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Regulation of ATP-binding cassette transporter A1 in cholesteryl ester storage disease Bilbey, Nicolas James 2009

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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 medium ECM extracellular matrix ERC endocytic recycling compartment FAFA fatty acid free albumin FBS fetal bovine serum hr hours HDL high density lipoproteins HDL-C high density lipoprotein cholesterol HMGR 3-hydroxy-3-methyl-glutaryl-CoA reductase ICAM-1 intercellular adhesion molecule- 1 IDL intermediate density lipoproteins viii LAL lysosomal acid lipase LAMPs lysosome-associated membrane proteins LCAT lecithin:cholesterol acyltransferase LDL low density lipoproteins LDL-C low density lipoprotein cholesterol LPDS lipoprotein deficient serum LPL lipoprotein lipase LRP LDL receptor-related protein LXR liver X receptor MCP-1 monocyte chemoattractant protein-i mm minutes MPR mannose-6-phosphate receptors mRNA messenger ribonucleic acid nCEH neutral cholesteryl ester hydrolase NL1 normal human fibroblast cell line 1 NL2 normal human fibroblast cell line 2 NPC Niemann-Pick Type C disease oxLDL oxidized LDL PBS phosphate-buffered saline PDI protein disulfide isomerase PM plasma membrane RCT reverse cholesterol transport RT-PCR reverse transcription polymerase chain reaction SCAP SREBP cleavage activating protein SMC smooth muscle cells SREBP sterol regulatory element binding protein SR-BI scavenger receptor class B, type I TD Tangier disease TLC thin layer chromatography UC unesterified cholesterol VCAM-1 vascular adhesion molecule-i VLDL very low density lipoprotein ix ACKNOWLEDGEMENTS I would like to thank my supervisor Dr. Gordon Francis for accepting me as a graduate student and for his guidance and support over the course of program. I would like to thank my supervisory committee members Dr. John Hill and Dr. Cheryl Wellington for their support throughout my graduate studies. I would also like to thank my external examiner, Dr. Susanne Clee for her involvement in the final stages of my program. Thank you to Dr. Hong Du, Cincinnati Children’s Hospital, for providing the adenovirus expressing full-length human lysosomal acid lipase. Thanks also to Dr. Rene Jacobs, University of Alberta, for providing the adenovirus expressing GFP. I would also like to give thanks to Teddy Chan for exceptional technical support and for providing me with the training and skills necessary to be successful in the lab. A special thanks to Leanne Bilawchuk for providing outstanding technical support and for completing the quantitative RT-PCR experiments. I would also like to thank Dr. Emmanuel Boadu, Dr. Hong Choi and Kristin Bowden for their helpful advice and insights. Special thanks to Priscilla Gao for cholesterol mass determinations. Thank you to the MCBL group, and to the members of the MCBL group for your support during the initial stages of my program. Thanks also to the James Hogg iCAPTURE Centre technicians and students for their support, guidance and direction. A special thanks to the funding agencies that supported the research for this project: CIHR Operating Grant MOP-79532 and a Graduate Studentship of the Strategic Training Program in Stroke, Cardiovascular, Obesity, Lipids and Atherosclerosis Research (SCOLAR). x DEDICATION I dedicate this to my parents, James and Marie-Christine, who provided unwaivering love and support over the course of my studies. Without your encouragement and guidance this would not have been possible. xi TtJOLL3f1UOIIMI:j 1.1 CARDIOVASCULAR DISEASE 1.1.1 Canadian and Global Burden of Cardiovascular Disease Cardiovascular diseases (CVD) related to atherosclerosis, specifically heart attacks and strokes, are the leading causes of death worldwide. In 2005, Statistics Canada reported that CVD was responsible for the deaths of 71,338 Canadians, including 30% of all male and 31% of all female deaths’. On a global scale, cardiovascular deaths are expected to increase from 16.7 million in 2002 to 23.3 million in 2030, outweighing estimated cancer-related deaths, which are expected to increase from 7.1 million in 2002 to 11.5 million in 20302. Moreover, coronary artery disease (CAD), which is the leading cause of cardiovascular mortality worldwide, is now also the leading cause of death in developing countries36.The costs associated with morbidity from CVD are assuming an ever-increasing portion of total health care budgets worldwide. In addition to population-based strategies to reduce CVD, including smoking cessation programs, additional strategies are needed to reduce the morbidity of CVD and to prevent premature CVD death. In the past three decades a major focus in CVD prevention has been in lowering levels of low-density lipoprotein cholesterol (LDL-C). However, it has been consistently demonstrated that lowering LDL-C reduces CAD events by no more than 30 to 40%. In recent years, the focus regarding cholesterol and heart disease has shifted toward strategies for increasing high density lipoprotein (HDL) levels as a potential therapy to prevent and treat CVD10’ Multiple studies from various parts of the world have shown that low levels of HDL cholesterol (HDL-C) are a major independent risk factor for premature atherosclerosis, and that HDL appears to have a preventive role in the onset of this disease. For every 1 mg/dL increase in plasma HDL-C, the incidence of coronary events declines by 2 to 3%11 12 Plasma HDL-C has been found to be the strongest predictor of CVD, even greater than LDL-C, smoking, and high blood pressure’3.Despite this strong inverse correlation between level of HDL-C and risk of CVD, currently there are no very effective therapies to raise HDL. The best agent currently available to raise HDL, vitamin B3 or niacin (nicotinic acid), is limited in its usefulness due to its tendency to cause skin flushing in the majority of people who use it, in addition to its ability to raise HDL-C on average only 20 to 30%. There is a clear need to develop alternative therapies that increase HDL formation and blood HDL-C levels in order to increase our ability to prevent and treat CVD. 2 1.2 ATHEROGENESIS Atherosclerosis is a disease of the arterial wall that is initiated by endothelial injury and the excessive retention of lipids, their oxidation and modification within the intima, and the eventual induction of chronic inflammation and thrombosis. The result is a progressive narrowing of the lumen by an atherosclerotic plaque that leads to constrictive remodeling of arteries4. 1.2.1 Artery Wall Architecture The normal artery wall is composed of a single layer of endothelial cells on the luminal side and three additional distinct concentric layers: the innermost is the intima, the middle layer is called the media and the outermost layer is the adventitia (Figure 1.l)’. Under normal conditions, the intima is an acellular layer composed of connective tissue consisting of collagen, laminin and heparin sulphate proteoglycans, and underlain by the internal elastic lamina’5’16 Beneath the intima is the medial layer (“media”), composed primarily of vascular smooth muscle cells (SMCs) and a matrix containing type I fibrillar collagen, glycoprotein fibronectin and chondroitin sulphate proteoglycans’5.The SMCs allow the artery to contract and expand, while adjusting the lumenal diameter. The adventitia, the outermost layer, contains connective tissue, collagen and elastic fibers, which allows the arteries to accommodate changing pressures on the walls induced by varying blood flow. The adventitia also contains nerves, lymphatics, and blood vessels (vasa vasorum) that nourish the cells of the arterial wall. Separating the medial and adventititial layers, and functioning as a boundary, is the external elastic lamina, a layer of interwoven elastin fibrils. Figure 1.1 Normal arterial wall layers. The (A) endothelial cells, in contact with blood, overlay the (B) intima. The (C) media is composed of smooth muscle cells that allow the artery to contract and expand. The outer layer, or (D) adventitia is composed of connective tissue (Adapted by permission from Macmillan Publishers Ltd: [Nature] (14), copyright © [19931. - 3 1.2.2 Initiation and Progression ofAtherosclerosis Atherosclerosis is characterized by the thickening, hardening, and the eventual loss of elasticity of blood vessels. Although not clear, the process leading to the development of the atherosclerotic lesion is believed to be initiated as a response to endothelial injury’719.Factors that may result in endothelial damage include mechanical factors (hypertension and reduced shear stress in the artery), elevated plasma cholesterol levels, oxidative stress, infectious agents, diabetes and smoking’6’17, 20 Injury to the endothelium leads to increased expression of cell surface adhesion molecules such as selectins, vascular cell adhesion molecule-i (VCAM- 1) and intercellular adhesion molecule-i (ICAM- 1 )2O 21 As a result, there is increased adherence of cells such as monocytes, macrophages and T-cells. These cells migrate to the subendothelium where monocytes differentiate into macrophages, which ingest aggregated or oxidized low density lipoproteins (0xLDL) and eventually develop into foam cells and contribute to formation of fatty streaks22. The initial migration of leukocytes is aided by several chemoattractant molecules, such as monocyte chemoattractant protein-i (MCP-1). In addition, the recruited leukocytes and other cell types release a variety of cytokines, vasoactive agents, and growth factors, which initiate and propogate an inflammatory response16.Activated T-cells, together with vascular wall cells, secrete cytokines such as tumor necrosis factor-f3 and interferon-y, fibrogenic mediators and growth factors that can promote the migration and proliferation of SMCs and the construction of a dense extracellular matrix (ECM). The macrophage-foam cells, T-cells and SMCs also release proinflammatory cytokines, including interleukin-1 (IL-i), tumor necrosis factor (TNF) and C-reactive protein (CRP), which induce expression of cellular adhesion molecules, mediating increased leukocyte adhesion to the endothelium21.The activation of an inflammatory response results in the development of the atherosclerotic lesion, which is characterized by a migration and proliferation of vascular smooth muscle cells into the intimal space from the medial layer, concurrent with a loss of contractility, as well as collagen accumulation. The macrophages recruited to the arterial wall express scavenger receptors for modified lipoproteins, such as SR-A and CD36, under the influence of macrophage-colony stimulating factor (M-CSF), which allows them to take up oxLDL23. The non-LDL-receptor mediated mechanism of endocytosis is not downregulated with increasing cell cholesterol content, allowing unregulated sterol accumulation and foam cell formation 21 These macrophage foam cells are also capable of secreting potent growth factors, such as platelet-derived growth factor (PDGF), which function as mitogens for vascular smooth muscle cells and encourage a proliferative response in the arterial wall. 4 An early event in the development of atherosclerosis is the retention and modification of apolipoprotein B containing low density lipoproteins (LDLs) in the arterial intima ECM. LDL particles diffuse in from the plasma via gaps between endothelial cells or via transcytosis and are subsequently retained by proteoglycans in the intimal space. Proteoglycans in the ECM, such as chondroitin sulfate proteoglycans, bind lipoproteins via ionic interactions between the negatively charged sulfate and carboxylic groups on the glycosaminoglycan chains and positively charged lysine and arginine residues on the apolipoproteins24.The retention of LDL results in oxidative modification of the particle by mechanisms believed to involve vascular cells such as endothelial cells, SMCs and macrophages. Although the exact in vivo mechanisms involving oxidation of LDL within the artery wall are unclear, several potential methods have been investigated. Lipoxygenases (15-lipoxygenase) expressed by fibroblasts, NAD(P)H oxidases produced by vascular cells, xanthine oxidases produced by endothelial cells, nitric oxide released by endothelial cells, and myeloperoxidase produced by macrophages are potential mechanisms believed to be involved in LDL modification within the artery wall25’6 As the lesion develops, macrophage foam cells, inflammatory cells, and cells of the intima accumulate and further propagate the inflammatory response. The continual accumulation of extracellular lipids leads to the formation of a necrotic cellular core, leading to a progressive destruction of the plaque architecture. Fibrous tissue also begins to form a cap over the lipid-rich necrotic cores at the blood interface (Figure 1.2). Ultimately, clinical complications arise when a plaque ruptures at its’ weaker shoulder regions and a thrombus forms that can potentially occlude the entire artery, leading to myocardial infarction. Figure 1.2 Formation of an advanced plaque.Note the fibrous cap forming over the plaque. The fibrous cap covers a mix of leukocytes, lipid, and debris or necrotic core. The figure was obtained with permission from Ross, R. (19). Copyright © [1999] Massachusetts Medical Society. All rights reserved. Macrophage accumulation Formation of Fibrous-cap formation necrotic core 5 1.2.3 Advanced Plaque and Rupture In the later stages of plaque development, the fibrous cap may undergo erosion or uneven thinning, often at the shoulders of the lesion where macrophages enter, accumulate, and apoptosis may occur. Weakening at the shoulder region of plaques may result from metalloproteinases such as collagenases, elastases, and stromelysins27. Activated T-cells stimulate metalloproteinases by macrophages in the lesion. These changes may also be accompanied by the production of tissue-factor procoagulant and other hemostatic factors, further increasing the risk for thrombosis. Upon rupture, clot or thrombus formation occurs, obstructing the flow of blood by extending into the arterial lumen28. Lesion rupture and thrombosis can be followed by healing and replacement of fibrous tissue, but the lesions are susceptible to repeated rupture and thrombus formation28. 1.3 LIPOPROTEINS 1.3.1 Lipoprotein Classes and Physiology Neutral lipids, in particular triglycerides and cholesteryl esters, are insoluble in water and must be transported in plasma by amphipathic molecules29.Lipoproteins are particles that are capable of transporting various lipids and proteins in aqueous solutions from the tissues where they are synthesized (primarily the liver and small intestine) and/or stored (i.e. the artery wall) to tissues that require or clear them. Hydrophobic neutral lipids form the core of the lipoproteins, while amphipathic lipids, including phospholipids and unesterified cholesterol, form the surface of the particles. Among the lipids and lipophilic molecules delivered by lipoprotein particles are triglycerides, phospholipids, unesterified and esterified cholesterol, fat-soluble vitamins (including beta-carotene, which gives lipoproteins their yellow colour), and antioxidants. Lipoproteins are categorized into distinct subclasses based on their density and their lipid and apolipoprotein composition29.The major classes of lipoproteins, based on their respective physical-chemical characteristics, are chylomicrons (CM), very-low-density lipoproteins (VLDLs), intermediate-density lipoproteins (IDLs), low-density lipoproteins (LDLs) and high density lipoproteins (HDLs)30Table 1.1). CMs are synthesized in the small intestine and are responsible for the transport of dietary lipids to tissues. VLDLs are synthesized in the liver and are required for transport of endogenous lipids, while DL and LDL are produced from the progressive hydrolysis of VLDL in the circulatory system. LDL, which is formed from the hydrolysis of DL, is required for delivery of cholesterol to peripheral tissues and to the liver. HDL particles are initially synthesized by the 6 liver and small intestine, and are required for the removal of excess cholesterol from cells and transport to the liver for recycling or excretion29’3132 Table 1.1 Physical characteristics of the primary lipoprotein classes. Adapted from Endocrinology and Metabolism Clinics of North America, Vol 27, Ginsberg, H.N., from Lipoprotein Physiology, Pages 503-5 19, Copyright © (1998), with permission from Elsevier (29). Lipoprotein Density (g/dL) Lipid Content (%) Protein Content (%) TG Chol PL Chylomicrons 0.95 80 . 95 2 - 7 3 - 9 1 — 10 VLDL 0.95-1.006 55-80 5-15 10-20 1—10 IDL 1.006-1.019 20-50 20-40 15-25 10—20 LDL 1.019 - 1.063 5 - 15 40 - 50 20 - 25 20 — 50 HDL 1.063-1.21 5-10 15-25 20-30 20—50 CM and VLDL, which have a diameter ranging from 75 to l200nm and 30 to 8Onm and densities of < 0.94gIml and 0.94 to 1 .OO6gIml, respectively, undergo continual hydrolysis by enzymes such as lipoprotein lipase (LPL), bound mainly to vascular endothelium overlying skeletal muscle, adipose tissue, and the heart, and hepatic lipase (HL) at the hepatocyte surface33. Interactions with these enzymes results in alterations of their density, size and composition. Also capable of exchanging soluble apolipoproteins, CM and VLDL are constantly undergoing structural modifications. The hydrolysis of VLDL by LPL produces DL, which have a density of 1.006 to l.019g/ml and a diameter of 25 to 35nm29.Following one of two potential fates, the DL particle may be taken up by the liver via interaction between apolipoprotein E (apoE) and the LDL receptor or, alternatively, may undergo additional hydrolysis by ilL, forming LDL particles. Initially identified using separation techniques such as ultracentrifugation and electrophoresis, LDL particles have been found to range in density from 1.019 to 1 .063g/ml and in diameter ranging from 18 to 25nm9’31. Apolipoprotein A-I (apoA-I), the primary apolipoprotein of HDL, may form 3 types of structures with lipids: small lipid-poor complexes, flattened discoidal particles containing primarily polar lipids (phospholipids and cholesterol), and spherical particles containing both polar and nonpolar lipids34.The lipid-poor apoA-I, or pref3i-HDL, contributes to approximately 2 to 5% of plasma HDL, with a molecular weight of 60 to 7OkDa and a diameter of 5 to 6nm35. Importantly, the lipid poor pre3i-HDL particle has a greater ability to absorb cholesterol from peripheral cells compared to its more lipid-rich brethren (pre32-HDL particles and a-HDL). Pre32-HDL is present at approximately 2 to 3% in plasma and, compared to pref3i particles, it is rich in phosphatidylcholine and contains a smaller proportion of sphingomyelin and 7 cholesterol36’7.Although the prel3-migrating HDL subclass tends to be present in lower concentrations in plasma, compared to the abundant (i-migrating subclass, it is critical as it represents the nascent form of the HDL particle and is necessary for the effective removal of excess cholesterol from the peripheral tissues. Finally, the a-HDL is the predominant species of HDL particles in plasma. Based on ultracentrifugation and gradient gel electrophoresis, a-HDL particles may be divided into three subclasses, HDL3 (density 1.12 to 1.219 gIml), HDL2 (density 1.063 to 1.12 g/ml) and HDL1 (density < 1.063 gIml), with diameters ranging from 5 to 12nm38’ Importantly, the lipid content and proteins associated with HDL are highly variable and are subject to modifications depending on interactions with tissues and other lipoproteins in the circulatory system, as well as changes in metabolic conditions40. 1.3.2 Apolipoproteins The apolipoproteins associated with the various lipoproteins may be classified into two basic groups, those that are non-exchangeable and those that are exchangeable, or soluble (capable of being free in plasma or associated with other lipoproteins). The non-exchangeable apolipoproteins are apolipoprotein B 100 (apoB 100) and apolipoprotein B48 (apoB48), the primary apoliproteins of LDL, VLDL and CM30. Physiologically they are large, water-insoluble proteins that are synthesized in the endoplasmic reticulum of liver or intestinal cells. They remain bound to the same lipoprotein particle as they circulate in the plasma undergoing metabolic transformations until they are cleared via specific receptors. Conversely, the exchangeable apolipoproteins, which include apoA-I, apoA-II, apoCs, and apoE, have smaller molecular masses and are relatively soluble in their lipid-poor states. They are able to transfer between lipoprotein particles while in the circulatory system (Table 1.2)41. 8 Table 1.2 Physical characteristic and function of the primary apolipoprotein classes Adapted from Endocrinology and Metabolism Clinics of North America, Vol 27, Ginsberg, H.N., from Lipoprotein Physiology, Pages 503-5 19, Copyright © (1998), with permission from Elsevier (29). MolecularApolipoprotein Lipoprotein FunctionWeight ApoA-l 28,016 HDL, CM • Structural component of HDL • LCAT activator ApoA-ll 8,707 HDL, CM • Unknown ApoB48 264,000 CM Assembly + secretion of CMfrom the small intestine • Assembly + secretion of VLDL from the liverApoBlOO 540,000 VLDL, IDL, LDL • Structural protein of VLDL, IDL, LDL • Ligand for LDL receptor ApoC-l 6,630 CM, VLDL, IDL, HDL • Inhibit hepatic uptake of CM and VLDL remnants ApoC-Il 8,900 CM, VLDL, IDL, HDL • Activator of LPL ApoC-Ill 8,800 CM, VLDL, IDL, HDL • Inhibitor of LPL • Ligand for binding of lipoproteins to ApoE 34,145 CM, VLDL, IDL, HDL the LDL receptor and LDL receptor- related receptor Apolipoproteins are bound to the surface of lipoproteins and, in addition to the activator and receptor functions noted below and in Table 1.2, serve to aid in the solubilization of neutral lipids in the circulation. They are able to bind to phospholipid/water interfaces, and can form particles with phospholipids, providing structural stability. They also fulfill a variety of functional roles with regards to regulating lipoprotein metabolism. 