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Regulation of ATP-Binding Cassette Transporter Al in Cholesteryl Ester Storage Disease

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Regulation of ATP-Binding Cassette Transporter Al in Cholesteryl Ester Storage Disease Regulation of ATP-Binding Cassette Transporter Al In Cholesteryl Ester Storage Disease by NICOLAS JAMES BILBEY B.Sc (Honours), Thompson Rivers University, 2006 A THESIS SUBMiTTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Experimental Medicine) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) June 2009 © Nicolas James Bilbey, 2009 ABSTRACT Previous studies from the Francis laboratory have determined that regulation of ABCA1 expression is impaired in the lysosomal cholesterol storage disorder Niemann-Pick type C (NPC) disease, the presumed reason for the low plasma HDL-cholesterol (HDL-C) levels found in the majority of NPC disease patients. Cholesteryl ester storage disease (CESD) is another lysosomal cholesterol storage disorder, resulting from deficiency in lysosomal acid lipase (LAL). CESD patients develop premature atherosclerosis, possibly related to their known low plasma HDL-C levels. We hypothesized that in CESD the reduced activity of LAL also leads to impaired ABCA1 regulation and HDL formation due to the decrease in release of unesterified cholesterol from lysosomes. Our results show that human CESD fibroblasts exhibit a blunted increase in ABCA1 mRNA and protein in response to addition of low density lipoprotein (LDL) to the medium when compared to normal human fibroblasts. Efflux of LDL-derived cholesterol radiolabel and mass to apolipoprotein A-I-containing medium was markedly reduced in CESD fibroblasts compared to normal fibroblasts. Cellular radiolabeled cholesteryl ester derived from LDL and total cell cholesteryl ester mass was increased in CESD compared to normal cells. Delivery of an adenovirus expressing full length human lysosomal acid lipase (Ad-hLAL) results in correction of LAL activity and an increase ABCA1 protein expression, as well as correction of cholesterol and phospholipid release to apoA-I and normalization of cholesteryl ester levels in the CESD fibroblasts. These accumulated results suggest ABCA 1 expression is dependent on lysosomal acid lipase activity, and provide additional support for a major role of the lysosomal pool of unesterified cholesterol as a regulator of ABCA1 expression and HDL formation in humans. 11 TABLE OF.CONTENTS ABSTRACT ii TABLE OF CONTENTS iii LIST OF TABLES v LIST OF FIGURES vi LIST OF ABBREVIATIONS viii ACKNOWLEDGEMENTS x DEDICATION xi CHAPTER 1: INTRODUCTION 1 1.1 CARDIOVASCULAR DISEASE 2 1.1.1 Canadian and Global Burden of Cardiovascular Disease 2 1.2 ATHEROGENESIS 3 1.2.1 Artery Wall Architecture 3 1.2.2 Initiation and Progression ofAtherosclerosis 4 1.2.3 Advanced Plaque and Rupture 6 1.3 LIPOPROTEINS 6 1.3.1 Lipoprotein Classes and Physiology 6 1.3.2 Apolipoproteins 8 1.4 CHOLESTEROL TRANSPORT 11 1.4.1 Lipid Transport 11 1.4.2 Regulation ofEndogenous Cholesterol Synthesis 12 1.4.3 Forward Cholesterol Transport 13 1.4.4 Reverse Cholesterol Transport (RCT) 14 1.4.5 Lipidation ofApoA-I: ATP-Binding Cassette Cholesterol Transporters 15 1.5 ATP-BINDING CASSETTE TRANSPORTER Al (ABCA1) 