development of atherosclerosis has generated significant interest in the determinationn of its function, and in the potential for the development of therapeutics aimed att reducing the incidence of atherosclerosis. As such, we developed ABCA1 over expressing mousee models in order to determine if raising ABCA1 levels was a viable alternative for the developmentt of anti-atherosclerotic therapeutics, and to validate ABCA1 as a therapeutic target. Wee chose to use BAC transgenic technologies to generate our mouse models because it is mostt biologically relevant to have ABCA1 expression driven by the endogenous promoter. The usee of the endogenous promoter removed any concerns about non-physiological regulation or expressionn of the ABCA1 gene, and ensured that ABCA1 was expressed in the right cellular milieuu in response to the appropriate cellular signals. Although several differences exist between micee and humans with respect to lipid metabolism and atherosclerosis (Table 2), we chose to generatee mouse models because this would permit the determination of other genes involved inn atherosclerosis and their interaction with ABCA1. Several well characterized mouse models doo exist either over expressing or not expressing many genes involved in lipid metabolism, thuss providing valuable tools to further dissect the pathways involved in lipid homeostasis. Inn Chapter 2 of this thesis, we describe the generation and characterization of the ABCA1 BAC transgenicc mouse model. Although the role of ABCA1 in atherosclerosis was not determined in thiss study, we discovered the presence of three alternative ABCA1 transcripts that were present inn the BAC transgenic mice and in human tissues. The tissue abundance and distribution of thesee transcripts were determined in Chapter 3 of this thesis, and the response of each of the transcriptss to feeding of a diet rich in fat was also examined. The role of over expressed ABCA1 inn atherosclerosis was addressed in Chapter 4, where we crossed the ABCA1 BAC transgenic micee to the ApoE-/- mice, and showed that ABCA1 is able to reduce atherosclerosis independent off plasma lipid levels. This led to our next study, detailed in Chapter 5, which had the goal of determiningg if ABCA1 over expression in macrophages alone was sufficient for the reduction off atherosclerosis. We did this by performing bone marrow transplantation from ABCA1 BAC transgenicc mice into the LDLr-/- mice.

Sincee ABCA1 is found at highest levels in the liver, it likely has an important function in the liver,, perhaps in the maintenance of total body sterol levels. In order to determine the role of hepaticc ABCA1 in maintenance of HDL-C levels, we performed our next study, which addressed thee role of overexpressed ABCA1 in the liver of mice. This study was performed by infecting

Tablee 2. Differences in lipoprotein metabolism and atherosclerosis between humans and mice

Humann Mouse Plasmaa CETP

C i r i _ u i d t ' ! ' l C jj ML.

Mainn cholesterol carrier HDL L LDL L Apo(a) ) ApoBB editing Atherosclerosis s Present t L O W W LDL L 50mg/dl l 150mg/dl l Present t Intestine e Susceptible e Absent t High h HDL L 50rng/dl l 10mg/dl l Absent t

Liverr and intestine Resistant t

thee recipient mice with adenoviral ABCA1, and subsequently determining its effect on plasma lipidd levels. This study is described in Chapter 6.

Inn addition to the mouse models, we also performed several other biochemical studies in order too gain further insight into the mechanism of action of the ABCA1 gene. In Chapter 7, we performedd a review of every single TD and FHA patient described in the literature, with the intentt of gaining insight into the phenotypic variability that is observed in both Tangier disease andd FHA patients. This led to the generation of some of the missense mutations in vitro, in orderr to perform genotype/phenotype correlations and also in order to further understand the biochemicall basis for the disease phenotypes observed in these patients. Chapter 8 describes thee generation of these variants in vitro, and details the biochemical assays that were used to determinee the defects in function caused by each of these missense mutations. In Chapter 9 wee describe a naturally occurring animal model for the deletion of ABCA1, the W H A M chicken. Thesee chickens have a mutation in their Z chromosome of the ABCA1 gene that causes the aminoo acid substitution E89K. The mutation was generated in vitro and biochemical analyses weree performed in order to determine the functional defect underlying the phenotype observed inn these chickens. Further biochemical characterizations of patient mutations are described in Chapterr 10, where the effect of truncation mutations on patients' phenotypes was determined too act through a dominant-negative mechanism.

Inn Chapter 11 the results of ali these studies are reviewed, and the insights gained into the role off ABCA1 are discussed. The implications that arise from this thesis for the development of therapeuticc compounds aimed at raising ABCA1 levels are also detailed.

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Chapter r

Humann ABCA1 BAC transgenic mice show increased

HDL-CC and ApoAl dependant efflux stimulated by an

internall promoter containing Liver X Receptor

Responsee elements in intron 1

Roshnii R. Singaraja

, Virginie Bocher

, Erick R. James

, Susanne M. Gee

,

Lin-Huaa Zhang

, Blair R. Leavitt

, Bing Tan

, Angela Brooks-Wilson

, Anita Kwok',

Nagatt Bissada

, Yu-zhou Yang

, Guoqing Liu

, Sherrie R. Tafuri

,

Catherinee Fievet

, Cheryl L. Wellington

, Bart Staels

and Michael R. Hayden\

::

Centre for Molecular Medicine & Therapeutics, Department of Medical Genetics and Children'ss and Women's Hospital, University of British Columbia, 980 West 28:" Avenue,

Vancouverr BC, V5Z 4H4, Canada. Institutt Pasteur de Lille and Faculté de Pharmacie, Université de Lille II - U545, 1 rue du Pr. Calmette-BP245,, 59019 Lille, Cedex, France. Xenonn Genetics, Inc., Suite 100 - 2386 East Mall, Vancouver BC, V5Z 1Z3.

::

Cardiovascular Molecular Science and Technologies, Pfizer Global Research and Development,, 2800 Plymouth Road, Ann Arbor, Ml, 48105,USA

Abstract t

Usingg BAC transgenic mice, we have shown that increased human ABCA1 protein expression resultss in a significant increase in cholesterol efflux in different tissues, and marked elevation in HDL-CC levels associated with increases in ApoAl and ApoAII. Three novel ABCA1 transcripts containingg three different transcription initiation sites that utilize seguences in intron 1 have beenn identified. In BAC transgenic mice there is an increased expression of ABCA1 protein, but thee distribution of the ABCA1 product in different cells remains similar to wild type mice. An internall promoter in human intron 1 containing LXREs is functional in vivo and directly contributess to regulation of the human ABCA1 gene in multiple tissues and to raised HDL-C, ApoAll and ApoAII levels. A highly significant relationship between raised protein levels, increased efflux,, and level of HDL elevation is evident. These data provide proof of principle that increased humann ABCA1 efflux activity is associated with an increase in HDL levels in vivo.