1.3.2.1 ApoBlOO ApoB 100 is the main structural protein of VLDL, DL, LDL and Lipoprotein (a). Synthesized in the liver, it is a large hydrophobic protein (4563-aa) with a molecular weight of 54OkDa. The hydrophobic lipid-binding regions of apoB 100 interact with the hydrophobic lipids in the lipoprotein core, while the hydrophilic sequences interact with the polar head groups of the surface phospholipids and the aqueous environment of the plasma. ApoB 100 contains an LDL receptor binding domain that mediates uptake of plasma LDL, VLDL and DL. Unlike other apolipoproteins ApoB 100 does not exchange between lipoproteins, and is required for the initial assembly and secretion of VLDL by the liver30’33• 1.3.2.2 ApoB48 Synthesized in the small intestine in humans, ApoB48 is transcribed from the gene for apoB 100, however the mRNA is edited so that a codon for glycine becomes a stop codon. 9 Therefore, the translation of apoB 100 mRNA stops prematurely at this codon in the human intestine producing the truncated apoB48, which is the N-terminal 48% portion of apoB 100. Found on CMs, apoB48 is required for intestinal assembly and secretion of these lipoproteins and does not have a binding site for the LDL receptor29’3033• 1.3.2.3 ApoC’s There are three types of apoC proteins (apoC-I, apoC-Il and apoC-Ill) synthesized in the liver, each with differing metabolic roles. ApoC-I is a minor component of CMs, VLDL, DL and I{DL, whose role includes that of binding of chylomicrons and VLDL to the LDL receptor and to the LDL receptor-related protein (LRP). ApoC-fi is found on CMs, VLDL, DL, and HDL and is a critical activator of LPL. ApoC-Ill is the major component of VLDL, but is also present on CMs, DL and HDL. Its function appears to be to inhibit both LPL and HL action, as well as hepatic uptake of CM and VLDL remnants29’3033• 1.3.2.4 ApoE ApoE is synthesized in the liver and by macrophages, and is found on all lipoproteins, except LDL. It has a critical role in the removal of remnant lipoproteins from plasma by interacting with several receptors in the liver, including the LDL receptor and LRP. ApoE also plays an integral role in sterol transport in the central nervous system. Synthesized by astrocytes and microglia, it is the primary apolipoprotein on HDL-like particles in the brain42. 1.3.2.5 ApoA-I ApoA-I is the primary structural protein of HDL and is synthesized in both the liver and small intestines. It functions as an activator of lecithin-cholesterol acyltransferase (LCAT), which esterifies free cholesterol on the HDL particle, and promotes cellular lipid efflux via interaction with ATP Binding Cassette Transporter Al (ABCA1). ApoA-I is usually present in one to four copies in each HDL particle in normal plasma. Preproapolipoprotein A-I, which in humans originates in the liver and in the small intestine, is initially secreted as a 249-aa proprotein that is cleaved by a plasma protease producing a mature 243 aa polypeptide34’ It consists of 22-aa repeating segments, which are usually spaced with helix-breaking proline residues34’46 The repeats form amphipathic alpha-helices, with charged residues facing the medium and nonpolar residues facing the acyl chains of the phospholipid bilayer in discoidal HDL or phospholipid monolayer on spherical HDL37.ApoA-I may also be stabilized by charge charge interactions between adjacent repeats. 10 1.4 CHOLESTEROL TRANSPORT Cholesterol fulfills multiple roles in mammalian cells. It is an essential component of eukaryotic membranes, making up a large percentage of the plasma membrane (PM) and organelle membranes, and also functions to reduce the fluidity of membranes by counteracting the action of unsaturated fatty acids in phospholipids, which act to increase it47. In addition, it is a precursor for oxysterol synthesis, bile acids and steroid hormones and has an effect on signal transduction and trafficking of membrane proteins and lipids. Importantly, excess cholesterol in cells in the intimal layer of arteries is a key biochemical feature of atherosclerosis48 1.4.1 Lipid Transport The cholesterol utilized by cells may be obtained from two primary sources, diet and de novo cholesterol synthesis from acetyl-coenzyme A (acetyl-C0A) in the endoplasmic reticulum49. Exogenous cholesterol from diet is absorbed by intestinal enterocytes and packaged into CMs29’ 50, 51 As the CMs, also laden with triglycerides produced from dietary long-chain fatty acids, navigate through the circulatory system they encounter LPL bound to the endothelium. LPL converts lipoprotein triglyceride into free fatty acids and mono- and diglycerides, allowing uptake of fatty acids by peripheral tissues52.LPL is expressed at high levels in adipose tissue and skeletal muscle, as well as the heart and lactating breast tissue, and is transported to the surface of capillary endothelial cells overlying these tissues, where, bound to heparan sulfate proteoglycans, it interacts with triglyceride-rich lipoproteins52.The resulting CM remnants are cleared by the liver via receptors, such as LDL receptors and LRP50’1. Intrahepatic cholesterol derived from dietary sources or from de novo synthesis is then repackaged and secreted with triglycerides as VLDLs29.As the VLDLs circulate in the peripheral tissue, the majority of their triglycerides are hydrolyzed by LPL, allowing the remnant particle to be endocytosed by the liver or to remain in the circulatory system as IDLs. The IDL particle still contains triglycerides, as well as the majority of the original cholesteryl esters and unesterified cholesterol, in addition to apoB 100 and apoE. Additional interactions with LPL in peripheral tissues, as well as HL on the hepatocyte surface, leads to the production of LDL particles, which carries approximately two-thirds of the cholesterol in human plasma, and which may subsequently be endocytosed via the LDL receptor51(Figure 1.3). Although LDL may be internalized by peripheral cells via the LDL receptor, the hepatic LDL receptor is necessary for clearing the majority of the LDL cholesterol from circulation. Sterols may then be recycled into new lipoprotein particles or degraded into bile acids. 11 Figure 1.3 Pathways for transporting and synthesizing cholesterol. Chylomicrons (CM) may be hydrolyzed by lipoprotein lipase (LPL) to produce remnant CM. Cholesterol and triglycerides may then be repackaged secreted as very low density lipoproteins (VLDLs) which undergo hydrolysis by lipoprotein lipase (LPL) to form low density lipoproteins (LDLs). The LDL particles may then be taken up in the peripheral tissues by receptor-mediated endocytosis. 1.4.2 Regulation ofEndogenous Cholesterol Synthesis The endoplasmic reticulum (ER) is an important regulatory compartment in cholesterol homeostasis, and is the site of cholesterol synthesis and esterification5356.The synthesis of cholesterol at the ER is a tightly controlled mechanism involving sterol regulatory element binding proteins (SREBPs). There are 3 known SREBP proteins, SREBP-la, SREBP-lc and SREBP-2. It has been found that SREBP-2 is primarily involved in the activation of genes involved in cholesterol synthesis, while SREBP-la and SREBP-lc are involved with genes that regulate fatty acid synthesis57. These proteins are initially synthesized as inactive ER transmembrane proteins which, when cholesterol is available, remain in the ER associated with the escort protein SCAP (SREBP cleavage activating protein) and the ER retention protein Insig58.When cholesterol levels begin to decrease, a conformational change occurs in the sterol sensing domain of SCAP, leading to the dissociation of Insig and allowing the SREBP-SCAP complex to reach the Golgi59. In the Golgi, two proteases release the active form of SREBP, which subsequently translocates to the nucleus to activate transcription of its target genes, including HMG-CoA reductase (HMGR) and the LDL receptor. Cholesterol synthesis may also Remnant Chylomicron LPL _________ CE , TG Chylomicron Dietary Cholesterol Small Intestine — CE 1.• ? TG •___I I VLDL Cholesterol LPL{ / LDL Receptor LDL Peripheral Tissue 12 be regulated posttranscriptionally. High levels of cholesterol increase degradation of HMGR, the rate-limiting enzyme in cholesterol synthesis, by promoting association of its sterol-sensing domain with Insig60. Under conditions of high cellular cholesterol, there is inhibition of endogenous cholesterol synthesis but also activation of acyl-coenzyme A:cholesterol acyltransferase (ACAT), an ER- localized enzyme, by the presence of excessive unesterified cholesterol6’.The cholesteryl esters that are formed are stored with triglycerides in the cores of the cytosolic lipid droplets. The reverse pathway involves neutral cholesteryl ester hydrolase (nCEH), which hydrolyzes cholesteryl esters in the lipid droplets61.The enzymes required for nCEH activity vary among cell types and are distinct from acid lipase in the endocytic pathway. For example, in steroidogenic cells, the major nCEH is hormone-sensitive lipase, which also hydrolyzes triglycerides62.Following release of unesterified cholesterol and fatty acids from the lipid droplet, the cholesterol may be transported to the ER and/or other compartments or re-esterified by ACAT. 1.4.3 Forward Cholesterol Transport The LDL particles, which bind to cell surface LDL receptors, are internalized via endocytosis into clathrin-coated vesicles that subsequently fuse with early sorting endosomes63.Many membrane proteins and lipids are then returned to the cell surface, mainly via a second element of the early endosomal system, the endocytic recycling compartment (ERC). The early endosomes subsequently acquire the properties of a late endosome, including a reduced pH, a change in the types of Rab protein associated with the membrane, and the acquisition of various acid hydrolases53.The lowering of the pH in the early endosomes promotes dissociation of LDL from the LDL receptors, allowing the recycling of the receptors and other proteins53. The recycled components bud off in vesicles destined for the ERC. Vesicles from the ERC return LDL receptors and other proteins and lipids to the PM, while non-recycled contents within the early endosomes continue to late endosomes. The majority of the acid hydrolases are delivered to late endosomes from the trans-Golgi network (TGN) by binding to mannose-6-phosophate receptors (MPR), which move between the TGN and the late endosomes. The presence of MPR is considered to be one of the hallmarks of late endosomes. Indigestible material and some membrane proteins, particularly heavily glycosylated lysosome-associated membrane proteins (LAMPs), are delivered from late endosomes to lysosomes, which are acid hydrolase-rich organelles that lack MPR and whose function is to digest endocytosed extracellular material53. 13 An essential hydrolase localized to the lysosomal compartment via the MPR system is lysosomal acid lipase (LAL), an enzyme required for hydrolysis of triglycerides and LDL-derived cholesteryl esters64. Following hydrolysis, LDL-derived unesterified cholesterol leaves late endosomes and is subsequently transported to other compartments, such as the ER and the PM53. LAL will be described in greater detail in subsequent sections. 1.4.4 Reverse Cholesterol Transport (RCT) Reverse cholesterol transport (RCT) involves the delivery of cholesterol from peripheral tissues to the liver34. HDLs function as a shuttle for excess unesterified cholesterol and cholesteryl esters from the peripheral tissues to the liver for excretion in bile, and carry approximately one-third of the cholesterol in human plasma. The primary protein of the HDL particle is apoA-I, synthesized by the liver and intestine, and secreted as a non-lipidated or lipid- poor particle into the circulatory system. The nascent apoA-I particle acquires phospholipids and cholesterol via cellular lipid efflux processes and by transfers from other lipoproteins, such as CMs and VLDL. Nascent HDL particles are initially formed as discoidal particles with a bilayer of phospholipids and apoA-I wrapped around the sides of the disc as a band65. Unesterified cholesterol intersects the region between the phospholipid head groups and their nonpolar fatty acid tails. Within the plasma, lecithin cholesterol acyltransferase (LCAT), made in the liver and associated with the HDL particle surface, esterifies HDL surface cholesterol to nonpolar cholesteryl esters. The cholesteryl esters migrate to the core of the particle, producing an increasingly spherical HDL with a surface monolayer of phospholipids. The mature HDL particles undergo remodeling by cholesteryl ester transfer protein (CETP), also synthesized in the liver, and which mediates the exchange of triglycerides and cholesteryl esters in VLDLs, CMs, remnants and LDLs for cholesteryl esters and phospholipids in HDL. Following interactions with CETP, HDL triglyceride can be catabolized by hepatic lipase located on the surface of hepatocytes34’51 Through interactions with scavenger receptor Bi (SR-B 1) the HDL particles may deliver their cholesteryl esters into cells, such as in the liver and steroidogenic tissue (Figure 1.4), with HDL- and SR-BI-derived cholesterol being preferentially shunted into bile. These HDL-remodeling processes regenerate lipid-poor apoA-I that recycles through the RCT pathway51. 14 Figure 1.4 Reverse cholesterol transport (RCT). The pathway involves the movement of cholesterol from the peripheral tissues to the liver for recycling or excretion. The figure was obtained from the American Physiological Society, ©2005, adapted by permission (51). 1.4.5 Lipidation ofApoA-I: ATP-Binding Cassette Cholesterol Transporters The delivery of cholesterol is regulated by membrane transporters of the ATP-binding cassette (ABC) superfamily. To date there are 48 members of the human family of ABC transporters, which are further subdivided into 7 families, designated ABC A to G51’66. These transporters couple energy from adenosine triphosphate (ATP) hydrolysis to the translocation of substrates across membranes67. The initial lipidation of the apoA-I particle with peripheral tissue-derived cholesterol requires the ATP-binding cassette transporter Al (ABCA 1), which mediates efflux of cellular cholesterol and phospholipids to lipid-poor apoA-I, but not HDL particles68. Another member of the transporter family is ATP-binding cassette transporter Gi (ABCG1), also known as human white gene69’ ABCG1 mediates cholesterol efflux to both small (HDL3) and larger (HDL2) subclasses but not to lipid-poor apoA-171.Therefore, ABCA1 promotes net cholesterol efflux to lipid-poor apoA-I, while ABCG1 facilitates net cholesterol efflux to HDL particles71’72• The primary mechanism by which ABCA1 promotes efflux of phospholipids and cholesterol to lipid- poor apoA-I is hypothesized to occur primarily by the creation of a lipid-rich microdiomain in the plasma membrane, where apoA-I can bind and be released as nascent HDL particles. However, it has also been shown that ABCA1 can interact directly with apoA-I to mediate Small Intestine I CAT Excr:tn UC Receptor 0 Nascent HDL SR-Bl LDL ABCA1 nCEH •CE Peripheral TissueLiver 15 cholesterol and phospholipid efflux73.As ABCA1 is the focus of this study, it will be discussed in greater detail in subsequent sections. In contrast, ABCG1 promotes efflux of cholesterol onto a variety of lipoprotein particles, including HDL, LDL, and phospholipid vesicles74’. Although active mechanisms involving transporters play a critical role in cholesterol efflux, passive mechanisms of efflux to acceptors have also been described. One form of passive efflux involves diffusion of cholesterol from the cell surface onto various extracellular acceptors including HDL, LDL, albumin and protein-free phospholipid vesicles76.Net cholesterol efflux via this process requires a concentration gradient between the cell membrane and the various extracellular acceptor particles. A gradient may be reached by extracellular cholesterol esterification by lecithin cholesterol acyltransferase (LCAT). The cholesterol exchange rate between the cell membrane and lipoproteins can be enhanced by PM receptors, such as ABCG 1 and scavenger receptor BI (SR-BI), which tether lipoproteins to the cell surface and induce redistribution of cholesterol in the PM7780. 1.5 ATP-BINDING CASSETTE TRANSPORTER Al (ABCA1) 1.5.1 General Mutations in ABC genes cause a variety of diseases, including cystic fibrosis, Startgardt’ s macular degeneration, and Tangier disease81. Structurally, ABCs may be categorized as either whole transporters or half-transporters. The whole transporters have 2 similar structural units joined covalently, while half-transporters are composed of single structural unit that form active heterodimers or homodimers66.Four members of the family have been shown to have a major impact on lipoprotein metabolism and cell cholesterol biology: ABCA 1, a whole transporter that mediates export of cellular cholesterol, phospholipids, and other metabolites to lipid-poor HDL apolipoproteins; ABCG 1, a homodimeric half-transporter that mediates cellular cholesterol export to lipidated lipoprotein particles; and ABCG5 and ABCG8, which form heterodimers that regulate intestinal absorption and promote biliary excretion of sterols51. ABCA1 was first cloned in 1994 and has been shown to play a critical role in the transport of cholesterol and phospholipids out of cells47. Human ABCA1 has been shown to be widely expressed in a variety of different tissues, with highest expression in the liver, small intestine, adrenal gland, and lung, and medium expression in the heart, aorta, and spleen82’3 As shown by studies in mice with a targeted knockout of hepatic ABCA 1, the lipid efflux from liver cells mediated by ABCA 1 is a rate-limiting step in HDL assembly and is required for the maintenance 16 of normal plasma HDL cholesterol concentrations, at least in mice84’ 85 Mutations in ABCA 1 have been associated with HDL deficiency and premature atherosclerosis86 87, indicating the critical role for ABCA1 in HDL production88,and in atherosclerosis prevention at the arterial wall. 1.5.2 Structure Full ABC transporters have two nucleotide-binding domains (NBDs) exposed to the cytoplasm and two transmembrane domains, each containing six to eleven membrane-spanning ct-helices89.The nucleotide-binding folds contain characteristic Walker A and B motifs that are 90-120 amino acids apart. Half transporters are those that contain one transmembrane domain and one nucleotide-binding domain that can combine into homo- or heterodimers to form functional full transporters. ABCA1 is a 2261 amino acid integral membrane protein that comprises 2 halves of similar structure. Each half has a transmembrane domain containing 6 helices and a NBD containing two Walker A and Walker B motifs, which are present in many proteins that use ATP, and a Walker C signature unique to ABC transporters (Figure 1.5). ABCAI is predicted to have an N terminus oriented into the cytosol and two large extracellular loops that are highly glycosylated and linked by 1 or more cysteine bonds51.The human ABCA1 gene has been mapped to chromosome 9q3 1 and is composed of 50 exons90. Figure 1.5 Structure of ABCA1 protein. Indicates the N-terminus oriented toward the cytosol and the nucleotide binding domains (NBD5). Also present are the six helices located on each domain. The figure was obtained from the American Physiological Society, ©2005, adapted by permission (51). 