16 1.5.1 General 16 1.5.2 Structure 17 1.5.3 ABCA1 Regulation 17 1.5.4 ABCA1 and Lipid Efflux 19 1.5.5 ABCA1 Cellular Distribution 19 1.5.5 ABCAJ-mediated Cholesterol Efflux: NPC and Tangier Disease 20 1.6 CHOLESTERYL ESTER STORAGE DISEASE (CESD) 25 1.6.1 Overview and Background 25 1.6.2 CESD Patient Tissue Lipid and Lipoprotein Levels 27 1.6.3 Abnormal Lipid Trafficking In CESD 27 1.6.4 CESD Genetic Background 29 111 1.6.5 Lysosomal Acid Lipase (LAL): Structure and Trafficking 30 1.6.6 CESD Mouse Model 32 1.7 HYPOTHESIS AND SPECIFIC AIMS 37 CHAPTER 2: MATERIALS AND METHODS 39 2.1 MATERIALS 40 2.2 METHODS 40 2.2.1 Preparation ofLipoproteins 40 2.2.2 Cell Culture 40 2.2.3 Labeling of Cellular Cholesterol Pools and Phospholipids 41 2.2.4 Adenoviral Delivery to Normal and CESD Fibroblasts 41 2.2.5 Lysosomal Acid Lipase Activity Assay 42 2.2.6 LDL Cholesterol Efflux 42 2.2.7 Phospholipid Efflux 43 2.2.8 Real Time-PCR Analysis ofABCAJ mRNA 44 2.2.9 Western Blot Analysis 45 2.2.10 Cholesterol Mass Assay 45 2.2.11 Statistical Analysis 46 CHAPTER 3: RESULTS 47 3.1 Reduced ABCA 1 mRNA Response to LDL Loading in CESD Fibroblasts 48 3.2 Reduced ABCA 1 Protein Levels in CESD Fibroblasts 51 3.3 Impaired ApoA-I-dependent Cholesterol Efflux in CESD Fibroblasts 53 3.4 Impaired Phospholipid Efflux to ApoA-I from CESD Fibroblasts 57 3.5 Reduced Cholesterol Mass Efflux to ApoA-I From CESD Fibroblasts 59 3.6 LXR Agonist Upregulates ABCA1 mRNA and Protein in CESD Fibroblasts 61 3.7 LXR Agonist Increased Phospholipid Efflux in CESD Fibroblasts 64 3.8 LXR Agonist Increased Cholesterol Efflux in CESD Fibroblasts 66 3.9 Adenovirus Delivery Optimization: ABCA1 and hLAL Expression 68 3.10 Increased ABCA1 and hLAL Protein Levels Following Delivery of Ad-hLAL 70 3.11 Increased ABCA1 Expression Following Delivery of Ad-hLAL 72 3.12 Increased LAL Activity Following Delivery of Ad-hLAL 74 3.13 Lipofectamine Affects Phospholipid Efflux in CESD Fibroblasts 76 3.14 Lipofectamine Causes Increased LDL Uptake by Cultured Fibroblasts 78 3.15 Effects of Reduced Lipofectamine Concentration on Phospholipid Efflux 80 3.16 Lipofectamine Necessary For Delivery of Ad-hLAL in Cultured Fibroblasts 82 3.17 Ad-hLAL Delivery With Lipofectamine and Varying Adenoviral MOI 84 3.18 Effect of Lipofectamine Concentration on LDL Uptake by Cultured Fibroblasts 86 3.19 Increased LAL Activity with Altered Ad-hLAL Delivery Protocol 88 3.20 ABCA1 Expression Following Delivery of Ad-hLAL 90 3.21 Increased Cholesterol Efflux with Ad-hLAL Delivery in CESD Fibroblasts 93 3.22 Increased Phospholipid Efflux with Delivery of Ad-hLAL in CESD Fibroblasts 96 CHAPTER 4: DISCUSSION 99 REFERENCES 107 iv LIST OF TABLES Table 1.1 Physical characteristics of the primary lipoprotein classes 7 Table 1.2 Physical characteristic and function of the primary apolipoprotein classes 9 Table 1.3 Plasma lipid levels in wild-type, mutant, and Ad-hLAL infected mutant mice 35 Table 3.1 Increased uptake of LDL in the presence of Lipofectamine 79 Table 3.2 Absence of affect on LDL uptake using lower Lipofectamine concentration 87 V LIST OF FIGURES Figure 1.1 Normal arterial wall layers 3 Figure 1.2 Formation of an advanced plaque 5 Figure 1.3 Pathways for transporting and synthesizing cholesterol 12 Figure 1.4 Reverse