1.5.3 ABCA1 Regulation Excess cholesterol has detrimental effects on cells, including membrane destabilization and ER stress, requiring cells to have stringent mechanisms for controlling intracellular cholesterol levels48. In addition to the SREBP-dependent mechanisms outlined above, another mechanism by which cells detect excess cholesterol is via nuclear receptors liver-X-receptor a and 13 (LXRct and LXRI3), which bind with the retinoic-X-receptor (RXRct) as obligate heterodimers to NBD1 17 promoter regions of genes such as ABCA1 and ABCG191. LXRa is highly expressed in macrophages and liver, with lower expression in intestines, adipose tissue, and kidneys92. Conversely, LXRI3 is ubiquitously expressed93.Although sterol loading has been shown to activate the LXR target genes93, cholesteryl esters and unesterified cholesterol cannot directly function as ligands for LXRs. Instead, the conversion of sterols to oxysterols is necessary to induce transcriptional activation9496.Activation of ABCA 1, and ABCG 1, in macrophages, liver, intestine, neuronal cells, and Sertoli cells occurs with 9-cis retinoic acid as an RXR agonist and the oxysterols 20-hydroxycholesterol, 22-hydroxycholesterol, 24-hydroxycholesterol and 27- hydroxycholesterol91’97101• However, 20-hydroxycholesterol and 22-hydroxycholesterol are limited in their tissue distribution to steroidogenic organs 102 24-hydroxycholesterol, a secreted product of cholesterol metabolism in the central nervous system, may traverse the blood-brain barrier to reach the circulation or is delivered to the circulation via the cerebrospinal fluid, and is subsequently metabolized to bile acids in the liver. The primary endogenous ligand for LXR appears to be 27-hydroxycholesterol, the most abundant oxysterol in tissues and plasma, which is active as a regulatory molecule and a bile acid substrate. Production of 27-hydroxycholesterol is mediated by sterol-27-hydroxylase (CYP27AJ) in mitochondria’°3Patients with the genetic disorder cerebrotendinous xanthomatosis (CTX), due to mutations in CYP27AJ, exhibit massive accumulation of cholesterol in multiple tissues indicating a defect in sterol transport’°4. Moreover, skin fibroblasts from a CTX patient were unable to upregulate ABCA1 expression in response to sterols, while overexpression of CYP27A1 induced ABCA1 expression’°5.Therefore, 27-hydroxycholesterol correlates with cellular cholesterol levels, emphasizing the positive feed- forward regulation of ABCA 1-mediated cholesterol efflux by oxysterols. Increasing levels of cellular cholesterol leads to the formation of 27-hydroxycholeseterol, which binds to LXRs and induce ABCAlexpression and thereby increasing cholesterol efflux’°5.27-hydroxysterol is also the predominant oxysterol in the artery wall’°6 and in macrophage-derived foam cells’07’ 108 emphasizing the importance of this oxysterol in regulation of LXR in the artery wall109. Activation of LXRs is also modulated at the transcriptional level by agonists of peroxisome proliferator-activated receptors alpha, gamma and delta (PPARa, PPARy, PPARö)”°’ “ Activation of various transcription factors (zinc finger protein ZNF2O2, SP3, USF1/USF2/Fra2, thyroid and glucocorticoid hormone receptors) also suppresses ABCA1 gene transcription”2.In addition, interferon gamma (INF7), oncostatin and angiotensin II down-regulate ABCA1 expression112 As well, the promoter region of ABCA1 contains binding motifs for cyclic AMP (cAMP), whose signal cascade is activated by apoA-I, leading to increased expression and 18 phosphorylation of ABCA1 in macrophages”3’15.Janus kinase 2 (JAK2) also phosphorylates ABCA1, a process that increases apoA-I binding and cholesterol efflux’16. With an approximate half-life of 1 hour, ABCA1 has a high turnover rate. Binding of apoA-I to ABCA1 stabilizes the transporter and slows its degradation. In the absence of apolipoproteins, phosphorylation of threonine residues within the ABCA1 PEST motif (a tetrapeptide motif that targets proteins for degradation) is a signal for calpain-mediated proteolytic degradation of ABCA1117’118 Other signals leading to ABCA1 degradation are exerted by unsaturated fatty acids via a phospholipase D2-dependent pathway, which induces proteasomal degradation’19 1.5.4 ABCA1 and Lipid Efflux Lipid-poor and lipid-free apolipolipoprotien Al (apoA-I) functions as a mediator for the removal of unesterified cholesterol from peripheral tissues120. However, it was found that the efflux of unesterified cholesterol and phospholipids by apoA-I was dysfunctional in Tangier disease cells’21’122 The inability of apoA-I to remove unesterified cholesterol and phospholipids provided a potential reason for the low HDL levels observed in Tangier patients, and indicated a critical role for apoA-I-dependent cholesterol efflux in regulating circulating plasma HDL levels’22’4.In 1999, it was discovered that mutations in ABCAJ were the cause of Tangier disease’258 Based on these findings, the ABCA1 protein was believed to be required for the lipidation of non-lipidated and lipid-poor HDL apolipoproteins, and thus necessary for the formation of nascent HDL particles. Although these findings have provided insight into HDL formation, the precise function of ABCA1 in promoting the trafficking of unesterified cholesterol and phospholipids to apoA-I remains unclear. 1.5.5 ABCA1 Cellular Distribution The localization of ABCA1 was initially elucidated using cell-surface labeling and immunoprecipitation, which showed that increased expression of ABCA1 in the PM led to increased lipid efflux’28. The relative amount of ABCA1 on the PM was determined using biotinylation studies in human fibroblasts and macrophages”3’128 The localization of ABCA1 in the PM was also studied using immunofluorescence confocal microscopy that observed ABCA1- FLAG-transfected Human Embryonic Kidney cells (HEK293) on the cell surface’29.Fluorescent studies using organelle-specific markers have shown that ABCA1 also localizes in intracellular compartments’30’ 131 including late endosomes/lysosomes, the trans-Golgi network, and endoplasmic reticulum”8’130, 132-136 A key study by Neufeld et al. in 2001 elucidated the movement of ABCA1-GFP in Chinese hamster ovary (CHO) cells using live cell imaging’30. 19 Their results suggested that ABCA1 in the PM and late endosomes/lysosomes transits between the PM and intracellular compartments. The trafficking between the late endosome/lysosome and the cell surface by ABCA1 is hypothesized to occur for the removal of unesterified cholesterol from late endosomes/lysosomes, and suggests cholesterol in this compartment forms a portion of the substrate pool for ABCA1 in nascent HDL formation. 1.5.5 ABCAJ-mediated Cholesterol Efflux: NPC and Tangier Disease Cholesterol found in lysosomes is derived from hydrolysis of cholesteryl esters delivered from endocytosis of LDL and hydrolyzed by lysosomal acid lipase (LAL)137,as well as de novo synthesized cholesterol delivered from the plasma membrane to lysosomes during membrane recycling138.In recent years there has been increasing evidence that lysosomal cholesterol may function as a substrate pool for ABCA1-mediated cholesterol efflux to apoA-173’ 139 To investigate whether the endosomes/lysosomes represent a source of cholesterol for ABCA 1- mediated cholesterol efflux, Chen et al. 68 used murine peritoneal macrophages treated with LXRJRXR ligands 22(R)-hydroxycholesterol and 9-cis-retinoic acid to measure efflux to apoA-I. They incubated macrophages with[3H]-cholesterol- acetyl LDL (acLDL) to accumulate cholesterol in late endosomes/lysosomes, and compared them with macrophages incubated with [3H]-cholesterol/lO% FBS, which is not preferentially stored in the late endosomes/lysosomes. They found that levels of cholesterol release were 2.5-fold higher in cells labeled with [3H]- cholesterol-acLDL compared with cells labeled with{3H]-cholesterol/lO% PBS. The same lab also explored cholesterol mass and isotopic efflux to apoA-I utilizing macrophages from NPC’ mice. After labeling the cells with either[3H]-cholesterol/1O% PBS,[3H]-cholesterol/ AcLDL, or [3H]-cholesterol/cyclodextrin (radiolabel that resides primarily in the PM), they found that there was greater isotopic and mass efflux of cholesterol in cells labeled with[3H]-cholesterol/AcLDL, consistent with the idea that the late endosomal/lysosomal cholesterol pool functions as a substrate source for cholesterol efflux by ABCA168. Research into the late endosomal/lysosomal compartment as a substrate pool, and as a regulator, for ABCA 1-mediated efflux have also utilized lysosomal cholesterol storage disorders as model systems for studying this particular compartments role in ABCA 1-dependent HDL formation’40’141 Niemann-Pick type C (NPC) disease is one such model. NPC disease is a neurovisceral disorder caused by mutations in NPCJ, or to a lesser extent NPC2, characterized by the accumulation of unesterified cholesterol and other lipids in late endosomes/lysosomes due to impaired cholesterol trafficking to other cell compartments. Choi et al.14° using NPC1- 20 deficient fibroblasts, showed that before and after loading cells with non-lipoprotein cholesterol or LDL there was reduced expression of ABCA1 mRNA and protein compared to normal fibroblasts (Figure 1 .6A,B). Functional studies using[3H]-cholesteryl linoleate-labelled LDL indicated that removal of radiolabeled cellular cholesterol to apoA-I-containing media was also markedly reduced in NPC1-deficient fibroblasts (Figure l.6C). In addition, the NPCF’ fibroblasts showed accumulation of[3H]-unesterified cholesterol. These results indicate that the LDL-derived unesterified cholesterol that accumulates in NPC’ fibroblasts may have a regulatory role with regards to ABCA1 expression. Figure 1.6 (A) Reduced ABCA1 mRNA and (B) protein expression in human NPC1-deficient fibroblasts. Fibroblasts incubated with[3H]-cholesteryl linoleate-labelled LDL showed that NPC 1-deficient fibroblasts had a markedly reduced release of [3H] unesterified cholesterol (UC) to apoA-I compared to normal fibroblasts. (C) Cell cholesteryl ester (CE) levels wer also markedly reduced, in addition to an accumulation of cellular UC in the lysosome. The figure was obtained from the Journal of Biological Chemistry, ©2003, with permission (140). A. C 16 — 14 2 4 12 Cl) r—, 10 = I4 2 0 NPCI genotype +1+ -1-l .1- Cholesterol - 4- - + - 4 I ABCAI mRNA 1 CyclopNln ____________________ RT-PCR ABCA1 niRNA * 1 Northern 26S rRNA — — 1_ — — — ABCAI:2SSRNA 025 025 00 053 0.01 000 ABCAI protein LIU — 1 34 1.2 36 0.5 1.2 LOL - - 4 - Wetem ABCAI protein 1 2.1 0.7 0.0 0.6 0.6 01020 3 4050 01020304050 01020304050 Hours 21 20 18 0 I.. o. 16 14 U 0 12 e 10 - 8 0 0 6 2 2 0 In addition, Boadu et at. 141 have shown that increasing ABCA1 expression in NPC fibroblasts reduced the accumulation of unesterified cholesterol in late endosomaLflysosomal compartments, suggesting ABCA1 was able to bypass the NPCJ mutation and directly influence the removal of the accumulated unesterified cholesterol (Figure 1 .7A,B)’41 Figure 1.7 Upregulation of ABCA1 reduces sterol accumulation in NPC fibroblasts. (A) Medium unesterified cholesterol (UC) and cell cholesteryl esters (CE) and UC cholesterol mass in NPC1’ and NPCT’ fibroblasts following a 24hr incubation with I Oj.tglml ApoA-I. (B) Increased phosphatidylcholine (PC) and LDL-derived cholesterol efflux following 24hr incubation with 10 tg/ml apoA-I from LXR agonist-treated NPCT human fibroblasts. The figure was obtained from the Journal of Biological Chemistry, ©2006, with permission (141). 120A. 100 80 ** :fli NPC1’ NPC1 500 B .0 400 22 30 300 Ui 20’E —i’? — U 100 10 a Another rare genetic disorder of cholesterol metabolism used to study the role of late endosomes and lysosomes in cellular cholesterol trafficking is Tangier disease (TD). TD, caused by mutation in both ABCA1 alleles, is characterized by a near or complete absence of circulating HDL and by the accumulation of cholesteryl esters in many tissues, including tonsils, lymph nodes, liver, spleen, thymus, intestine, and Schwann cells124.The ability of lipid-poor apoA-I and other HDL apolipoproteins to interact with the cell surface to remove excess cholesterol and phospholipid is severely impaired in TD subjects. Using Tangier fibroblasts as a model, Francis et at. 121 showed that apoA-I has an impaired ability to remove unesterified cholesterol and 22 phospholipids, potentially due to a defective interaction between apoA-I and cell-surface binding sites, leading to low plasma HDL levels in Tangier disease (Figure 1.8). Figure 1.8 Impaired lipidation of apoA-I in TD fibroblasts. (A) Effects of HDL and apo A-I on[3H]cholesterol efflux from cholesterol-loaded normal (NL) and Tangier (TG) fibroblasts. (B) Effect of HDL and apo A-I on unesterified and esterified[3HJ-cholesterol and esterified cholesterol mass in cholesterol-loaded normal and Tangier fibroblasts. The figure was obtained from the Journal of Clinical Investigation, © 1995, with permission (121). 10 0 C.) I I. A. B. I 10 I0 jeo Jeo Houra Recent studies by Neufeld et al. 142 using Tangier disease fibroblasts, have shown that the ABCA1 deficiency results in the accumulation of unesterified cholesterol in late endosomes/lysosomes, which was not mobilized following exposure to apoA-I (Figure 1.8)142. These results suggest that ABCA1 activity is necessary for the removal of unesterified cholesterol from late endocytic compartments. This was further supported by their results showing that expression of ABCA1 as ABCA1-GFP fusion protein in Tangier disease fibroblasts corrected apoA-I-stimulated mobilization of unesterified cholesterol from late endosomal/lysosomal compartments (Figure 1 .9A,B)’42 Hours 0 24 48 0 24 48 23 Figure 1.9 Impaired ABCA1 function in TD fibroblasts. (A) The motility of Tangier disease late endocytic vesicles is impaired. Late endosomes and lysosomes in wild type (a) and Tangier disease (b) fibroblasts were labeled by incubation with DiI-LDL for 24 h. There is perinuclear accumulation of DiILDL-labeled late endocytic vesicles in Tangier disease (b) fibroblasts. Wild type (c-f) and Tangier disease (g—j) fibroblasts were incubated 50 .tg/ml LDL followed by incubation with medium in the absence (c,d and g,h) or presence (e,f and i,j) of lOig/m1 apoA-I for 24 h. Fibroblasts were subsequently fixed and stained with filipin to reveal the cellular distribution of cholesterol (d, f, h, and j) and immunostained LAMP2 (green, c, e, g, and i). There is an accumulation of cholesterol (h and j) in Tangier disease late endocytic vesicles (g and i). (B) ABCA1-GFP expression in Tangier disease fibroblasts restores apoA-I-mediated cellular cholesterol efflux. Wild type and Tangier disease fibroblasts infected with adenovirus expressing ABCA1-GFP (ABCG1-GFP). Expression of ABCA1-GFP enhances sterol efflux in wild type cells and corrects the defective efflux in Tangier disease fibroblasts. The figure was obtained from the Journal of Biological Chemistry, ©2004, with permission (142). B. % FVLUX WILDTVPE TANGIERWILOTYPE ACA1.O1’P 24 1.6 CHOLESTERYL ESTER STORAGE DISEASE (CESD) As discussed above, studies using normal and Tangier fibroblasts as model systems have helped elucidate the distribution and function of ABCA i’d” 142 As well, NPC disease fibroblasts have provided insight into the late endosomal/lysosomal cholesterol pooi as a regulator of ABCA 1 expression’41.Another lysosomal storage disorder, used as a model system in this study, is cholesteryl ester storage disease (CESD). CESD results from deficiency in LAL, with patients developing premature atherosclerosis potentially due to their known low plasma HDL-C levels64. 1.6.1 Overview and Background There are currently two known autosomal recessive disorders that result in the extensive accumulation of cholesteryl esters and triglycerides within the lysosomal compartments. The first, Wolman disease, is the more severe condition, with abnormal lipid metabolism observed soon after birth, and death occuring by 6 to 12 months64. Hepatosplenomegaly, steatorrhea, abdominal distention, adrenal calcification, and failure to thrive are observed within the first week’43.Massive intracellular storage of both cholesteryl esters and triglycerides are observed in the liver, adrenal gland, and intestine. Conversely, CESD is a milder version, sometimes compatible with survival into later adulthood’43’144 Lipid deposition is widespread although hepatomegaly may be the only clinical manifestation. Other clinical manifestations may include digestive abnormalities, steatorrhea, and in a large percentage of patients development of premature atherosclerosis. Phenotypic difference between the two disorders is due to higher levels of residual LAL activity in CESD, which can be evidenced by in situ loading tests on living cultured cells64. Studies by Goldstein et al. 137 demonstrated that cultured fibroblasts from CESD patients exhibit reduced LAL activity compared to normal fibroblasts and a reduced ability to hydrolyze cholesteryl esters (Figure 1.10). Reduced LAL activity has been confirmed by Du et al)45 in fibroblasts from two CESD patients (P1, P2) when compared to normal fibroblasts (HF)(Figure 1.11). 25 E•3QLDo 0 in 0 w 0 m 0” V i02 Potential treatment options for CESD patients involve the use of HMG-CoA reductase inhibitors (statins), which have been shown to be well tolerated during long term treatment, and may help prevent premature atherosclerosis 146-149 Ezetimibe, a lipid-lowering medication that prevents the absorption of cholesterol and plant sterols at the small intestinal brush border by interfering with activity of NPCJL1 receptor, has been shown to be a potential treatment in CESD patients’49.Gene therapy to replace LAL activity in the liver at least is a potential but not yet clinically viable treatment for CESD patients. Figure 1.10 CESD human fibroblasts exhibit reduced LAL activity. (A) Reduced Hydrolysis of [3H] cholesteryl linoleate bound to LDL by cell-free extracts of CESD when compared to normal fibroblasts at varying pH. (B) Reduced hydrolysis of the cholesteryl ester, but not apolipoprotein components of LDL, by cell-free extracts from CESD as compared to normal fibroblasts. The figure was obtained from the Journal of Biological Chemistry, ©1975, with permission (137). A. B. Strain • Norr’oILA CE.S] 1 A Hydrolysis of Cholesferyl Lroleafe 40 - B Hydrolysis f Apolipoprotein 30 0 0 L - - 0’ or i:i 20 120 pH HFP1 P2 Figure 1.11 Reduced hEAL protein expression in CESD fibroblasts. HF, normal human fibroblasts; P1 and P2 are CESD patient fibroblasts. Reprinted from Molecular Genetics and Metabolism, Vol 64, Du H., Sheriff, S., Bezerra J., Leonova T., Grabowski, GA., Molecular and Enzymatic Analyses of Lysosomal Acid Lipase in Cholesteryl Ester Storage Disease, pg 126-134, Copyright © (1998), with permission from Elsevier (145). 105 70.8 — 43.6 — } 26 1.6.2 CESD Patient Tissue Lipid and Lipoprotein Levels CESD is characterized by hypercholesterolemia, abnormal lipid deposition in many organs, and HDL deficiency’50.The excessive accumulation of cholesteryl esters and triglycerides in CESD is seen in tissues including liver, intestine, spleen, lymph node, aorta, and cultured skin fibroblasts64. Studies of tissue lipid levels in CESD patients have shown an increase in cholesteryl esters ranging from approximately 120 to 350 times normal. Tissue accumulation of triglycerides is not nearly as significant when compared to cholesteryl esters, with only a two fold increase observed on averageTM’ 151153 The differences in accumulation may be due to greater catalytic efficiency of LAL for triglycerides when compared to cholesteryl esters154’57. The plasma lipid levels in all CESD patients shows hypercholesterolemia, which may also be associated with hypertriglyceridemia’58.Patients may have increases in LDL cholesterol and variable increases in VLDL. Patients also have low levels of HDL cholesterol, with concentrations typically varying from 0.2 to 0.7 mmol/L of plasmaTM.These HDL-C levels are typical of patients with inherited disorders affecting HDL-C levels, as opposed to secondary causes of low HDL-C, such as diabetes, which typically cause milder reductions in plasma HDL C (G. Francis, personal communication). 1.6.3 Abnormal Lipid Trafficking In CESD In CESD, the activity of LAL in cultured fibroblasts, using synthetic substrate derivatives such as 4-methylumbelliferyl oleate (4-MUO), have been shown to be less than 12 percent of controls in patients and approximately 50 percent in heterozygotes64’157 LAL is present in nearly all cells, except for erythrocytes, and is required for the degradation of LDL-derived sterols137. Cells take up LDL via receptor-mediated endocytosis, which once bound, are endocytosed into clathrin-coated vesicles that eventually fuse with lysosomes. Within the acidic lysosomal compartment, apolipoproteins, cholesteryl esters, and other lipid components of the LDL particle are hydrolyzed by lysosomal ymes’59’160 Under normal conditions, unesterified cholesterol and fatty acids are released from the lysosomes, which may be directed to several locations, such as the ER, the Golgi or the PM. The portion of the unesterified cholesterol shuttled to the ER is responsible for initiating three critical regulatory mechanisms. Endogenous cholesterol synthesis is inhibited by suppressing activity of HMG-CoA reductase, the rate limiting step in cholesterol synthesis. In addition, expression of the LDL receptor is downregulated, leading to a reduction in the number of receptors on the cell surface and thus a reduced uptake of LDL. Finally, intracellular esterification of unesterified cholesterol is increased by microsomal membrane bound acyl-CoA:cholesterol acyltransferase (ACAT). In this reaction, the unesterified 27 cholesterol is reesterified with primarily oleic (C 18:1) acid. The esterified cholesterol subsequently accumulates as cytoplasmic lipid droplets (Figure 1.12). Conversely, in CESD fibroblasts the hydrolysis and subsequent regulatory effect of cholesteryl esters is abnormal. Due to the significant reduction in LAL activity, the reduced production and therefore flux of UC out of lysosomes results in higher endogenous cholesterol synthesis and LDL receptor expression, and reduced ACAT activity than in normal fibroblasts in response to LDL loading’37’161 These lead to a relative increase in uptake of LDL into the lysosomal pathway, which leads to a cyclical accumulation of cholesteryl esters in the lysosomes, in addition to increased endogenous cholesterol synthesis’62’7(Figure 1.12, Figure 1.13). Figure 1.12 Increased LDL uptake in CESD fibroblasts compared to normal fibroblasts. Increased LDL uptake observed in CESD fibroblasts as a result of increased LDL receptor expression, compared to normal fibroblasts. The figure was obtained from Journal of Biological Chemistry, © 1975, with permission (137). 1600 1200 juJ 800 40O 16 RD U S 28 Figure 1.13 Normal and CESD cell metabolism. Under normal conditions, LDL is taken up via endocytosis into early endosomes/lysosomes (Early EIL). Within the late endosomal/lysosomal compartment (Late EIL), LAL hydrolyzes cholesteryl esters (CE) and triglycerides resulting in the release of unesterified cholesterol (UC) from the lysosomes. The UC is directed to several subcellular locations, such as the mitochondria, plasma membrane (PM), the endoplasmic reticulum (ER), where it is responsible for suppressing activity of HMG-CoA reductase and transcription of the LDL receptor gene, while cellular formation of CE is stimulated through activation of acyl CoA:cholesterol acyltransferase (ACAT). In CESD, LDL particles are endocytosed normally, however due to a significant deficiency in LAL activity there is an accumulation of CE within the lysosome, and reduced liberation of UC which results in increased expression of HMG-CoA reductase, increased transcription of the LDL receptor gene, and reduced ACAT activity. 1.6.4 CESD Genetic Background As described previously, the inherited deficiency of LAL activity results in two rare autosomal recessive disorders, Wolman disease and CESD. Wolman patients have a complete deficiency in active LAL, while CESD patients typically display approximately 3 to 12% residual enzymatic activity64.In CESD and Wolman cells, the significant reduction or complete absence of functional LAL indicates that the acid lipase deficiency in the patients is a result of mutations interfering with the catalytic activity of the enzyme or may be due to inappropriate intracellular processing and targeting of the enzyme. Since cloning of the cDNA in 1991 by Anderson and Sando’68, the location and genomic organization of the gene (LIPA) encoding human LAL has been found to reside on chromosome 10q23.2-q23.3 and to consist of ten exons 29 over a 38.8-kb region’69471.Since its discovery, many deleterious mutations in the LIPA gene have been identified, most of which were found to affect mRNA splicing and/or amino acid substitutions. Two CESD patients were initially found to be compound heterozygotes for a G-to A transition at position -1 of the exon 8 splice donor (E8SJM, Exon 8 Splice Junction Mutation), which leads to an in-frame deletion of exon 8, producing a protein that is 24 aa shorter and has no residual enzyme activity, but does not produce the Wolman phenotype. The vast majority of CESD patients have the E8SJM mutation150’72,which results in a small fraction of correctly spliced, active LAL enzyme173”45’174 The E8SJM mutation has been reported to be present along with L179P, AAG3O2, a null mutation, and G66V in CESD patients. Another version of the exon 8 skipping mutation has shown a 72-bp in-frame deletion due to the already described G to A substitution (G934 to A substitution), compound heterozygote with a C to T substitution at position 233 in exon 3 or deletion of a C (nucleotide 673, 674, or 675) in exon 6175176. Other mutations identified in CESD patients include either homozygosity for T2671 or compound heterozygosity with Q64R. The mutants T2671 and Q64R and the previously reported L273S, G66V and H274Y CESD substitutions, over-expressed in stable clones, were found to have an LAL activity 3-8% of that of normal fibroblasts177.Another patient revealed a T insertion in exon 6 at codon 178 that shifted the reading frame and caused a premature termination at codon 190 (FS A128-X190) in conjunction with a G-to-A change at the last nucleotide of exon 8 (Q277) on the second allele, which resulted in a silent mutation at the amino acid level’49. Currently, there is a wide spectrum of homozygous and compound heterozygous mutations associated with the development of CESD, which were identified in patients from varying age groups and ethnicities. 1.6.5 Lysosomal Acid Lipase (LAL): Structure and Trafficking LAL contains a hydrophobic 21 amino acid long leader sequence, belonging to a family of signal peptides, necessary for directing the newly synthesized enzyme to the PM, secretory vesicles, and lysosomes64’ 168 From the initial site of synthesis at the rough endoplasmic reticulum, the lipase is transported to the trans-Golgi network where proteins are shuttled to their respective destinations. Once the enzyme reaches the cis-Golgi network, it is glycosylated by an N-acetylglucosaminylposphotransferase, which joins N-acetylglucosarnine phosphate to -1,2 mannose residues’78’82.Subsequently, a phosphodiesterase cleaves the N-acetylglucosamine to produce mannose 6-phosphate (Man-6-P), which is responsible for binding the acid lipase to the Man-6-P receptors on the Golgi’83’5.Following binding with the Man-6-P receptor, the enzyme 30 exits the trans-Golgi network in a weakly acidic prelysosomal compartment’78. This compartment then fuses with a late endosome containing substrates for LAL. Once fusion is complete between the prelysosomal compartment and the late endosome, the Man-6-P receptor is released and recycled back to the Golgi’78. LAL is initially synthesized as a propeptide with 49 additional amino acids necessary for protein transport and stabilization, which are later cleaved in the endosome’86.The presence of the propeptide explains the presence of two active forms of LAL with different molecular weights. Human LAL purified from various sources may have different molecular sizes, such that human fibroblasts express two molecular forms of 41 and 49 kDa, and LAL purified from human liver showed two molecular forms of 41 and 56 kDa187’89.In human fibroblasts, the LAL with the larger molecular mass represents the secreted form, while the smaller molecular mass represents the intracellular enzyme’68. Interestingly, the secreted form may be taken up by neighbouring cells, and becomes active once in an acidic environment156. The enzymatic activity of LAL appears to be regulated by the presence of substrates. For example, when uptake of LDL was stimulated, such as by providing a cholesterol-rich diet, the activity was observed to increase 2 to 3 fold, based on increased substrate availability’90’191 The LAL promoter sequence in monocytes, characterized by Ries et al., is GC-rich and contains a TATA-box, which is regulated by transcription factors Spi and AP-2. During monocyte differentiation into macrophages, these factors bind at the promoter sequence and induce expression of LAL192. Importantly, unlike ABCA1, the LAL promoter sequence does not contain an LXR response element. The purification of LAL has been studied in a variety of human tissues such as the liver, placenta, aorta, leukocytes, fibroblasts, and cardiac myocytes64’188, 193 The purified form of the enzyme was found to be water soluble, has a high affinity for hydrophobic surfaces and is salt- sensitive. The hydrolytic specificity of the purified form was found to include a broad range of sterols, including triolelyglycerol, dioleylglycerol, monooleylglycerol, cholesteryl oleate and cholesteryl linoleate’94’ 195 The positional specificity of purified LAL was studied with trioleylglycerols, dioleylglycerols, and monooleylglycerols, which showed a preference for 1(3)- ester bonds193’ 196 Furthermore, it was found that cationic phospholipids such as phosphatidylcholine increase the activity of purified LAL’97200. The full-length cDNA of lysosomal acid lipase has been isolated and sequenced, and has been shown to have 58 and 57% homology with gastric and rat lingual lipase, respectively. Both of these enzymes are involved in the preduodenal hydrolysis of ingested triglycerides. Although 31 the sequence does not show any significant homology with other lipases such as hormone- sensitive lipase, lecithin-cholesterol acyltransferase (LCAT), and the gene family that includes lipoprotein lipase (LPL), it does share the presence of the esterase associated amino acid sequence motif —Gly-X-Ser-X-Gly with most other lipases. Studies using X-ray crystallography have shown that the serine in this sequence appears two times within the human LAL sequence (residues 97 to 101 and 151 to 155), serines are the active-site catalytic residues in human pancreatic lipase, hepatic lipase, and pancreatic cholesterol esterase201203.Structurally, seven cysteine residues are present within the human acid lipase amino acid sequence, including one that is located in the leader sequence. The cysteine residue at position 240, which is also present in the gastric and rat lingual lipases, may be functionally related to the active site of the enzyme. 1.6.6 CESD Mouse Model Mouse and human LAL are encoded by approximately 3.2-kb and 2.5-kb cDNA, respectively, with a 95% similarity of the deduced amino acid sequences. The mouse and human LAL genes encompass 10 exons spanning 37 and 47.7 kb of genomic DNA with the same exon/intron organization204.The expression patterns of mouse LAL mRNA and protein have similar tissue distribution, with high-level expression found in liver parenchymal cells and Kupffer cells, splenic macrophages, and epithelial cells of the small intestine. Using gene targeting by homologous recombination, a mouse model with an LAL null mutation was created by Du et al. 205, 206 and was found to be biochemically similar to Wolman disease, yet physiologically similar to CESD. The homozygote knockout mice (laL’/laT”) produce no LAL mRNA, protein or exhibit any enzyme activity (Figure 1. 14A). They appear normal at birth, survive to sexual maturity and can produce progeny. However, as they mature they accumulate approximately 30-fold greater amounts of triglycerides and cholesteryl esters in several organs, compared to their wild type littermates207.Within 21 days, the liver becomes a yellow-orange color and is approximately 1.5 to 2.0 times larger than normal (Figure 1. 14B,C). The spleen of the lar’/lal mice also showed a pale yellow color and appeared slightly larger in size (Figure 1. 14B). There were no gross abnormalities observed in other tissues from lar/lal mice including the kidneys, brain and adrenal glands. Heterozygote LAL mice (la(’/lar) have approximately half normal LAL activity and do not show abnormal lipid accumulation205. 32 Figure 1.14 Reduced enzyme activity and enlarged liver and spleen in LAL knockout mouse model. (A) LAL enzyme activity assay of mouse liver extract using radiolabelled cholesteryl oleate as substrate. (B) Enlarged and discolored liver and spleen from lalilal (a), laltflat (b) and lalt’lat (c) mice. The homozygote mutant liver is the only one to exhibit the yellowish discoloration from massive lipid accumulation. The spleen located below the liver also appears to be discolored. The figures were obtained from Du H, Duanmu M, Witte D, Grabowski GA., Targeted disruption of the mouse lysosomal acid lipase gene:long-term survival with massive cholesteryl ester and triglyceride storage, Human Molecular Genetics, ©1998, Vol 7, pg 1347-54, by permission of Oxford University Press (205). (C) Image of control (left) and LAL-deficient mouse (right) at 24 weeks of age. The LAL deficient mouse shows an enlarged and yellow liver (yellow arrow) compared to normal (white arrow). The figure was obtained from the Journal of Lipid Research, ©2001, with permission (204). 1• 120 A. 100 80 60 0 E 40 20 0— laI+flal.i- Pal-hal Further evaluation of tissue lipids in la1/lal mice showed that at 6-weeks of age the total cholesteryl esters in the 1a1/lal mice liver was approximately 32-fold greater than the wild- types, while the heterozygote mouse was similar to the levels in the wild-types. The liver triglyceride levels were found to be approximately 35-fold greater in the knockout mice compared with the wild-types. Cholesteryl esters also accumulated in spleen and small intestine, with approximately 10- and 2-fold higher levels in the knockout mice, respectively205. The average lifespan of lal/1aL” mice is 8 months, compared to —24 months in wild type mice of similar background (l29Sv and CF-i), with death resulting from massively enlarged livers and spleens and potentially malabsorption due to intestinal infiltration by macrophages overloaded with CEs and TGs204. In mutant mice, the progressive phenotype leads to 33 hepatosplenomegaly that may account for 10-35% of body weight, severe macrophage infiltration of the small intestine, and lymph nodes that are engorged with lipid-filled macrophages207. An adenovirus expression vector carrying full-length human lysosomal acid lipase (Ad hLAL) was used to evaluate tissue delivery and expression, as well as phenotypic effects in the mice207. lal/lal’ mice injected with Ad-hLAL showed a reduction in liver weight of 9.4% at 6 days following infection and approximately 6% at 20 and 47 days (Figure 1.1 5A). Hepatic LAL activities were compared in PBS- and Ad-hLAL-treated lat’/lal mice. In the adenovirus-treated mice LAL activity increased 243-fold at 6 days post-injection and 161-fold at 47 days postinjection, or -12 and 9-fold higher than endogenous LAL activity in wild-type mouse liver, respectively (Figure 1.15B). Furthermore, reduction in lipid storage in macrophages of the small intestine began at 2 months and was progressive with increasing age, but was completely corrected at both 20 and 47 days following delivery of an adenovirus expressing LAL207. Figure 1.15 Decreased liver size and increased LAL activity from Ad-hLAL-injected lal’ mice. (A) Liver to body weight percentage in Ad-hLAL-infected mice or PBS-injected mice at 47 days following infection or injection. (B) Hepatic LAL activity, using[3C]-cholesteryl oleate as substrate. The figure was obtained from Human Gene Therapy, ©2002, with permission (207). A. B. 20 I EPBS Arus ri q1 IEL___I__1__I IaI÷l+ 6 20 47 (days) 0 __________________________ Ial+i+ 6 20 47 (days) IaI-i 34 With regards to plasma lipid levels, Du et al. 207 showed that the Ad-hLAL-treated mice had reduced plasma CE, while plasma TG levels were not significantly different (Table 1.3). Reduced IDL and LDL cholesterol levels were also observed, however no significant change in HDL occurred (Figure 1.16)204. Table 1.3 Plasma lipid levels in wild-type (lal+/+), mutant mice (lat’) and Ad-hLAL infected mutant mice. The table was obtained from Human Gene Therapy, 02002, with permission (207). Plasma lipids Inig/dl) Free Cholestervl hulesterol esters Triglycerides lah 16±2 50±2 48±9 lalIPBS 39±18 64±6 34±7 !aL /virus6 25±4 56±6 20±2 days laL’Ivirus20 16±04 31±5 31±8 days lal ‘virus 47 26 ± 1 34 ± 1 26 ± 1 days Figure 1.16 Serum lipoproteins in lat’ (open circle) and lai’ (closed circle) mice. Mouse serum from mice was applied to fast protein liquid chromatography (FPLC). The insert shows the agarose gel electrophoretic patterns of serum lipoproteins from la1 (lanes I and 4) and 1a1 (lanes 2 and 3) mice. The figure was obtained from the Journal of Lipid Research, 02001, with permission (204). 60 3 34 50 a30 ‘4 20 10 t ,-ftt • 0010 20 30 4050 VLDL LDI. HDL Fractions Infection with adenovirus expressing a gene of interest requires virus interaction with the coxsackie-adenovirus receptor (CAR) mediating virus attachment to the cell surface, and on interaction with a133- and uv135- integrins mediating virus entry into the cell 208-211 These results 35 indicate that expression of the receptors required for adenoviral infection are sufficiently wide spread for effective infection and that the virus is correctly shuttled to the appropriate regions of accumulation. Thus, adenovirus-mediated gene transfer may be a viable potential therapy for a variety of enzymatic disorders. However, although animal models may provide useful insight into pathological conditions, results obtained may not be directly applicable to humans. For example, studies using NPC’ mice hepatocytes have shown that they exhibit an increase in ABCA1 protein but not mRNA levels, increased apoA-I lipidation and increased HDL cholesterol levels 212-214 in contrast, studies using human NPC’ fibroblasts have shown that they have impaired regulation and activity of ABCA1, resulting in decreased efflux of cell phospholipid and cholesterol and formation of HDL particles in vitro. In addition, the majority of NPC patients display low plasma HDL-C levels 140 Furthermore, analysis of plasma lipid levels in lal mice did not show significant differences in HDL levels compared to normal mice207,while CESD patients exhibit low plasma HDL levels64. The discrepancies observed indicates the necessity for using human derived cells for studying biochemical pathways and pathologic conditions as animal models cannot be directly applied to human systems in this situation. Based on the known differences between mouse and human CESD phenotypes and mouse and human HDL metabolism, the present study used human skin fibroblasts obtained from CESD patients as a model system. 36 1.7 HYPOTHESIS AND SPECIFIC AIMS In summary, it has already been demonstrated that patients with CESD have an increased risk of premature atherosclerosis, and that their plasma HDL-cholesterol (HDL-C) levels are approximately half normal. Our lab previously demonstrated that another lysosomal cholesterol storage disease, Niemann-Pick disease type C, results in impaired regulation of ABCA1 and HDL particle formation at the cellular level, and low plasma FTDL-C in the majority of NPC disease patients. Based on these findings, we hypothesize that in CESD, the reduced activity of LAL leads to a decrease in the release of unesterified cholesterol from lysosomes, resulting in impaired ABCA1 regulation and HDL formation.. This project will elucidate whether the low plasma HDL-C seen in CESD is a consequence of impaired regulation of ABCA1, and further determine the importance of lysosomally-derived cholesterol as a regulator of ABCA1 expression. The objectives of this thesis are: 1. To determine whether ABCA1 mRNA and protein levels in human CESD skin fibroblasts are reduced when compared to normal skin fibroblasts. 2. To determine whether there is reduced LDL-derived cholesterol and phospholipid efflux to apoA-I containing media from CESD fibroblasts compared to normal fibroblasts. 3. To determine whether there is an accumulation of cholesteryl ester mass and reduced unesterified cholesterol in apoA-I containing media in CESD fibroblasts compared to normal fibroblasts. 4. To determine whether delivery of an adenovirus expressing full length human lysosomal acid lipase (Ad-hLAL) in normal and CESD fibroblasts alters ABCA1 expression and activity in these cells 5. To determine whether delivery of Ad-hLAL to CESD fibroblasts alters LDL-derived cholesterol and phospholipid efflux to apoA-I containing media. To also determine whether delivery to normal fibroblasts can improve LDL-denved cholesterol and phospholipid efflux to apoA-I containing media. 37 6. To determine whether delivery of Ad-hLAL to CESD fibroblasts can reduce the cholesteryl ester mass accumulated within the lysosome and improve efflux of unesterified cholesterol to apoA-I containing media. 38 6E SUOHJ1ITGNVS’1VflIL1VI’ 4J:zIIIJVH3 2.1 MATERIALS Liver X receptor agonist TO-90 1317 and essentially fatty acid-free bovine serum albumin (BSA) were purchased from Sigma-Aldrich. [Cholesteryl -1, 2, 6, 7-3Hj cholesteryl linoleate (60-100 Ci/mmol) was purchased from American Radiolabeled Chemicals. [methyl - 3H] choline chloride (66.7 Cilmmol) was purchased from PerkinElmer. Dulbecco’s modified Eagle’s medium (DMEM) was purchased from BioWhittaker and fetal bovine serum (PBS) and lipoprotein deficient serum (LPDS) were purchased from Cocalico Biologicals, Inc. Nitrocellulose membranes, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) reagents, and pre-stained protein molecular mass markers were purchased from Bio-Rad. The adenovirus expressing full length human lysosomal acid lipase (Ad-hLAL) was a gift from Dr. Hong Du207 (Cincinnati Children’s Hospital Research Foundation) and adenovirus expressing GFP was a gift from Dr. Rene Jacobs215 (University of Alberta). Cholesteryl oleate, unesterified cholesterol, 1- monooleoyl-rac-glycerol, 1 ,2-distearoyl-rac--glycerol, triolein, oleic acid, used for the complete carrier, were purchased from Sigma-Aldrich. Phosphatidyicholine (PC) and sphingomyelin (SM), used for the PC/SM carrier, were purchased from Sigma-Aldrich. The PE-S1L G plastic backed flexible plates and LS 6500 Multi-Purpose Scintillation Counter used for thin-layer chromatography (TLC) analysis were purchased from Whatman and Beckman Coulter, respectively. 2.2 METHODS 2.2.1 Preparation ofLipoproteins Low density lipoproteins (LDL) were isolated by density gradient ultracentrifugation from plasma donated by fasting healthy donors216. Radiolabeling of LDL using [1,2,6,7-3H]- cholesteryl linoleate was performed as described 217, to a specific activity of 16 to 44 cpmlng of LDL protein. 