cholesterol transport (RCT) 15 Figure 1.5 Structure of ABCA1 protein 17 Figure 1.6 Reduced ABCA1 mRNA and protein expression in human NPCF’ fibroblasts 21 Figure 1.7 Upregulation of ABCA1 reduces sterol accumulation in NPC’ fibroblasts 22 Figure 1.8 Impaired lipidation of apoA-I in TD fibroblasts 23 Figure 1.9 Impaired ABCA1 function in TD fibroblasts 24 Figure 1.10 CESD human fibroblasts exhibit reduced LAL activity 26 Figure 1.11 Reduced hLAL protein expression in CESD fibroblasts 26 Figure 1.12 Increased LDL uptake in CESD fibroblasts compared to normal fibroblasts 28 Figure 1.13 Normal and CESD cell metabolism 29 Figure 1.14 Reduced enzyme activity and enlarged liver/spleen in LAL KO mouse model 33 Figure 1.15 Decreased liver size and increased LAL activity in Ad-hLAL-injected lal mice.. 34 Figure 1.16 Serum lipoproteins in lar and lal+k mice 35 Figure 3.1 Reduced ABCA1 mRNA response to LDL-loading in CESD fibroblasts 50 Figure 3.2 Inhibited upregulation of ABCA1 protein in CESD fibroblasts 52 Figure 3.3 Impaired cholesterol efflux to apoA-I in CESD fibroblasts 55 Figure 3.4 Reduced phospholipid efflux to apoA-I in CESD fibroblasts 58 Figure 3.5 Reduced release of UC to apoA-I and CE accumulation in CESD fibroblasts 60 Figure 3.6 LXR agonist upregulates ABCA1 mRNA and protein in CESD fibroblasts 62 Figure 3.7 Increased phospholipid efflux with LXR agonist in normal and CESD fibroblasts .. 65 Figure 3.8 Increased cholesterol efflux with LXR agonist in normal and CESD fibroblasts 67 vi Figure 3.9 Increased ABCA1 and hLAL expression with delivery of Ad-hLAL 69 Figure 3.10 Increased hLAL protein levels with delivery of Ad-hLAL 71 Figure 3.11 Increased ABCA1 protein expression with delivery of Ad-hLAL 73 Figure 3.12 Increased LAL activity with delivery of Ad-hLAL 75 Figure 3.13 Lipofectamine affects phospholipid efflux in CESD fibroblasts 77 Figure 3.14 Optimization of Lipofectamine conditions for phospholipid efflux 81 Figure 3.15 Minimal increase in hLAL protein level in absence of Lipofectamine 83 Figure 3.16 Increased hLAL protein levels with delivery of Ad-hLAL: modified conditions.... 85 Figure 3.17 Increased LAL activity with delivery of Ad-hLAL: modified conditions 89 Figure 3.18 No change in AECA 1 mRNA but increased protein in response to Ad-hLAL 92 Figure 3.19 Increased cholesterol efflux with delivery of Ad-hLAL in CESD fibroblasts 95 Figure 3.20 Increased phospholipid efflux with delivery of Ad-hLAL in CESD fibroblasts 98 vii LIST OF ABBREVIATIONS ABCA1 ATP-binding cassette transporter Al ABCG1 ATP-binding cassette transporter Gl ACAT acyl-C0A:cholesterol acyltransferase Ad-hLAL adenovirus expressing full-length human lysosomal acid lipase Ad-GFP adenovirus expressing full-length green fluorescent protein ApoA-I apolipoprotein A-I ApoA-II apolipoprotein A-Il ApoB apolipoprotein B ApoC apolipoprotein C ApoE apolipoprotein E CAD coronary artery disease CAR coxsackie-adenovirus receptor CD1 CESD human fibroblast cell line 1 CD2 CESD human fibroblast cell line 2 cDNA complemeitary deoxyribonucleic acid CE cholesteryl esters CESD cholesteryl ester storage disease CETP cholesteryl ester transfer protein CM chylomicrons CVD cardiovascular disease DMEM dulbecco’s modified eagle’s
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