2.2.2 Cell Culture Normal human skin fibroblasts were purchased from the American Type Culture Collection (Manassas, VA). Cholesteryl ester storage disease (CESD) human skin fibroblasts were obtained via skin biopsy from an adult patient of Dr. Gordon Francis (CESD1)(University of British Columbia). Genetic sequencing of CESD1, completed in the laboratory of Dr. Robert Hegele (Robarts Research Institute, London, ON) indicated a heterozygous splicing mutation (Q277), which leads to a G-to-A change at the last nucleotide of exon 8, in one allele. The second allele 40 did not contain any previously described LAL mutations, however, it is assumed that there is a disease causing mutation in the second allele, as the patient exhibits a classic histologic phenotype of CESD based on prior liver biopsy. A second CESD human skin fibroblast cell line from an adult patient was generously provided by Dr. John S. Parks (CESD2)(Wake Forest University School of Medicine)218.CESD2 is known to have the common E8SJM mutation which leads to excision of exon 8, in conjunction with a CT deletion in exon 4 which leads to a frameshift four codons downstream at codon 137 (c.397-398delCT; FS 1 37X)218 The fibroblasts from the subjects with cholesteryl ester storage disease have been shown to be deficient in lysosomal acid lipase activity. All cells were grown in monolayers and were used between the 5th and 25th passage. Cell lines were maintained in a humidified incubator (5% C02) at 37°C in 75- cm2 stock flasks containing 10 ml of growth medium consisting of DMEM containing 10% FBS, supplemented with penicillin (50 units/mi). Cells were plated at 75,000 cells/35-mrn well or 150,000 cells/60-mm dish and grown to approximately 60% confluence in DMEM containing 10% FBS. Cells were subsequently grown to 100% confluence in DMEM containing 10% LPDS’41. To load cells with low density lipoprotein (LDL) cholesterol, confluent cells were rinsed twice with phosphate-buffered saline (PBS) containing 1mg/mi fatty acid free albumin (FAFA) and incubated for 24hr in DMEM containing 50 jig/mI LDL. 2.2.3 Labeling of Cellular Cholesterol Pools and Phospholipids Radiolabeling LDL-derived cellular cholesterol pools involved incubating fibroblasts in DMEM containing 10% FBS until cells reached 60% confluence, and were subsequently incubated in DMEM containing 10% LPDS until fibrobiasts were confluent, encouraging the up-regulation of LDL receptor expression. Following a 4hr adenoviral infection if required, the fibroblasts were then incubated for 24hr with 50jig/mI[3H]-cholesteryl linoleate-labeled LDL. Cells were then rinsed 2 times with PBS containing 1mg/mi FAFA prior to addition of apoA-I. Choline-containing phosphoiipids were labeled in cells for 24hr in the presence of 50jig/ml LDL by addition of 5j.tCi/ml[3H]-choline chloride to the DMEM medium during a 24hr incubation period 140, 141 Cells were rinsed 5 times with PBS/FAFA prior to addition of lOjig/mi ApoA-I. 2.2.4 Adenoviral Delivery to Normal and CESD Fibroblasts Cells were seeded at 75,000 cells/35-mm well or 150,000 cells/60-mm dish and grown to approximately 60% confluence in DMEM containing 10% FBS. Cells were subsequently grown to 100% confluence in DMEM containing 10% LPDS. After reaching confluence, cells were washed two times with warm DMEM and incubated with 600 jiL or 1000 jiL of Optimem media 41 in 35-mm wells or 60-mm dishes, respectively. Fibroblasts were subsequently incubated with a multiplicity of infection (MOl) of 600 of adenovirus expressing full-length GFP (Ad-GFP) as a control or adenovirus expressing full-length human lysosomal acid lipase (Ad-hLAL) in the presence of 1.4% Lipofectamine 2000 for 4hr at 37°C. Varying MOIs and adenoviral incubations were utilized to optimize the delivery of adenovirus. For the first 2hr of infection the dishes or plates were rocked gently every 30mins to effectively disperse the adenovirus. Following infection period cells were incubated for 24hr in DMEM containing 50 jig/mi LDL. Concentration of LDL were determined by Lowry assay, based on protein content219. 2.2.5 Lysosomal Acid Lipase Activity Assay Fibroblasts were seeded and infected as previously described. Following infection, cells were harvested by trypsinization, washed three times in PBS, and suspended in 5 to 10 volumes of distilled water. Cells were then sonicated in an ice water bath for 30 seconds with an Ultrasonic Cleaner FS6O (Fisher Scientific) and centrifuged at 21,000 x g for 30mins at 4°C. The supernatant was collected and stored at -80°C until ready for assay. The hLAL activities were estimated with the fluorogenic substrate 4-methylumbelliferyl oleate (4-MUO), which fluoresces following lipase induced ester hydrolysis, releasing the fluorophore and forming 4- methylumbelliferone 157, 220 A solution of 25 mmol of 4-MUO in hexane and 40 mmol of L-cL phosphatidylcholine in chloroform were evaporated under N2, and subsequently resuspended in 2.4 mM sodium taurodeoxycholate and sonicated at SOW using ice-bath Ultrasonic Cleaner FS6O (Fisher Scientific). The resultant homogeneous dispersions were used as the substrates. Under the assay conditions, 50 jil of 4-MUO/L-a-phosphatidylcholine liposome substrate were added to 400j.d of 0.2 M sodium acetate at pH 5.5 (pH of lysosomal compartment), incubated at 37°C with 20j.tg cell supernatant (quantitated by Biorad assay) and recorded fluorescence intensity at an excitation wavelength of 335nm and an emission wavelength of 455nm using the Safire2 plate reader (Tecan). 2.2.6 LDL Cholesterol Efflux Following the labeling protocol, cells were incubated for 24hr in DMEM containing 10 jig/nil apoA-I. At the completion of the indicated incubation period, effluxed media were collected and centrifuged at 3,000 rpm for lOmins at 4°C to remove cell debris. Radioactivity in the medium was then measured directly (for cells labeled with[3H]-cholesteryl linoleate-labelled LDL). Cells were rinsed twice with iced PBS/FAFA and twice with iced PBS. Cells were stored at -20°C until lipid extraction. Extracted cellular lipids were separated by thin-layer chromatography (TLC) 42 and assayed for radioactivity as previously described 123 Cell samples were incubated with lmlI35mrn well Hexane:isopropanol (3:2, v/v) for 30mins at room temperature for extraction of cellular lipids. Samples were then pipetted into glass test tubes, containing 20u1/tube complete carrier, using borosilicate glass Pasteur pipets (VWR). Complete carrier, which is composed of cholesteryl oleate, cholesterol, 1 -monooleoyl-rac-glycerol, 1 ,2-distearoyl-rac-glycerol, triolein and oleic acid, functions as a marker used to identify lipids of interest following separation of sterols during TLC. Sample extracts were subsequently dried down on a Dry Bath incubator (Fisher Scientific). Extracts were then resuspended in 1 lOj.tL chloroform spotted onto PE-SIL G plastic backed flexible plates (Whatman). TLC tank was equilibriated with a Hexane:Diethyl Ether:Acetic Acid mixture (130:40:1.5, vlv/v), and following separation of sterols, lipid spots were obtained using an iodine chamber. Radioactivity was measured using LS 6500 Multi Purpose Scintillation Counter (Beckman Coulter). Protein content of extracted cell layers was collected using 0.lM NaOH and determined using a Lowry assay and bovine serum albumin as standard 219 2.2.7 Phospholipid Efflux Following the labeling protocol, cells were incubated for 24hr in DMEM containing 10 jig/mI apoA-I. At the completion of the indicated incubation period, medium was collected and centrifuged at 2000 rpm for lOmins at 4°C and cells were rinsed twice with iced PBSIFAFA and twice with iced PBS. Cells were then stored at -20°C until lipid extraction. Centrifuged media samples were then collected in screw-top tubes (1 3x 100 with Teflon liners) that contained 2OjiL of PC/SM carrier, to which 4mL of Folch mixture (chloroform:Methanol, 2:1) was added. Samples were subsequently vortexed and centrifuged at 2000 rpm for lOmins at 4°C and the upper aqueous phase was aspirated off. A imi aliquot of dH2O was then added and tubes were inverted gently, followed by centrifugation at 2000 rpm for lOmins at 4°C. The addition of a imi aliquot of dH2O was repeated, and the top aqueous phase was aspirated off and the lower phase was dried down. Cell samples were then incubated with 2mL/35mm well with Hexane:Isopropanol and incubated for 30mins at room temperature. After the incubation, 2OjiL/tube of PC/SM carrier was added into each glass test tube (l3xlOOmm) followed by the lipid extracts. The PC/SM carrier, which contains phosphatidylcholine (PC) and sphingomyelin (SM), functions as a marker used to identify phospholipids of interest following separation during TLC. The TLC chambers were then equilibrated with filter paper for 2hr with chloroform:methanol: acetic acid: water mixture (1 OOml: 60m1: 1 6m1: 6m1). The dried lipid extracts 43 were resuspended in 1 lOiiL of chloroform and 100 jiL/sample was spotted onto PE-SIL G plastic backed flexible plates (Whatman). The plates were then developed in the TLC chambers for approximately 2hr. Following separation of sterols, lipid spots were obtained using an iodine chamber. Radioactivity was then measured to determine levels of radiolabeled phosphatidyicholine and sphingomyelin 221,123 Protein content of extracted cell layers was collected using 0. 1M NaOH and determined using a Lowry assay and bovine serum albumin as standard 219 2.2.8 Real Time-PCR Analysis ofABCA1 mRNA Total RNA was isolated from cells using Trizol Reagent extraction (Invitrogen). The concentration of RNA was measured spectrophotometrically at a wavelength of 260nm, and 2 ig of RNA was treated with DNase I (Invitrogen) following the protocol outlined by the manufacturer. First strand eDNA synthesis was performed using 500 nM of oligo(dT) primer and RNa5eOUTTM Recombinant Ribonuclease Inhibitor (Invitrogen). The mixtures were incubated at 45 °C for 90mins followed by incubation at 95 °C for 3mins (Whatman Biometra T-gradient thermocycler) and then immediately stored on ice. Amplification of ABCAI and cyclophilin mRNAs was performed concurrently to ensure equivalent amounts of starting cDNA for each sample. Diethyl pyrocarbonate-treated water, lx PCR buffer (20mM Tris-HC1, pH 8.4, and 50mM KC1), 1.5 mM MgC12, 0.1 mM dNTPs, and cDNA were added to 200 1 thin walled PCR tubes and mixed, and one-half volume was transferred to another PCR tube. Subsequently, 1 unit of Taq DNA polymerase (Invitrogen) and 2 t1 of 10 .tM forward and reverse primers (ABCA1 or cyclophilin) were added to complete the reaction mixture. ABCA1 amplification was performed by initially denaturing DNA at 95 °C for 3mins. Thereafter, denaturing was at 95 °C for 20 seconds, annealing at 58 °C for 20 seconds, and extension at 72 °C for 40 seconds for at total of 40 cycles 141 SYBR Green was used to detect and quantitate the PCR products from quantitative RT-PCR. SYBR Green binds double-stranded DNA, and upon excitation emits light. Thus, as a PCR product accumulates, fluorescence increases. Human cyclophilin amplification was performed using the same conditions. The reactions were completed using the Realplex2 Mastercycler (Eppendorf). The primers used are as follows: human ABCA1, 5’-GAC ATC CTG AAG CCA ATC CTG (forward), 5’-CCT TGT GGC TGG AGT GTC AGG T (reverse); human cyclophilin, 5’- ACC CAA AGG GAA CTG CAG CGA GAG C (forward), 5’-CCG CGT CTC CTT TGA GCT GTT TGC AG (reverse). The real-time PCR analysis was completed by Leanne Bilawchuk, a technician in the Francis laboratory. 44 2.2.9 Western Blot Analysis Cells in 60-mm dishes were harvested with 300iiL of extraction buffer containing 20mM Tris, 5mM EDTA, 5mM EGTA, 0.5% Maltoside and lx Complete Mini Protease Inhibitor (Roche). Cells were scraped using disposable Cell Lifters (Fisher Scientific) and homogenized with a Teflon pestle. Samples were then centrifuged at 2500 rpm for lOmins at 4°C to pellet nucleic acids. An aliquot of the supematant was subsequently used for BioRad protein assay. Samples to be used for human LAL (hLAL) analysis were boiled for 5mins at 100°C. Fifteen to thirty micrograms of proteins were separated by a 5% and 15% gradient SDS-PAGE under reducing conditions and transferred to nitrocellulose membrane. Immunoblotting was performed according to standard protocols using polyclonal rabbit anti-human ABCA 1 antibody (1:1000 dilution)(a gift from Dr. Shinji Yokoyama, Nagoya City University222),a polyclonal rabbit anti hLAL antibody (1:2000 dilution, Seven Hills Bioreagents) and a goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (1:10,000 dilution, Sigma). Chemiluminescence was detected using Super Signal West Femto Maximum Sensitivity Substrate (Pierce Protein Research Products) and the Chemigenius Biolmaging System (Syngene). 2.2.10 Cholesterol Mass Assay Cells were seeded in 6-well plates, grown to confluence and incubated with 50 jig/mI LDL for 24hr, as previously described. After the incubation, cells were washed twice with PBS/lmg/ml FAFA and incubated 24hr in the presence of lOjig/mi ApoA-I. After incubation, the media was collected and cells were washed twice with iced PBS/lmg/ml FAFA and twice with iced PBS. After the wash steps, 2m1 PBS was added to the wells and cells were scraped using disposable Cell Lifters (Fisher Scientific), collected and homogenized by sonication. Medium samples were centrifuged at 3000 rpm for lOmins at 4°C and transferred to a 15m1 glass test tube. Phospholipids from media or cell homogenates were digested using phospholipase C to remove the polar head groups, and samples were subsequently vortexed for 2hr at 30°C. Total lipids were extracted in the presence of tridecanoin as the internal standard. Organic and aqueous phase where then separated by spinning at 2000 rpm for lOmins at 4°C, and the upper phase removed by vaccum. Next, 3m1 of theoretical upper phase (methanol/water/chloroform/acetic acid; 480:470:30:9.6), was added, the sample vortexed, centrifuged at 2000 rpm for lOmins at 4°C, and the upper phase was aspirated off. The addition of the theoretical upper phase was repeated once more, and subsequently passed the lower layer phase through a Pasteur pipette column charged with anhydrous sodium sulfate, and collected samples. Samples were derivatized with Sylon BFT (Supelco) and analyzed by gas chromatography (Agilent Technologies, 6890 Series equipped 45 with a Zebron capillary column (ZB-5, 15 m x 0.32 mm x 0.25 jim) and connected to a flame ionization detector; Zebron, Palo Alto, CA). The oven temperature was raised from 170 to 290°C at 20 °C/min, and then to 340 °C at 10°C/mm where the temperature was kept for 24mins. Helium was used as the carrier gas. The gas chromatography was operated in constant flow mode with a flow rate of 4.5 ml of helium/mm. The injector was operated in the split mode and was kept at 325°C, and the detector was kept at 350 °C 223 Separation of sterols was identified by comparing their retention times with standards, and calculation of sterol mass in samples was based on the internal standard. 2.2.11 Statistical Analysis Results are expressed as mean ± S.D. Significant differences between experimental groups were determined using one-way analysis of variance (ANOVA) with Tukey’s HSD as a post-hoc test for pairwise comparisons. 46 Lfr sriiisrn: 3.1 Reduced ABCA1 mRNA Response to LDL Loading in CESD Fibroblasts As discussed previously, the ATP Binding Cassette Transporter Al (ABCA1) is required for the lipidation of non-lipidated or lipid-poor apolipoprotein A-I (apoA-I), and as such is a critical determinant of HDL particle formation73.Therefore, the formation of nascent HDL particles is dependent on adequate expression of ABCA1 to transfer intracellular unesterified cholesterol (UC), as well as phospholipids, to apoA-I. Based on the Francis laboratories’ previous finding of impaired ABCA 1 regulation in the lysosomal cholesterol storage disease Niemann-Pick Disease Type C (NPC)140, we hypothesized that the low plasma HDL-cholesterol (HDL-C) levels observed in cholesteryl ester storage disease (CESD) patients is a result of dysregulation of ABCA1. In order to determine whether impaired regulation of ABCA1 expression occurs at a transcriptional level, quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed on mRNA samples from two normal (NL1, NL2) and two CESD (CD1, CD2) human skin fibroblast cell lines. Cells were initially incubated in lipoprotein deficient serum (LPDS) until reaching confluence, and were subsequently incubated under non-cholesterol-loaded or low density lipoprotein (LDL)-loaded conditions for 24hr. In order to specifically examine the role of the lysosomal cholesterol pooi in the regulation of ABCA1 expression in CESD cells, the fibroblasts were incubated with LDL, which is taken up via receptor-mediated endocytosis and requires lysosomal acid lipase (LAL) for hydrolysis. The levels of ABCA1 mRNA were determined under non-LDL-loaded and LDL-loaded conditions, and the fold increase in ABCA1 mRNA observed following incubation with LDL was calculated and corrected for a housekeeping gene, cyclophilin. In normal fibroblasts, NL1 and NL2, there was an 8.9 ± 0.07- fold and 9.4 ± 0.58-fold increase in ABCA1 mRNA, respectively, following addition of LDL (Figure 3.1). These results are consistent with previous findings from our lab showing LDL derived unesterified cholesterol functions as a regulator of ABCA1 expression at the mRNA level in human fibroblasts’40.Conversely, the CESD fibroblasts displayed a significant reduction in their ability to upregulate ABCA1 mRNA levels following incubation with LDL. The CD1 cell line exhibited a 1.9 ± 0.51-fold increase in ABCA1 mRNA, whereas the CD2 cell line exhibited a 3.0 ± 0.31-fold increase (Figure 3.1). These results suggest that the CESD mutation, resulting in a significant depletion of active LAL, a massive accumulation of cholesteryl esters, and a reduced flux of unesterified cholesterol from the lysosome, leads to a reduced ability of the diseased fibroblasts to upregulate ABCA1 following endocytosis of LDL. The modest increases that are observed in the CESD fibroblasts are likely due to the known residual lipase activity 48 within the lysosomal compartment, and slowed but not total loss of release of UC from this compartment’37. 49 0 ci) I.(I) ci) 0-.. OE U0 —J- 0 4-, (0D CUG) -I-,wo I 0 20 LL - C-) Figure 3.1 Reduced ABCA1 mRNA response to LDL (LDL-C)-loading in human CESD fibroblasts compared to normal fibroblasts. Confluent normal (NL1, NL2) and CESD (CD 1, CD2) fibroblasts were incubated in the absence or presence of 50 igIm1 LDL for 24hr, and cell mRNA was isolated for determination of ABCA1 expression. Quantitative reverse transcription PCR results are displayed as a fold increase of ABCA1 mRNA with addition of LDL, relative to the non-LDL condition, corrected for cyclophilin. ABCA1 mRNA fold increase after LDL-loading is reduced in CD 1 and CD2 fibroblasts relative to normal (NL1, NL2) fibroblasts. Results are the mean ± S.D. of triplicate determinations and are representative of three experiments with similar results. 12 10 8 6 4 2 0 NL1 NL2 CD1 CD2 Cell Line 50 3.2 Reduced ABCA1 Protein Levels in CESD Fibroblasts In order to confirm whether reduced ABCA1 mRNA levels in response to LDL loading in CESD cells is also associated with reduced ABCA1 protein levels, Western blot analysis was performed. In the normal human fibroblasts, NL1 and NL2, there was a significant upregulation in ABCA1 protein levels following incubation with LDL, which corresponded to an increase of 1 to 2.4 ± 0.15 (2.4 ± 0.15-fold) and 0.5 ± 0.05 to 2 ± 0.05 (4 ± 0.12-fold) in arbitrary densitometry units, respectively (Figure 3.2A,B), with all bands normalized to the density of the non-LDL loaded ABCA1 band in NL1. However, similar to the reduced ABCA1 expression observed in NPC disease fibroblasts following cholesterol loading’40,CESD human fibroblasts displayed a marked reduction in their ability to upregulate ABCA1 protein following LDL-loading. Although similar basal ABCA1 protein levels were observed between normal and CESD fibroblasts, following incubation with LDL, CD1 and CD2 increased from 0.64 ± 0.13 to 1.1 ± 0.13 (1.7 ± 0.32-fold) and from 0.8 ± 0.02 to 1.5 ± 0.16 (1.8 ± 0.13-fold) in arbitrary densitometry units, respectively (Figure 3.2A,B). The greater difference in mRNA response to LDL-loading between normal and CESD cells, compared to the response observed at a protein level, may be due to differences in post-transcriptional regulation. The increases in ABCA1 protein, following addition of LDL, in the CESD cells were approximately 40 to 50% that of the ABCA1 expression observed in the normal fibroblasts. These results suggest that the significant reduction in LAL activity, known to be present in all CESD patients and responsible for the accumulation of cholesteryl esters within the lysosome, is a factor resulting in dysregulation of ABCA1 in these cells. Importantly, the impaired upregulation of ABCA1 in the CESD fibroblasts may be the cause of the low plasma HDL-C levels observed in CESD patients64. 51 .1-’ D S 0 C’, 0 C-) Figure 3.2 (A) Inhibited upregulation of ABCA1 protein in response LDL loading in human CESD fibroblasts compared to normal fibroblasts. Confluent normal (NL1, NL2) and CESD (CD1, CD2) fibroblasts were incubated in the absence or presence of 50 .tg/m1 LDL for 24hr, and protein isolated for determination of ABCA1 expression. Results are representative of four experiments with similar results. Numeric densitometry values represent the intensities of (B) ABCA1 normalized to non-LDL-loaded NL1 ABCA1 band. The loading control used for Western analysis was protein disulfide isomerase (PDI). , p<O.OO1, ABCA1 protein increase after LDL loading is inhibited in CESD1 (CD1) and CESD2 (CD2) fibroblasts compared to normal (NL1, NL2) fibroblasts. Results are the mean ± S.D. of four experiments with similar results. A. LDLChoI Nil (—) NLI (+) NL2 {.-) ABCAI NL2 (.1.) CDI (4 CDI (4.) CD2 (—) PD, CD2 (4) B. 3.0 2.5 2.0 1.5 1.0 0.5 0.0 NLI NL2 CDI CD2 Cell Line Non-LDL Cholesterol Loaded LEEI LDL Cholesterol Loaded 52 3.3 Impaired ApoA-I-dependent Cholesterol Efflux in CESD Fibroblasts In order to investigate the functional consequences of the impaired ABCA1 mRNA and protein responses to LDL-loading observed in CESD fibroblasts (Figure 3.1, 3.2), we conducted cholesterol efflux studies using[3H1-cholesteryl linoleate-labeled LDL as a substrate. Following incubation with the radiolabelled LDL, the cells were exposed to apoA-I for 1 to 48hr. As expected, the two CESD cell lines showed a marked reduction in their ability to release LDL derived UC to apoA-I compared to the two normal cell lines (Figure 3.3A). NL1 and NL2 released 23.6 ± 2.70% and 18.8 ± 0.69% of total cell plus medium[3H]-sterols to the medium after 481w, respectively. Conversely, CD1 and CD2 released only 7.4 ± 0.67% and 10.3 ± 1.04% of their total[3H]-sterols after 481w, respectively. CD 1 released 3.1 ± 0.21- and 2.5 ± 0.13-fold less LDL-derived UC than NL1 and NL2 after 481w, respectively, while CD2 released 2.3 ± 0.22- and 1.8 ± 0.14-fold less. In addition, cholesteryl ester (CE) accumulation was measured in normal and mutant fibroblasts. As expected, CD1 and CD2 exhibited a significant accumulation of CEs, initially with 85.2 ± 1.38% and 79.5 ± 0.98% of total[3H]-sterol being in CE form, respectively, which was depleted to 65.9 ± 1.38% and 57.9 ± 0.14% after 481w, respectively (Figure 3.3B). In contrast, NL1 and NL2 initially had 35.1 ± 0.98% and 44.25 ± 1.12% of total [3H]-sterol in CE form, respectively, which gradually decreases during the 48 hr time course incubation with apoA-I to 24.8 ± 3.16% and 36.1 ± 0.83% of total[3H]-sterol, respectively (Figure 3.3B). Therefore, CD1 initially accumulated 2.4 ± 0.05- and 1.9 ± 0.04-fold more LDL derived CE than NL1 and NL2, respectively, which changed increased to 2.7 ± 0.15- and 2.3 ± 0.13-fold, respectively, after 481w. CD2 initially accumulated 1.9 ± 0.04- and 1.8 ± 0.03-fold more LDL-derived CE than NL1 and NL2, respectively, which changed to 1.8 ± 0.04- and 1.6 ± 0.03-fold, respectively, after 48hr (Figure 3.3B). The cellular LDL-derived UC was shown to represent 63.2 ± 1.18% and 55.1 ± 1.09% of total[3H1-sterol in NL1 and NL2, respectively, and gradually decreased to 46.2 ± 6.87% and 45.1 ± 0.13% after a 481w incubation with apoA-I, respectively (Figure 3.3C). As expected, CD1 and CD2 displayed a reduced pooi of LDL-derived UC compared to the normal fibroblasts, with 14.3 ± 1.39% and 19.8 ± 0.78% of the total [3H]- sterol being in the UC form, respectively, but gradually increased to 26.6 ± 0.73% and 31.8 ± 1.14% after 48 hr incubation with apoA-I, respectively (Figure 3.3C). Initially, CD1 displayed 4.4 ± 0.11- and 3.1 ± 0.12-fold less cellular LDL-derived UC than NLI and NL2, respectively, while CD2 exhibited 3.9 ± 0.06- and 2.8 ± 0.06-fold less. However, after a 481w incubation with apoA-I, CD1 displayed 1.7 ± 0.17- and 1.5 ± 0.04-fold less cellular LDL-derived UC than NL1 and NL2, respectively, while CD2 exhibited 1.7 ± 0.15- and 1.4 ± 0.02-fold less (Figure 3.3C). 53 The gradual decrease in cellular LDL-derived CE and increase in LDL-derived UC is thought to be due to the residual LAL activity observed in CESD patient fibroblasts64’137 The impaired function of ABCA1 observed in CESD fibroblasts were supported by results showing that CESD fibroblasts released significantly less cholesterol following incubation with apoA-I, compared to normal fibroblasts. The experiment involved the same protocol as discussed above, however instead of a timecourse fibroblasts were incubated with apoA-I for a single 24hrs time point. CD1 and CD2 released 5.8 ± 0.64% and 10.6 ± 0.25% of their total [3H] sterol following 24hr incubation in apoA-I containing media, compared to NL1 and NL2, which released 19.7 ± 0.12% and 24.4 ± 0.42% of their total {3H] sterol, respectively, to apoA-I containing media (Figure 3.3D). In addition, CD1 and CD2 also exhibited accumulation of CE and reduced cellular LDL-derived UC, compared to normal fibroblasts (Figure 3.3E,F). Taken together, these results indicate that the LAL mutations found in CD 1 and CD2 fibroblasts are capable of reducing the activity of the enzyme sufficiently to significantly reduce release of UC for eventual efflux to apoA-I, produce a massive CE accumulation within the lysosomes, and to reduce the amount of intracellular LDL-derived UC. The results also suggest, in conjunction with the reduced ABCA1 niRNA and protein in CESD fibroblasts, that the reduced release of UC to apoA-I is caused by impaired ABCA1 expression and therefore activity. 54 Figure 3.3 Impaired cholesterol efflux to apoA-I in CESD fibroblasts. (A) ApoA-I-mediated efflux of[3H] LDL derived cholesterol from human normal (NL1, NL2) and CESD (CD 1, CD2) fibroblasts for 1 — 48 hr (B) LDL derived[3HJ-cholesteryl esters after incubation with apoA-I for 1 to 48 hr and (C) Cellular LDL-derived {3H]- unesterified cholesterol levels after incubation with apoA-I for 1 to 48hr. (D) Reduced efflux of LDL-derived unesterified cholesterol (UC) to apoA-I containing media from CESD fibroblasts at 24hr and (E) elevated LDL derived cellular cholesteryl esters (CE) in CESD fibroblasts at 24hr and (F) Reduced LDL-derived cellular UC in CESD fibroblasts at 24hr. Confluent normal (NL1, NL2) and CESD fibroblasts from two unrelated CESD patients (CD1, CD2) were incubated with 50 jtg/ml[3H]-cholesteryl linoleate-labeled LDL for 241w, followed by a ito 481w or a 241w incubation in the presence ((+) ApoA-I) or absence ((-) ApoA-I) of 10 .tg/ml ApoA-I. Efflux results are expressed as percent of total medium plus cell[3H]-cholesterol in the medium.[3H]-cholesteryl esters are expressed as percent of total medium plus cell[3H]-cholesterol in cell CE.[3H]-unesterified cholesterol are expressed as percent of total medium plus cell[3H]-cholesterol in cell. Mean ± S.D. of three determinations and representative of 3 experiments. , p < 0.00 1 relative to normal fibroblasts. 0 0 Cl) Co 0 I— 0 10 20 30 40 Time (hours) 0 10 20 30 40 50 60 Time (hours) B. Cellular CE 0 a) Cl) I Co 0 I— 100 - 80 - 60 - 40 - 20 - 0- —— NLI —0— NL2 —v-- CDI CD2 A. Media UC 30 - __________ 25 - 20 - 15 - 10 - 5- 0- 0 10 20 30 40 50 60 Time (hours) C. Cellular UC 80 60 0 I G) C’) zL_ 40 0 I— —— NL1 —0— NL2 —— CDI —..— CD2 20 0 50 60 55 9c auflio I -vodv(+) L -vodvC-)— aufliio O1N[iN 003[03[iN 003[03O1N[iN -10 0) C,, CD 0 017 09 09 i-vodv)+) i-vodvC-)— euriieo a0 6’ 0) I Co CD 0 00[ 311IflhT3I i-vodvC+)rzz i-vodvC-)— DO 6’ 0) I Co CD 0 3flnipapu J3JflfflJfO3•J 3.4 Impaired Phospholipid Efflux to ApoA-I from CESD Fibroblasts As indicated above, fibroblasts from Tangier disease patients were found to have a marked defect in efflux of cholesterol and phospholipids to apoA-I, suggesting that ABCA1 mediates or regulates the efflux of cellular cholesterol and phospholipids to the acceptor129’2 45 The ability of apoA-I to function as a cholesterol acceptor is believed to be dependent upon the initial or simultaneous phospholipidation by ABCA1, as apoA-I cannot solubilize cholesterol on its own224. To investigate ABCA 1-dependent phospholipid efflux in CESD fibroblasts, we utilized [3H]-choline to label the two major phospholipids present in HDL, phosphatidylcholine (PC) and sphingomyelin (SM), followed by efflux to apoA-L After a 24hr incubation with apoA-I, NL1 and NL2 released 8.75 ± 0.09% and 9.72 ± 0.22% of their total[3H]-PC, respectively (Figure 3.4A). The CESD fibroblasts, CD1 and CD2, showed a reduced ability to release PC to apoA-I, liberating only 3.08 ± 0.04% and 6.37 ± 0.06% of their total[3H]-PC, respectively (Figure 3.4A). CD1 released 2.84 ± 0.02- and 3.1 ± 0.03-fold less of their total[3H]-PC than NL1 and NL2, respectively, while CD2 released 1.4 ± 0.02- and 1.5 ± 0.03-fold less, respectively Figure 3.4A). With regards to SM, NL1 and NL2 released 7.02 ± 0.36% and 7.78 ± 0.05% of their total [3H]-SM, respectively (Figure 3.4C). CD1 and CD2 demonstrated a reduced ability to efflux SM to apoA-I containing media, releasing 4.31 ± 0.56% and 5.41 ± 0.25% of their total[3H]-SM, respectively (Figure 3.4C). CD1 released 1.6 ± 0.18- and 1.8 ± 0.14-fold less of their total [3H]- SM than NL1 and NL2, respectively, while CD2 released 1.3 ± 0.09- and 1.4 ± 0.05-fold less, respectively (Figure 3.4C). Although the reduction in PC and SM release to apoA-I were not as striking as the differences in cholesterol efflux when compared to normal fibroblasts, they were statistically significant. These results support the observations from the cholesterol efflux study (Figure 3.3), which indicated that the impaired regulation of ABCA1 mRNA and protein in the CESD fibroblasts (Figure 3.1, 3.2) translates to a functional dysregulation of ABCA 1. These results also support the concept of the lysosome as a major source of the ABCA1 cholesterol substrate pooi, whereas the drop in phospholipid efflux from CESD cells is more likely mains related to the reduced ABCA1 activity. 57 Figure 3.4 Reduced phosphatidylcholine (PC) and sphingomyelin (SM) efflux to apoA-I containing media (A, C) and accumulation of cellular PC and SM (B, D) in CESD fibroblasts.Confluent normal (NLI, N2) and CESD (CD 1, CD2) fibroblasts were incubated in the absence or presence of 5 p.Ci/ml[3H]-choline and 50 igIml LDL for 24hr, followed by a 24 hr incubation in the presence ((+) ApoA-I) or absence ((-) ApoA-I) of 10 ig/ml ApoA-I. Results are expressed as percent of total cell plus medium[3H]-PC or -SM in the medium (A and B, respectively), cell PC (C), and cell SM (D). Mean ± S.D. of three determinations and representative of 3 experiments. , p < 0.05 relative to normal fibroblasts. A. Media PC B. Cellular PC 14 12 10 C) I— 4 2 Cr, (I) 0 I C) 0 I.— Cell Line Cell Line (-)ApoA-l (-)ApoA-I (-I-) ApoA-l 2 (i) ApoA-I NL1 NL2 CD1 CD2 C. Media SM 10 D. Cellular SM 8 T : lu2 NL1 NL2 COl CD2 Cell Line (-I ApoA-I (*)ApoA.4 NLI NL2 CD1 CD2 Cell Line — l-(ApOA-I (.)ApoA-I 58 3.5 Reduced Cholesterol Mass Efflux to ApoA-I From CESD Fibroblasts In order to further investigate ABCA1 function in CESD fibroblasts, we conducted analysis of changes in cell and medium cholesterol mass in response to apoA-I incubation in LDL-loaded normal and CESD fibroblasts. To determine total cellular CE and cellular and medium UC mass in normal and CESD cells, we utilized gas chromatography (GC) to quantitate micrograms (jig) of UC or CE per milligram (mg) of cell protein. Similar to the functional cholesterol efflux study using[3H]-cholesteryl linoleate-labeled LDL, the medium showed a significant reduction in the mass of UC removed by apoA-I in the medium of CESD fibroblasts compared to the normal fibroblasts. The CESD cells released 1.6 ± 0.85 jig UC per mg of cell protein, a 4.0 ± 0.7-fold reduction compared to the 6.4 ± 1.1 jig UC per mg of cell protein observed in the normal fibroblasts apoA-I containing media (Figure 3.5A). In addition, the CESD fibroblasts also displayed a cellular CE mass of 35 ± 14.01 ug/mg cell protein, a significantly greater accumulation than the 6.3 ± 1.9 ug/mg cell protein observed in the normal fibroblasts (Figure 3.5B). These values represent a 5.5 ± 0.7-fold greater accumulation of CE in the CESD fibroblasts relative to the normal cells. Although the medium UC and cellular CE values corroborated the cholesterol efflux studies using radiolabelled LDL, the cellular UC levels were found to be dissimilar from those observed in the radiolabeled cholesterol efflux studies. Although the results from the radiolabel studies showed a reduced level of cellular LDL-derived UC compared to the normal fibroblasts, the mass assay using GC analysis indicated that there were no significant differences in total cellular UC content between the mutant and normal fibroblasts (Figure 3 .5C). The rationale for this result is that with radiolabel studies we quantitate LDL-derived labeled UC, as opposed to measuring total cell UC, which can originate from de novo synthesis or exogenous sources, and which the cell normally keeps at a constant level. 59 Figure 3.5 Reduced release of UC to apoA-I and CE accumulation in CESD fibroblasts. (A) Mass of LDL derived cholesterol released to apoA-I containing media from human normal and CESD fibroblasts. (B) Total cholesteryl ester mass in human normal and CESD fibroblasts. (C) Total cellular unesterified cholesterol mass in human normal and CESD fibroblasts. Confluent normal (NL 1, NL2) and CESD fibroblasts from three unrelated CESD patients (CD 1, CD2, CD3) were incubated with 50 .tg/ml unlabeled LDL for 24hr, followed by a 24 hr incubation with 10 j.ig/ml apoA-I. Cellular unesterified and esterified cholesterol and media unesterified cholesterol are expressed as .tg cholesterol/mg cell protein by gas chromatography. Results are mean ± S.D. of the indicated number of determinations. , p <0.001 relative to normal fibroblasts. p <0.005 relative to normal fibroblasts. A. Media UC = U) 0 0 U) C) 2 0) ii CU 0 U) C,) U) 0 0 0 U) U) a) U) = D B. Cellular CE C. Cellular UC C U) 0 0 U) 60 50 40 30 20 10 Normal (n4) CESD (n6) Normal (rm4) CESO (rr6) 70 60 50 40 30 20 10 0 Normal (ri4) CESD (n6) 60 3.6 LXR Agonist Upregulates ABCA1 mRNA and Protein in CESD Fibroblasts Previous studies from the Francis laboratory, using NPC disease human fibroblasts, have demonstrated low basal and cholesterol-stimulated levels of ABCA1 mRNA140,consistent with impaired oxysterol- and LXR-target gene regulation in this disease226. It was also found that addition of an exogenous non-oxysterol LXR agonist (TO-9013 17) resulted in increased ABCA1 protein levels in NPC i’ fibroblasts similar to those observed in cholesterol-loaded normal fibroblasts’41.Similar to NPC disease, we have found that fibroblasts from CESD patients also demonstrate reduced ABCA1 mRNA and protein levels following addition of a sterol substrate, in this case LDL. In order to investigate whether the addition of an LXR agonist would be able to correct the reduced ABCA1 rnRNA levels observed in CESD fibroblasts following LDL-loading, we also utilized the non-oxysterol LXR agonist TO-901317. Using quantitative RT-PCR, we found that following a 24hr incubation with TO-901317, both normal and CESD fibroblasts demonstrated increases in ABCA1 mRNA expression. The normal fibroblasts, NL1 and NL2, displayed a 14.5 ± 0.57-fold and 16.2 ± 0.09-fold increases in ABCA1 expression after incubation with TO-901317, respectively (Figure 3.6A). CD1 and CD2, which demonstrated reduced ABCA1 expression after addition of LDL compared to normals, had a 10.0 ± 0.52-fold and 11.0 ± 0.42-fold increase in expression following incubation with TO-901317, respectively, up to, and above, levels of ABCA1 mRNA like those seen in unstimulated but LDL-loaded normal fibroblasts (Figure 3.6A). In order to confirm whether increased ABCA1 mRNA levels in response to TO-9013l7 in CESD cells was also associated with increased ABCA1 protein levels, we again performed Western blot analysis of ABCA1. Using the same protocol of LDL-loading with and without TO-90 1317 for 24hr, we found that in normal fibroblasts the LXR agonist upregulated ABCA1 protein levels 2.7 ± 0.68-fold and 1.7 ± 0.37-fold in NL1 and NL2 fibroblasts, respectively (Figure 3.6B,C). Similarly, following CD1 and CD2 cells showed a 3.1 ± 0.82-fold and 2.7 ± 0.88-fold increase in ABCA1 protein levels, respectively (Figure 3.6B,C). Although incubation with an LXR agonist increased ABCA1 expression in both normal and CESD fibroblasts, these same conditions did not affect expression of lysosomal acid lipase. In the presence or absence of TO-9013 17, LAL protein levels did not change in normal or CESD fibroblasts (Figure 3.6B,D). These results suggest that transcriptional and translational regulation of ABCA1 is intact in CESD cells if an appropriate stimulus of ABCA1 expression is provided. These results also, therefore, suggest that oxysterol generation is impaired in CESD cells, which we expect to be the case due to the reduced flux of UC from the lysosomes, and has also already been demonstrated by Frolov et al.226 in NPC cells. 61 Figure 3.6 LXR agonist (TO-901317) upregulates ABCA1 mRNA and protein in normal and CESD fibroblasts.Confluent normal (NL1, NL2) and CESD (CD 1, CD2) fibroblasts were incubated for 4 hours in the presence of 1.4% Lipofectamine, followed by an additional 24 hour incubation with 50 jig/mi LDL, and for LXR agonist condition, 5 j.tM TO-901 317. (A) Cell mRNA was isolated for determination of ABCA1 expression. Real Time PCR results are displayed as a fold increase of ABCA1 mRNA with addition of LDL, compared to non-LDL loaded condition, corrected for cyclophilin. Results are the mean ± S.D. of triplicate determinations and are representative of three experiments with similar results. For western blot analysis, protein was isolated for determination of ABCA1 expression. (B) Cells were probed for hLAL and ABCA1 expression by Western blotting. Results are representative of three experiments with similar results. Numeric densitometry values represent the intensities of (C) ABCAI normalized to non-LXR treated normal (NL1) ABCA1 band, and (D) hLAL protein normalized to normal (NL1) non-LXR treated fibroblasts. The loading control used for Western analysis was protein disulfide isomerase (PDI). Results are the mean ± S.D. of three experiments with similar results. A. C 0 0 C-) 20 15 10 5 0 Ni N2 CESD1 CESD2 Cell Line ____ (4-) LDL-C I I (+) LDL-C + LXR Agonist 62 B. NLI NL2 CDI CD2 LXR Agonist (•-) (+) (—) (+) (—) (+) (—) (+) hLAL ABCAI poi C. D. 1.6 I 1.4 P j [2 I1.0 U) U) 0.8 I 0 F FL1 LI (-) (+) (-) (i-) (_) (+) (-) (+) 0 — — — NL1 NL2 CD1 CD2 Nil NL2 COl CD2 Cell Line Cell Line 63 3.7 LXR Agonist Increased Phospholipid Efflux in CESD Fibroblasts Studies from the Francis laboratory have shown that the addition of the LXR agonist TO- 9013 17 was able to bypass the NPC mutation and correct apoA-I-mediated efflux of PC’41. To determine whether the increase in ABCA 1 expression with TO-90 1317 results in increased phospholipids efflux from CESD cells, we again performed studies using[3H]-choline to label PC and SM but in the presence or absence of the LXR agonist. In normal fibroblasts, addition of the LXR agonist increased release of total[3H]-PC to apoA-I from 12.3 ± 0.7% to 15.8 ± 0.54% and from 9.4 ± 0.25% to 12.9 ± 2.2% in NL1 and NL2, a fold increase of 1.3 ± 0.09 and 1.4 ± 0.2, respectively (Figure 3.7A). In CD1 and CD2, the LXR agonist increased release of total [3H]-PC to apoA-I from 5.7 ± 0.52% to 8.1 ± 0.58% and from 5.4 ± 0.29% to 9.1 ± 0.61%, respectively (Figure 3.7A). Although the increase in PC efflux was not elevated to levels comparable to normal fibroblasts, there was a fold increase of 1.4 ± 0.16 and 1.7 ± 0.10 in CD1 and CD2, respectively (Figure 3.7A). Therefore, although addition of an LXR agonist was able to increase release of PC to apoA-I in normal and CESD fibroblasts, the medium percentage total [3H]-PC was still impaired in the mutant cells. Similar to PC efflux, following addition of an LXR agonist, release of SM to apoA-I containing media was increased in both normal and CESD fibroblasts. In NL1 and NL2, release of total[3H]-SM to apoA-I increased from 12.94 ± 0.32% to 19.2 ± 0.16% and from 13.4 ± 0.52% to 20.3 ± 0.87% following addition of an LXR agonist, respectively, an increase of 1.5 ± 0.03-fold and 1.5 ± 0.08-fold in SM efflux, respectively (Figure 3 .7B). The CESD fibroblasts, following addition of the agonist, increased release of total [3H]- SM to apoA-I from 5.6 ± 0.38% to 11.4 ± 0.47% and from 7.3 ± 0.58% to 13.9 ± 1.1%, respectively, an increase of 2.0 ± 0.1-fold and 1.9 ± 0.16-fold in SM efflux, respectively (Figure 3.7B). However, as with release of PC to apoA-I, the increase in SM efflux following addition of the LXR agonist was not as great as that observed in normal cells. 64 Figure 3.7 Increased phospholipid efflux following addition of an LXR agonist (TO-901317) to normal and CESD fibroblast.Confluent normal (NL1, NL2) and CESD (CD1, CD2) fibroblasts were incubated in media containing 1.4% Lipofectamine for 4hr. Cells were subsequently incubated with 5 .tCi/ml[3H]-choline and 50 jig/ml LDL for 24hr, followed by a 24hr incubated with 10 ig/ml ApoA-I ((+) ApoA-I) or 10 jig/mi ApoA-I with 5 jiM TO-90 1317 (LXR Agonist + ApoA-I). Results are expressed as percent of total cell plus medium[3Hj-PC or -SM in the medium (A and B, respectively), cell PC (C), and cell SM (D). Mean ± S.D. of three determinations and representative of 3 experiments. , p < 0.005 relative to normal fibroblasts. p < 0.05 relative to normal fibroblasts. 20 15 C-) 0 10 Co 0 I Co I Co C I— A. Media PC — (+)ApoA-l LXR Agonist + ApoA-I Cl, I Co 0 I C. Cellular PC (+)ApoA-l LXRAgonist+ApoA-I C-) 0 CU 0 I NL1 NL2 CD1 CD2 NLI NL2 CD1 CD2 Cell Line Cell Line B. Media SM D. Cellular SM 30 25 20 15 10 5 0 NLI NL2 CD1 CD2 Cell Line — (+) ApoA-l LXRAgonist+ApoA-l NLI NL2 CEJ1 CD2 Cell Line — (+) ApoA-l LXR Agonist + AA-l 65 3.8 LXR Agonist Increased Cholesterol Efflux in CESD Fibroblasts To determine whether the increase in ABCA1 expression with the LXR agonist TO-901317 results in increased cholesterol efflux from CESD cells, we conducted a study using [3H]- cholesteryl linoleate-labeled LDL cholesterol as a substrate and measured cholesterol release to apoA-I in the absence and presence of TO-90 1317. The NL 1 cell line exhibited an increase in total[3H]-sterol release to the medium, from 23.1 ± 0.26% to 33.9 ± 1.7%, following addition of the agonist, while NL2 exhibited an increase from 33.5 ± 1.1% to 41.5 ± 1.6% (Figure 3.8). This represents a 1.5 ± 0.06-fold and 1.3 ± 0.07-fold increase in total[3H]-sterol release to the medium by NL1 and NL2, respectively, following incubation with TO-901317. In CESD cells, addition of the LXR agonist increased UC efflux from 7.3 ± 0.34% to 14.1 ± 0.73% and from 10.3 ± 0.25% to 20.8 ± 0.62% in CD1 and CD2, respectively (Figure 3.8). The increases represented a 1.9 ± 0.09-fold and 2.0 ± 0.05-fold increase in efflux by CD1 and CD2, respectively, with the addition of the LXR agonist compared to the apoA-I alone condition. However, although a significant increase in release of UC to apoA-I was observed in the CESD cells following incubation with TO-9013 17, they still had blunted activity relative to normal fibroblasts. These results indicate that although the presence of the LXR agonist is capable of upregulating ABCA1 and increasing efflux in both CESD fibroblasts, it is unable to correct or circumvent the LAL mutation responsible for the blunted efflux observed in CD1 and CD2. These results provide further support for the role of lysosomal unesterified cholesterol as a key component of the ABCA1 substrate pool of cholesterol for apoA-I-dependent efflux. 66 Figure 3.8 Increased efflux of LDL-derived unesterified cholesterol (UC) to apoA-I containing media following incubation with LXR agonist (TO-901317).Confluent normal (NL1, NL2) and CESD (CD1, CD2) fibroblasts were incubated for 4hr in 1.4% Lipofectamine and were subsequently incubated with 50 jiglml [3H]- cholesteryl linoleate-labeled LDL for 241w, followed by a 24 hr incubation in the presence ((+) ApoA-I) of 10 tg/ml ApoA-I. LXR agonist condition cells were concurrently incubated with 5j.tM TO-9013 17 during 24hr incubation with apoA-I. Efflux results are expressed as percent of total medium plus cell[3H]-cholesterol in the medium. Mean ± S.D. of three determinations and representative of 3 experiments. ‘, p <0.001 relative to normal fibroblasts. 0 ci C’) z C., (0 .1-’ 0 F— 60 50 40 30 20 10 0 Cell Line NL1 NL2 CDI CD2 — (+)ApoA-l EEEI LXR Agonist + ApoA-I 67 3.9 Adenovirus Delivery Optimization: ABCA1 and hLAL Expression In order to investigate the impact of correcting LAL expression in CESD fibroblasts on ABCA1 expression and activity, we used an adenovirus expressing full-length human lysosomal acid lipase (Ad-hLAL) to introduce a functional enzyme into normal and CESD fibroblasts. Previous attempts in our laboratory to transfect human skin fibroblasts with cDNA using electroporation, Fugene6, calcium phosphate, Superfect, and Effectene had resulted in low transfection efficiency, therefore it was necessary to pursue alternate methods of delivery. Fibroblasts were incubated with varying multiplicities of infection (MOIs) of Ad-hLAL for 1 or 2 hours with 2.8% Lipofectamine and were subsequently incubated for 24hr with LDL. Delivery of the adenovirus in normal cells (NL1) produced an 8-fold and 16-fold increase in LAL expression at 100 MOI at 1 and 2hr incubation times, relative to the basal control levels (Ctl) (Figure 3.9A,B). However, in the normal cell lines the increased expression of hLAL did not translate to an increase in ABCA 1 expression (Figure 3 .9C). Similarly, following delivery of the CESD cell line (CD1), hLAL expression was upregulated 5-fold at 100 MOl with a 11w incubation, while an MOl of 100 and 200 MOl with a 21w incubation resulted in upregulation of hLAL 11- and 13-fold, respectively (Figure 3.9A,B). Interestingly, a 1.8-fold increase in ABCA1 expression in CD1 was observed at MOl 100 after a lhr incubation, relative to the control ABCA1 band (Figure 3.9A,C). Importantly, although expression of IiLAL was greatest at MOl of 100 and 200 with a 21w incubation, the ABCA1 expression only increased 1.25-fold and 1.3- fold, respectively (Figure 3 .9C). These results suggested that further optimization of our delivery conditions were required to see the potential changes in ABCA 1 expression. 68 Figure 3.9 Increased ABCA1 and hEAL expression in human CESD fibroblasts following delivery of adenovirus expressing full-length human lysosomal acid lipase (Ad-hLAL).Confluent normal (NL 1, NL2) and CESD fibroblasts from two unrelated CESD patients (CD 1, CD2) were infected with Ad-hLAL at an MOl of 50, 100 or 200 with incubation times of either 1 or 2 hours in the presence of 2.8% Lipofectamine. Lipofectamine exposure alone was used as a control (Ctl). Cells were subsequently probed for hLAL and ABCA1 expression by Western blotting. Numeric densitometry values represent the intensities of (B) hLAL protein and (C) ABCAI protein normalized to the normal (NL1) Lipofectamine control (Cnt). The loading control used for Western analysis was protein disulfide isomerase (PDI). Results are from a single experiment. A. B. 18 16 C 14 12 10 •NL1 . 8 cOOl 0 -l 0 C 1.6 1.4 = 1.2 01 •NL1 ‘ 08 cCDI 0.6 e Q4 C, 0.2 0 Incubatiuii Time MOl NLI 1 Hour 2 Hour CII 50 100 200 50 100 200 hLAL CDI 1 Hour CAt 50 100 200 ABCAI __ — — . — 2 Hour 50 100 200 — PDI — — — — — — Cont’oI 50 100 200 50 100 200 1 hour 2 hour Control 50 100 200 50 100 2C0 1 hour 2 hoiz 69 3.10 Increased ABCA1 and hLAL Protein Levels Following Delivery of Ad-hLAL As shown in Figure 3.9, delivery of Ad-hLAL led to increased hLAL protein at 100 MOl with a lhr and 2hr incubation period. However, as ABCA1 protein levels were not significantly affected we tested the effects of introducing a non-Lipofectamine control (Ctl) and utilized two normal cell lines (NL1, NL2) and two CESD cell lines (CD1, CD2). Using the same delivery protocol described in section 3.9, normal cells showed increased LAL expression at both MOIs and incubation times, however NL1 expressed a 5.8-fold increase in expression at 200 MOl with a lhr incubation, and a 7.4-fold and 4.1-fold increase at 100 and 200 MOl with a 2hr incubation, respectively, relative to the Lipofectamine control (LP)(Figure 3.10). The associated ABCA 1 band with each condition did not appear to change dramatically with each delivery condition relative to the control condition. Similarly, NL2 had increased hLAL expression in all infected conditions relative to basal expression, however the greatest upregulation was observed at 200 MOl with a lhr incubation with a 33.5-fold increase and at 200 M0112 hr incubation with a 61- fold increase (Figure 3.10). Interestingly, the ABCA1 band was reduced with each Lipofectamine condition in the NL2 cells (Figure 3.10). Tn the CESD cells a similar upregulation in hLAL expression was seen at all infected conditions. CD1 exhibited a 26-fold and 30-fold increase at 100 MOT and 200 MOl with a lhr incubation, respectively, and a 31-fold and 37-fold increase at 100 and 200 MOl with a 2hr incubation, relative to the Lipofectamine control (LP) (Figure 3.10). An increase in ABCA1 expression was observed with each delivery condition relative to the basal levels, however the greatest increase was observed at 100 MOl with a lhr incubation, which resulted in a 2.8-fold increase in CD1 (Figure 3.10). The CD2 cell line also had increased hLAL expression with all Ad-hLAL conditions, resulting in a 22.5-fold and 41- fold increase at 100 and 200 MOT with a lhr incubation, and a 28-fold and 200-fold increase with MOl 100 and 200 with a 2hr incubation, respectively (Figure 3.10). The greatest ABCA1 expression appeared to be with 100 or 200 MOl with a lhr incubation, which produced a 1.6-fold and 1.7-fold increase, respectively, while the 2hr incubation conditions maintained the ABCA1 expression similar to those of the non-infected conditions (Figure 3.10). The accumulated results from this experiment suggested that further optimization of our delivery conditions were required to see the potential changes in ABCA1 expression. Therefore subsequent experiments investigated increased MOIs and extended incubation times in the presence of Ad-hLAL. 70 Figure 3.10 Increased hLAL protein expression in normal and CESD fibroblasts following delivery of an adenovirus expressing full-length human lysosomal acid lipase (Ad-hLAL).Confluent normal (NL1, NL2) and CESD (CD1, CD2) fibroblasts were incubated with Ad-hLAL using an MOl of 100 or 200, with an incubation period of 1 or 2 hours in the presence of 2.8% Lipofectamine, followed by an additional 23 or 22 hours incubation with DMEM and 50 Ig/ml LDL. Cells were subsequently probed for hLAL and ABCA1 expression by Western blotting. Numeric values represent the intensities of ABCA1 protein and hLAL protein normalized to normal (NL1) Lipofectamine control (LP). A non-Lipofectamine, LDL-loaded control (control) was also present. The loading control used for Western analysis was protein disulfide isomerase (PDI). Results are from a single experiment. NLI CDI NL2 CD2 I 1401k 2 How 1 Hour 2 Hour 1 Hour 2 Hour I Hoia 2 How MOl CU LPIOO200100200CtI LP100200100200 CU LPIOO200IOO 200 Cli LP 100200100200 bLAL ABCAI — — — — — — PDI — — — — — — — — — — 71 3.11 Increased ABCA1 Expression Following Delivery of Ad-hLAL To determine the effect of delivery Ad-hLAL on ABCA1 expression in normal and CESD cells, relative to an adenovirus control expressing green fluorescent protein (Ad-GFP), fibroblasts were incubated with 2.8% Lipofectamine for 8hr. The previously discussed adenoviral delivery experiments had utilized 1 or 2hr incubation periods, however significant increases in ABCA1 protein levels were not consistently observed in the normal and CESD cell lines. In order to visualize increases in hLAL expression, and potential changes on ABCA1 expression, we extended the incubation period to 8hr. Incubation with Ad-hLAL increased hLAL expression in normal fibroblasts, increasing 7- and 14-fold in NL1 and NL2 relative to the control, respectively (Figure 3.1 1A,B). Similarly, incubation with Ad-hLAL increased LAL expression 20-fold in CD1 and CD2 (Figure 3.1 1A,B). Analysis of ABCA1 expression demonstrated that the control and Ad-GFP conditions had similar levels within each cell line. However, the CESD cell lines had approximately 1.6-fold less ABCA 1 expression in control and Ad-GFP conditions compared to NL1 and NL2 (Figure 3.1 1A,C). Incubation with Ad-hLAL lead to a 1.3-fold increase in ABCA1 expression in NL1 and NL2, while CD1 and CD2 demonstrated a 1.3- and 1.5-fold increase in expression relative to ABCA 1 levels in control cells (Figure 3.1 1A,C). These results suggest that this new adenoviral delivery protocol, using 2.8% Lipofectamine with an 8hr incubation, allowed increased expression of hLAL relative to basal levels in both normal and CESD fibroblasts. In addition, these conditions allowed us to visualize increased expression of ABCA 1, indicating that the release of unesterified cholesterol from the lysosomes following addition of LAL may have a regulatory effect on ABCA 1. 72 U) C D > U) E C U) C 1) U C 8) C 0 -J -J -C Figure 3.11 Increased ABCA1 protein expression in normal and CESD fibroblasts following delivery of Ad hLAL. Confluent normal (NL1, NL2) and CESD (CD 1, CD2) fibroblasts were incubated with either Ad-hLAL or Ad-GFP at an MOl of 100. Fibroblasts were incubated for 8 hour in the presence of 2.8% Lipofectamine, following by an additional 24 hour incubation in DMEM and 50 ig/ml LDL. (A) Cells were subsequently probed for hLAL and ABCA1 expression by Western blotting. Numeric densitometry values represent the intensities of (B) hLAL protein and (C) ABCA1 protein normalized to normal (NL1) and normal (NL2) control (Ctl) condition. The loading control used for Western analysis was protein disulfide isomerase (PDI). Results are from a single experiment. A. NLI CDI NL2 CD2 Ad- Ad- Ad- Ad- Ad- Ad- Ad- Ad Cti GFP hLAL CtI GFP hLAL CII GFP IiLAL Cu GFP hLAL hLAL{ ABCAI I ii’ p -— — — — - — — — B. 14 12 10 C. 4 2 1.6 1.4 C D >. 1.2 11.0 0.8 C 0.6 C a 0.4 C) U < 0.2 0.0 NL1 NL2 CD1 C02 CeN Line — ctl Ad-GFP Ad-h LAL IHI I NL1 NC) CD1 CD2 Cell Lines — ctl ___ Ad-GFP Ad-h LA L 73 3.12 Increased LAL Activity Following Delivery of Ad-bLAL In CESD there is a significant reduction in the amount of active LAL present in the lysososmal compartment207.In order to determine the enzyme activity of normal and CESD fibroblasts following Ad-hLAL delivery we utilized 4-methylumbelliferyl oleate (4-MUO), a substrate that fluoresces after being hydrolyzed220.Fibroblasts were incubated with Ad-hLAL at an MOl of 100 in 2.8% Lipofectamine with an incubation time of 8hr. As previous experiments had indicated that reduced incubation times may not be sufficient to affect ABCA1 expression (Figure 3.9, 3.10), we extended the incubation time to 8hr in the presence of Ad-hLAL. NL1 and NL2 incubated with Ad-hLAL resulted in a 2.0 ± 0.18-fold and 2.5 ± 0.23-fold increase in enzyme activity in whole cell homogenates, respectively, relative to the non-infected controls (Figure 3.12). CESD fibroblasts, CD1 and CD2, each had a reduced enzyme activity under control conditions relative to normal fibroblasts, as expected. However, following delivery of Ad-hLAL, 11.4 ± 0.24-fold and 20 ± 0.07-fold increases were observed in CD 1 and CD2, respectively (Figure 3.12). Following delivery of Ad-hLAL, the CESD fibroblasts exhibited enzyme activities above that of normal cell lie control, non-Ad-hLAL conditions. These results indicate that following Ad-hLAL delivery to fibroblasts the expressed LAL enzyme is active at a pH 5.5, equivalent to the acidity in the lysosomal compartment, and that hLAL activity is fully rescued when compared to normal fibroblasts. 74 U) a) D C E 0 c) C G) .+ C-) rz) D 0 c\1 D U > > C.) a) 2 >‘ N C w Figure 3.12 Increased LAL activity in cell homogenates of normal and CESD human fibroblasts infected with adenovirus coding for full length human LAL. Confluent normal (NL 1, NL2) and CESD (CD 1, CD2) fibroblasts were infected with Ad-hLAL for 8hr in 2.8% Lipofectamine using an MOl of 100. After an additional 24hr incubation in DMEM the cells were homogenized and exposed to 25 mol 4-methylumbelliferyl oleate (4- MUO) for 30 minutes to determine production of fluorescent unesterified 4-methylumbelliferone. , p < 0.001 relative to normal fibroblasts (NL1, NL2) control condition. Results are the mean ± S.D. of triplicate from a single experiment. 40000 30000 20000 10000 0 NL1 NL2 CD1 CD2 Control I I Ad-hLAL Cell Line 75 3.13 Lipofectamine Affects Phospholipid Efflux in CESD Fibroblasts As shown in Figure 3.4, CESD fibroblasts demonstrated impaired efflux of PC to apoA-I containing media. However, as the protocol involved incubation of adenovirus in the presence of the lipophilic reagent Lipofectamine 2000, we investigated the functional effect of Lipofectamine alone on phospholipid efflux. Normal and CESD fibroblasts were incubated in the absence or presence of apoA-I and in the absence or presence of 2.8% Lipofectamine. After the 24hr incubation in apoA-I containing media and non-Lipofectamine conditions, the CESD fibroblasts demonstrated impaired release of PC compared to normal fibroblasts (Figure 3. 13A). Under non-Lipofectamine conditions, addition of apoA-I resulted in a 13.3 ± 0.02% and 9.8 ± 0.16% release of total[3H]-PC in NL1 and NL2, respectively, compared to a release of 5.8 ± 0.64% and 5.15 ± 0.13% of total [3H]-PC in CD 1 and CD2, respectively (Figure 3.1 3A). Addition of 2.8% Lipofectamine to the incubation media did not affect the non-apoA-I condition in normal and CESD fibroblasts, however incubation with apoA-I in the presence of Lipofectamine altered efflux to apoA-I from normal cells, and removed the differences in efflux between normal and CESD fibroblasts. NL1 and NL2 exhibited a 10.1 ± 0.56% and 14.1 ± 0.08% release of total[3H]-PC, respectively, while CD1 and CD2 displayed a 9.5 ± 0.30% and 9.6 ± 0.89% release of total [3H]-PC to apoA-I, respectively (Figure 3.13C). Similar Lipofectamine induced effects were observed with sphingomyelin efflux in normal and CESD fibroblasts (data not shown). These results indicate that the addition of 2.8% Lipofectamine during adenoviral delivery was likely having an effect on phospholipid efflux, possibly related to changes in cell membrane permeability, that required us to identify an alternate approach in order to study the effect of increasing LAL expression on lipid efflux from CESD cells. 76 Figure 3.13 Loss of reduced phosphatidyicholine (PC) efflux to apoA-I containing media following incubation in the presence of 2.8% Lipofectamine. Confluent normal (NLI, NL2) and CESD (CD 1, CD2) fibroblasts were incubated in the absence or presence of 2.8% Lipofectamine for 8hr. Next, cells were incubated with 5 j.tCi/ml[3H]-choline and 50 tg/ml LDL for 24hr, followed by 24hr incubation with 10 jig/ml ApoA-I. (A) Media and (B) cellular PC levels without Lipofectamine (C) media and (D) cellular PC levels with Lipofectamine present. Efflux is expressed as percent of total cellular counts plus[3H]-PC counts in the medium. Results are mean ± S.D. of three determinations, representative of 3 experiments. , p <0.001 relative to normal fibroblasts. A. Media PC 16 14 12 C.) 10 I 0 I C) a I CO 0 I 6 C. Media PC (Lipofectamine) (-:APoA-l (.)ApnA-I C-) I (0 0 I— NL1 NL2 CDI CD2 NL1 NL2 CD1 CD2 Cell Line Cell Line (-) ApoA-I (+)ApoA B. Cellular PC 110 100 90 90 D. Cellular PC (Lipofectamine) C.) cC I (0 0 I l-IAPOA-I (*) ApoA-I NLI NC.) CD1 CD2 NL1 NC) CD1 CD2 Cell Line Cell Line (-) ApoA-I (*) ApoA-l 77 3.14 Lipofectamine Causes Increased LDL Uptake by Cultured Fibroblasts Lipofectamine 2000 is a cationic synthetic lipid that forms complexes due to ionic interactions between the head group of the lipid, with a strong positive charge, that neutralizes the negative charge on the surface of the adenovirus. The mixture results in the formation of structures that fuse and pass across the plasma membrane to deliver DNA into the cell. However, the concentration and length of incubation with the reagent can have deleterious affects on the condition of cultured cells. As shown in Figure 3.13, incubation with 2.8% Lipofectamine resulted in alterations of PC efflux to apoA-I-containing media. The effect of Lipofectamine treatment on uptake of[3H1-cholesteryl linoleate-labeled LDL was also determined. As shown in Table 3.1, the addition of 2.8% Lipofectamine for 8hr resulted in a 2 to 3-fold increase in total cellular [3H] cholesterol counts in normal fibroblasts (NL1). A similar fold increase was also observed in the CESD cell line, CDI. These results indicate that while the cells may be capable of maintaining a relatively high confluence compared to non-Lipofectamine condition cells, as observed by measuring protein levels using the Lowry assay, the concentration of Lipofectamine is high enough to induce physiological changes in LDL uptake as well as phospholipid efflux. This result required us to determine a concentration of Lipofectamine suitable for adenoviral delivery but that would not affect LDL uptake and our critical assays of cholesterol and phospholipid efflux. 78 Table 3.1 Increased uptake of LDL in the presence of Lipofectamine.Confluent normal (NLI, NL2) and CESD (CD 1, CD2) fibroblasts were incubated in the absence or presence of 2.8% Lipofectamine for 8hr. Next, cells were incubated with 50 tg/m1[3H]-cholesteryl linoleate-labelled LDL for 24hr, followed by 24hr incubation with or without 10 tg/ml ApoA-I. Results are the mean ± S.D. of triplicate determinations and are representative of two experiments with similar results. Lipofectamine [i-l] Cellular Cholesterol Counts (mean ± std)Cell Line Condition Present (dpm x lO5Img cell piotein) (-) ApoA-I (-) 7.8±0488 NLI (-i-)ApoA-I (-) 5.4 ± 0.616 (+)ApoA-I (+) 17.2±1.58 (-) ApoA-I (-) 20.7 ± 2.61 CDI (+)ApoA-I (-) 17.9±2.8 (+) ApoA-I (+) 54.2 ± 2.30 79 3.15 Effects of Reduced Lipofectamine Concentration on Phospholipid Efflux Due to the apparent detrimental affects caused by exposure of normal and CESD fibroblasts to 2.8% Lipofectamine, we next set out to optimize the adenoviral delivery protocol using lower concentrations of Lipofectamine. The optimization conditions involved incubating normal (NL1) and CESD (CD 1) fibroblasts with varying concentrations of Lipofectamine for 4hr, followed by a 24hr incubation with[3H]-choline and LDL in the absence of the transfection reagent. The reduced incubation time in the presence of Lipofectamine, from 8hr to 41w, was selected due to the aforementioned affects of the transfection reagent at 81w. The results indicated that a concentration of 0.7% and 1.4% Lipofectamine maintained a similar level of PC and SM efflux during subsequent apoA-I incubations from both normal and CESD fibroblasts when compared to cells not exposed to Lipofectamine. Efflux of[3H]-PC in normal fibroblasts with apoA-I was maintained at -10% of total [3H] PC while the CESD cells exhibited —6% efflux of [3H] PC at 0%, 0.7% and 1.4% Lipofectamine (Figure 3. 14A). However, the impaired ability of CESD fibroblasts to efflux PC was obscured when the concentration of Lipofectamine was increased to 2.1%, with NL1 [3H] PC efflux decreasing to 7.9 ± 0.46% and CD1 efflux increasing to 7.2 ± 0.37% (Figure 3.14A). The same trend was observed with SM efflux to apoA-I, with 5 ± 0.67% and 3.5 ± 0.19% [3H]-SM efflux in NL1 and CD1, respectively, with 0.7% and 1.4% Lipofectamine (Figure 3. 14B). However, incubation with 2.1% Lipofectamine resulted in increased SM release in CD1, increasing from 3.5 ± 0.19% to 4.8 ± 0.27%[3H1-SM efflux (Figure 3. 14B) Based on these results, it appeared that a concentration of 1.4% Lipofectamine could be used during adenoviral delivery without disrupting one of our key assays of ABCA1 function, cellular phospholipid efflux to apoA-I. 80 Figure 3.14 Optimization of Lipofectamine conditions for phosphatidyicholine (PC) and sphingomyelin (SM) efflux to apoA-I containing media in normal (NL1) and CESD (CD1) fibroblasts. Confluent normal (NL 1) and CESD (CD1) fibroblasts were incubated for 4hr with 0%, 0.7%, 1.7% or 2.1% Lipofectamine, following by a 24hr incubation with 5 iiCilml[3HJ-choline and 50tgIml LDL. Fibroblasts were then incubated for an additional 24hr in the presence ((+) ApoA-I) or absence ((-) ApoA-I) of 10 .tgJml ApoA-I. Results are expressed as percent of total cell plus medium[3H]-PC or -SM in the medium (A and B, respectively), cell PC (C), and cell SM (D). Results are the mean ± S.D. of triplicate determinations from a single experiment. A. Media PC 12 10 0 18 I (0 0 I. .\ A A A A A A AQc’ oP’ coP’ ,P’ go?’ O 0010k 0° 1 o1° 2.° 2.* Lipofectamine Conditions C. Cellular PC C.) I (1 95 S 0 I 100 (I,) I (0 g5 S 0 I-. go Lipofectamine Conditions NL1 CD T In In In pçO ppçP.1 p?o’ l°’ O 0° \p(o % D. Cellular SM 105 NL CD1 T I T B. Media SM Cl) (0 S I-. 0 kPQO 0°. 0° o1°’ . ,A L(.* .\(lOl Lipofectamine Conditions ir In In 85 oP 2.(,OI( *l01( l(t( Lipofectamine Conditions 81 3.16 Lipofectamine Necessary For Delivery of Ad-hLAL in Cultured Fibroblasts As preliminary experiments using Lipofectamine indicated that higher concentrations of the transfection reagent had adverse effects on cell culture, resulting in alterations in cell function, we also attempted to optimize the adenoviral delivery protocol by removing Lipofectamine altogether during addition of adenovirus. Previous studies had shown that it was possible to infect fibroblasts with adenovirus in the absence of reagents such as Lipofectamine, and still observe significant upregulation of the gene of interest227’8 Using a range of MOIs from 100 to 1000, we incubated normal (NL1) and CESD (CD1) fibroblasts for 8hr, followed by a 24 hr incubation period with LDL (LDL). However, Western blot analysis showed no changes in hLAL expression in normal or CESD fibroblasts (Figure 3.15). Interestingly, increased ABCA 1 was observed in NL1 at 800 and 1000 MOl, as well as in CD1 with increasing concentration of adenovirus present (Figure 3.15). However, as minimal to no increases in LAL expression were observed under these same adenoviral delivery conditions it is possible that the changes may be due to other factors. Based on the hLAL expression without the use of Lipofectamine, these results indicate that for efficient adenoviral delivery to fibroblasts used in the current study a transfection reagent, such as Lipofectamine, is still required. 82 Figure 3.15 Minimal increase in hLAL and ABCA1 protein expression in CESD and normal fibroblasts following incubation with adenovirus expressing full-length human lysosomal acid lipase (Ad-hLAL) in the absence of Lipofectamine. Confluent normal (NL 1) and CESD (CD 1) fibroblasts were infected with Ad-hLAL at an MOl of 100, 200, 400, 600, 800 or 1000. Fibroblasts were incubated for 8 hours with Ad-hLAL in the absence of Lipofectamine, followed by an additional incubation in DMEM and 50ig/ml LDL cholesterol for 24hr. Cells were subsequently probed for hLAL and ABCA1 expression by Western blotting. The loading control used for Western analysis was protein disulfide isomerase (PDI). Results are from a single experiment. Numeric densitometry values represent the intensities of (B) hLAL normalized to non-AdhLAL (Ctl) NL1 and CDI band. The loading control used for Western analysis was protein disulfide isomerase (PDI). A. B. NL1 CDI IAOI CII 100 200 4)0 600 800 1000 CII 100 200 400 600 800 1000 hLAL{ ABCAI — PDI — __ __ — — 12 I I I.NLII ‘ ID 1 ‘DCDII I I 0 U, 0.6 GA 0 ..i 02 -J 0 CtI 100 200 400 600 800 1000 83 3.17 Ad-hEAL Delivery With Lipofectamine and Varying Adenoviral MOl Based on the results obtained from the phospholipid efflux experiments with lower Lipofectamine exposure, we did further experiments using 1.4% Lipofectamine and varying adenovirus MOl in order to attempt to optimize LAL expression. MOIs of 200 to 1000 with a 41w incubation period and MOIs from 400 to 1000 with an 81w incubation period were tested. Delivery of Ad-hLAL to normal cells (NL 1) produced the greatest increase in LAL expression at 600 and 1000 MOl (41w incubation) with a 3- and 4.5-fold increase, and the highest at 600 MOl (81w incubation) with a 7.1-fold increase in expression (Figure 3.16A,B). Although not as highly expressed as in normal fibroblasts, the CESD cells (CD 1) also showed the highest upregulation of LAL at 600 and 1000 MOl (4hr incubation) with a 6.5- and 8.8-fold increase in expression. In addition, the 8hr incubation with MOI 400, 600 and 1000 exhibited a 12.4-, 15.5- and 16.7-fold increase in hLAL expression, respectively, in CD 1 (Figure 3.1 6A,B). However, it was observed that with the 81w incubation there was increased cell death, based on cell protein measurements using Biorad analysis. We also observed this the 1000 MOl condition using a 41w incubation. Although our primary goal was to observe increases in LAL expression, we also investigated the concurrent alterations in ABCA1 expression. The greatest upregulation in ABCA1 expression in NL1 was observed with MOl 600 with 41w incubation, with a 1.38-fold increase in ABCA1 expression (Figure 3.l6A,C). In CD1, expression of ABCA1 was increased with MOl 400, 600 and 1000 (4 hr incubation), with a 1.3-, 1.2- and 1.6-fold increase in expression (Figure 3. 16A,C). Interestingly, a reduction in ABCA1 expression was observed at MOl 600 and 1000 with 8hr incubation, indicating higher concentrations of virus and/or increasing incubation times in the presence of reduced levels of Lipofectamine may still have detrimental effects on fibroblast condition and functional abilities. Based on the LAL expression results, we adopted a protocol using an MOl of 600 in the presence of 1.4% Lipofectamine with a 4hr incubation before addition of LDL. 84 Figure 3.16 Increased hLAL protein expression in normal (NL1) and CESD (CD1) fibroblasts following delivery of Ad-hLAL. Confluent normal (NL1) and CESD (CD 1) fibroblasts were infected with Ad-hLAL at an MOl of 200, 400, 600, or 1000. Cells were incubated for either 4 or 8hr in the presence of 1.4% Lipofectamine. Two controls were used: control (Ctl) fibroblasts were incubated with 1.4% Lipofectamine for 8hr without adenovirus, or fibroblasts were infected at 200 MOl with Ad-hLAL in the presence of 2.8% Lipofectamine for 4hr. Following incubation for 4 or 8 hours, fibroblasts were incubated for an additional 24hr in DMEM and 50 jig/mI LDL. Numeric densitometry values represent the intensities of (B) hLAL protein and (C) ABCA1 protein normalized to LDL-loaded control (Ctl) band. The loading control used for Western analysis was protein disulfide isomerase (PDI). Results are from a single experiment. 4 C S 18 ________ = 16 14 r1 812 I I io 1r8 H [-4 —1 L II -J Cnt 200 200 400 (2.8%) (1.4%) (1.4%) 41w A. NLI CDI 4hnta flhn.z 4hour 8ha Incubation hadon hcubatiou bcubon •00 (1.4%, MOl Cd 201 204 440 Lofectnine (1.4%) (2.8%) (1.4%) (1.4%) hLAL{ — — ABCAI PDI 100 (1.4% 1000 400 400 1 (1.4%) (1.4%) (1.4%)(1.4%) joo 400 000 1004 ca 200 240 411 (1.4%) (1.4%) (1.4%) (1.4%) (1.4%) (2.8%) (1.4%) (1.4%) — — — — - — — B. oNL1 — — — — .. lI 200 (a8%) 20C 400 1.4%) (1.4’/,) cnt C. 6C0 (1.4%) : 4 hr 1000 400 600 (1.4%) 1.4%) (1.4%) 1000 ( 8 hr .5 0 = .5 4 C 600 1000 400 630 1000 (1.4%) (1.4%) (1.4%) (1.4%) (1.4%) 8 hr [:j p1.4 - 1 1.2 - - Cot 2C0 200 4C0 2.8%) (1.4%) 1.4%) 600 1000 400 6C0 1000 (1.4%) 1.4%) (1.4%, (14%) (1.4% 4 hr 8 hr 85 3.18 Effect of Lipofectamine Concentration on LDL Uptake by Cultured Fibroblasts As shown in Table 3.1, the addition of 2.8% Lipofectamine resulted in a 3 to 4-fold increase in total cellular [3H]-cholesterol counts in normal fibroblasts (NL 1). A similar fold increase was also observed in the CESD cell line, CD 1. These results indicated that this concentration of Lipofectamine alters LDL uptake. To determine if the changes in LDL uptake were corrected with the modified adenoviral delivery protocol using 50% less Lipofectamine as the previous method, we determined that the uptake of[3H]-cholesteryl linoleate-labeled LDL by normal (NL1) and CESD (CD1) fibroblasts incubated in the absence (-) or presence (+) of 1.4% Lipofectamine for 4hr in the absence or presence of apoA-I. The changes in LDL uptake were measured as the differences in total cellular disintegrations per minute (dpm), the number of atoms in a radioactive material that decay in one minute, for each condition. The results indicate that there are no significant differences in the{3H]-cholesterol counts between the non Lipofectamine and Lipofectamine conditions (Table 3.2). These findings further support the optimization experiments using phospholipid efflux studies that demonstrated at 1.4% Lipofectamine there were no major effects on our major cellular lipid efflux assays. 86 Table 3.2 Absence of affect on LDL uptake using lower Lipofectamine concentration. Confluent normal (NL 1, NL2) and CESD (CD 1, CD2) fibroblasts were incubated in the absence or presence of 1.4% Lipofectamine for 4hr. Next, cells were incubated with 50 Lg/ml[3H]-cholesteryl linoleate-labelled LDL for 24 h, followed by 24hr incubation with 10 j.tg/ml ApoA-I. Results are the mean ± S.D. of triplicate determinations and are representative of two experiments with similar results. Cell Line Condition Upofectamine [I1] Cellular Cholesterol Counts (mean ± std)Present (dpm x lO5mg cell protein) (—) ApoA—I (—) 1 .51 ± 0.1 1 I NLI (+)ApoA—I (-) 1.21±0.194 (+)ApoM (+) 1.26±0.119 (-) ApoA-I (-) 1.6 ± 0.238 CDI (+) ApoA—I (-) 1.23 ± 0.221 (+)ApoA-I (+) 1.41 ± 0.155 87 3.19 Increased LAL Activity with Altered Ad-hLAL Delivery Protocol To determine whether the modified adenovirus delivery protocol would maintain adequate infection efficiency as suggested by Western blot analysis of LAL protein levels, we conducted an LAL activity assay using 4-MUO as a substrate. The LAL assay was performed using an MOl of 600 instead of 100, and 1.4% Lipofectamine as opposed to 2.8%. The activity assay showed a 1.6 ± 0.09-fold and 2.0 ± 0.43-fold increase in LAL enzyme activity in NL1 and NL2, respectively, as measured by increased production of fluorescent unesterified 4- methylumbelliferone (Figure 3.17). As shown previously, CD1 and CD2 both showed reduced basal LAL activity, which increased by 14 ± 0.72- and 3.8 ± 0.06-fold, respectively, following delivery of Ad-hLAL (Figure 3.17). These results indicate that following adenoviral delivery of Ad-hLAL to fibroblasts with our altered infection conditions, the expressed LAL enzyme again showed normalization of activity to levels seen in control normal fibroblasts. 88 60000 30000 10000 0 Figure 3.17 Increased LAL activity in cell homogenates of normal and CESD human fibroblasts infected with adenovirus coding for full length human LAL (Ad-hLAL).Confluent normal (NL1, NL2) and CESD (CD 1, CD2) fibroblasts were incubated with an MOl 600 of Ad-hLAL in the presence of 1 .4%Lipofectamine for 4hr, followed by a 24hr incubation with 50 tg/ml LDL. The cells were subsequently homogenized and exposed to 25 mol 4-methylumbelliferyl oleate (4-MUO) for 30 minutes to determine production of fluorescent unesterified 4- methylumbelliferone. , p < 0.001 relative to normal fibroblasts. Results are the mean ± S.D. of triplicate determinations and is representative of two experiments. .E 50000 0 L. 40000 D LL >‘ > 20000 a) E N C w NL1 NL2 CD1 CD2 Cell Line — Control Ad-h LAL 89 3.20 ABCA1 Expression Following Delivery of Ad-hEAL In order to determine whether delivery of Ad-hLAL to CESD fibroblasts using our modified infection protocol could ameliorate the impaired regulation of ABCA 1 expression (Figure 3.1), quantitative RT-PCR was again performed on mRNA samples from normal and CESD human skin fibroblasts. The levels of ABCA1 mRNA were determined under non-LDL-loaded conditions, LDL-loaded conditions, in the presence of a non-oxysterol LXR agonist (TO 901317) plus LDL, and incubated with either Ad-hLAL or Ad-GFP followed by LDL-loading. The fold increase in ABCA1 mRNA, relative to non-LDL-loaded conditions, was observed following incubation with LDL was calculated and corrected for the housekeeping gene, cyclophilin. In normal fibroblasts, NL1 and NL2, no change in ABCA1 mRNA expression was observed following delivery of Ad-hLAL, however the LXR agonist was able to upregulate expression, as expected (Figure 3.18). Similarly, the CESD fibroblasts exhibited no change in ABCA1 mR.NA levels following incubation with Ad-hLAL (Figure 3.18). These results suggest that, correction of the CESD mutation, by introducing active LAL, does not result in correction of the reduced ABCA 1 mRNA levels seen in CESD fibroblasts, at least under the conditions used here. However, it is important to note that we observed a large reduction in the upregulation of ABCA 1 mRNA with LDL loading in the presence of Lipofectamine in normal fibroblasts (—2- fold increase)(Figure 3.18) compared to the same conditions without Lipofectamine (-8-fold increase)(Figure 3.1). These results suggest that Lipofectamine may be having additional adverse cellular affects, potentially directly or indirectly inducing RNA degradation. Therefore, the anticipated release of unesterified cholesterol from the lysosomal compartment following introduction of active LAL is not showing the expected increase in ABCA1 mRNA under the experimental conditions. In the CESD fibroblasts, as shown in Figure 3.6A, introduction of the non-oxysterol LXR agonist was able to upregulate ABCA1 mRNA expression to normal levels, indicating that ABCA 1 is still functional at a transcriptional level in the mutant fibroblasts. In order to determine whether delivery of Ad-hLAL to CESD fibroblasts could improve the impaired regulation of ABCA1 protein expression, we also performed a Western blot analysis on samples from normal and CESD human skin fibroblasts. Both normal and mutant cells infected with Ad-hLAL demonstrated increased expression of LAL (Figure 3. 18B). NL1 and NL2 exhibited a 5.9-fold and 5.0-fold increase in hLAL expression over basal levels, respectively, in arbitrary densitometry units. CD1 and CD2 had a 111-fold and 80-fold increase in hLAL expression over basal levels, respectively. Delivery of Ad-hLAL increased hLAL expression in CESD fibroblasts to, and even beyond, normal levels. In contrast to our ABCA1 mRNA data, 90 infection with Ad-hLAL did appear to increase ABCA1 protein levels in both normal and CESD fibroblasts (Figure 3.18B). NL1 and NL2 showed a 2.6-fold and 2.4-fold increase in ABCA1 expression, respectively, with Ad-hLAL incubation. However, the upregulation in NL1 was obscured by a 2-fold increase observed in the NL1 Ad-GFP condition. CD1 and CD2 exhibited a 2.7-fold and 2.9-fold increase in ABCA1 expression following delivery of Ad-hLAL, respectively. These results indicate that increasing the level of functional LAL was capable of influencing expression of ABCA1, potentially by releasing increased amounts of UC from the lysososmal compartment, which may function as a substrate for oxysterol formation. However, the expected increase in ABCA 1 protein following LDL-loading in normal fibroblasts was not observed, as seen in previous experiments that did not include Lipofectamine (Figure 3.2). Therefore, this result further supports potential adverse effects of Lipofectamine at both a transcriptional, and now, at a translational level. 91 Figure 3.18 No change in ABCA1 mRNA, but increased ABCA1 protein in response to Ad-hLAL Confluent normal (NL I, NL2) and CESD (CDI ,CD2) fibroblasts were incubated with Ad-hLAL or Ad-GFP at an MOl of 600 for 4hr in 1.4% Lipofectamine. Cells were subsequently incubated in the absence or presence of 50 jig/mi LDL for 24hr. LXR agonist condition incubated with 5mJvI TO-901 317 concurrently with LDL for 24hr (A) Quantitative RT PCR results are displayed as a fold increase of ABCA1 mRNA with addition of LDL, corrected for cyclophilin. No change in ABCA1 mRNA levels with Ad-hLAL delivery. Results are the mean ± S.D. of triplicate determinations and are representative of three experiments with similar results. The Western blot analysis was set-up parallel to the cholesterol efflux study (Figure 3.21) and proteins were isolated for determination of ABCA1 and hLAL expression and numeric values represent the intensities of (B) ABCAI normalized to loading control (PDI) bands, while hLAL normalized to non-LDL-loaded NL1 hLAL band. The loading control used for Western analysis was protein disulfide isomerase (PDI). ABCAI protein levels increased in normal and CESD cells following incubation with Ad-hLAL. Results are representative of 3 experiments with similar results. A. 10 0 0 8 . . - - - <.$ 400 Q) .- 0 00 2 0 o 0 IL NL1 CD1 Cell Line inI IHI NL2 CD2 — (+) LDL-C EEJ Ad-GFP + LDL-C Ad-hLAL + LDL-C LXR Agonist + LDL-C 92 3.21 Increased Cholesterol Efflux with Ad-hLAL Delivery in CESD Fibroblasts To determine ABCA1 function in CESD fibroblasts following delivery of Ad-hLAL, cells were incubated with[3H]-cholesteryl linoleate-labeled LDL for 24hr, followed by an additional 24hr incubation in the absence or presence of apoA-I. As in our initial results, addition of apoA-I to CESD fibroblasts (CD 1, CD2) resulted in reduced efflux of UC to apoA-I-containing media when compared to normal cells (Figure 3. 19A). In the CESD fibroblasts, delivery of Ad-hLAL was able to significantly increase efflux of UC to apoA-I to levels near that observed in normal fibroblasts. Efflux was increased from 7.3 ± 0.34% to 20.4 ± 0.78% and from 10.2 ± 0.25% to 27.9 ± 0.62% of total[3H1-sterol in CD1 and CD2, an increase of 2.8 ± 0.08-fold and 2.7 ± 0.05- fold, respectively (Figure 3.19A). Addition of the LXR agonist TO-901317 was also able to increase UC release to apoA-I, but to a lesser extent than Ad-hLAL treatment, to 14.1 ± 0.73% (1.9 ± 0.09-fold) and 20.8 ± 0.62% (2.0 ± 0.05-fold) of total[3H]-sterol in CD1 and CD2, respectively (Figure 3. 19A). In CESD cells, correction of LAL activity was capable of increasing efflux by releasing more UC from lysosomes, indicating that the LAL was active and localizing in the lysosomal compartment. Furthermore, direct activation of ABCA1 by addition of TO- 901317 was able to induce increased UC release to apoA-I, however not to levels observed with Ad-hLAL delivery. In normal cells, delivery of LAL did not increase release of UC to apoA-I in normal cells, while addition of TO-9013 17 was able to increase efflux from 23.1 ± 0.26% to 34 ± 1.7% and from 33.5 ± 1.1% to 41.5 ± 1.7% of total[3H]-sterol in NL1 and NL2, a 1.5 ± 0.06-fold and 1.2 ± 0.07-fold increase, respectively (Figure 3.19A). Cellular radiolabelled CE levels were also measured in normal and CESD fibroblasts. CESD cells showed a dramatic decrease in the accumulated CE, with a reduction from 76.1 ± 2.1% to 23.5 ± 0.89% and from 71.9 ± 1.1% to 16.5 ± 0.98 of total[3H]-sterol in CD1 and CD2, respectively (Figure 3.19B). These represent a 3.2 ± 0.07- and 4.3 ± 0.07-fold decrease in LDL derived UC in CD 1 and CD2 following delivery of Ad-hLAL, respectively. Addition of the TO- 9013 17 had minimal affect on CE levels in normal and CESD fibroblasts, however some reductions were observed in NL1, CD1 and CD2. Normal cells showed a small, but statistically significant, decrease in cellular CE following delivery of Ad-hLAL (Figure 3. 19B). Cellular radiolabelled UC levels were also measured. The CESD fibroblasts demonstrated a normalization of cellular UC levels following infection. Delivery of active LAL resulted in normalization of cellular UC levels, from 16.6 ± 2.12% to 56.13 ± 0.12% and from 17.7 ± 1.17% to 56.8 ± 1.4% of total[3H]-sterol in CD1 and CD2, an increase of 3.4 ± 0.13-fold and 3.2± 0.09- fold, respectively (Figure 3.l9C). In addition, incubation with TO-901317 resulted in a small 93 decrease in cellular UC levels in both normal and CESD fibroblasts. Small but significant increases in cellular UC levels were found following infection with Ad-hLAL in normal fibroblasts, suggesting some increased hydrolysis of CE (Figure 3.1 9C). Overall, these results suggest that infection with Ad-hLAL produces an active enzyme that localizes within the lysosomal compartment and is able to significantly increase efflux of UC to apoA-I in CESD cells, to a level similar to efflux of UC to apoA-I from normal cells incubated with Lipofectamine but without adenovirus. In addition, correction of LAL activity also normalized LDL-derived CE levels and cellular LDL-derived UC levels in CESD fibroblasts. Therefore, correction of LAL activity in CESD fibroblasts has produced significant changes in the impaired sterol metabolism, increased ABCA1 expression, and led to significant increases and normalization in efflux of UC to apoA-I and cellular UC levels, as well as normalization of hydrolysis of CE in the lysosomal compartment. However, increasing LAL expression did not appear to induce major changes in CE hydrolysis or release of UC to apoA-I in normal fibroblasts, suggesting these cells already have maximal LAL capacity. 94 Figure 3.19 Increased cholesterol efflux following delivery of Ad-hLAL. (A) Efflux of LDL-derived unesterified cholesterol (UC) to apoA-I containing media following delivery of Ad-hLAL to normal and CESD fibroblasts (B) LDL-derived cellular cholesteryl ester (CE) levels following delivery of Ad-hLAL in normal and CESD fibroblasts (C) LDL-derived cellular UC levels following delivery of Ad-hLAL in normal and CESD fibroblasts. Confluent normal (NL 1, NL2) and CESD (CD 1, CD2) fibroblasts were incubated with Ad-hLAL or Ad-GFP at an MOl of 600 for 4hr in 1.4% Lipofectamine. Cells were subsequently incubated with 50 tgIml[3Hj-cholesteryl linoleate-labeled LDL for 24hr, followed by a 24 hr incubation in the presence ((+) ApoA-I) or absence ((-) ApoA-I) of 10 p.g/ml ApoA-I. LXR agonist condition cells were concurrently incubated with 51.tM TO-9013 17 during 24hr incubation with apoA-I. Efflux results are expressed as percent of total medium plus cell[3H]-cholesterol in the medium. [3HJ- cholesteryl esters are expressed as percent of total medium plus cell[3H]-cholesterol in cell CE.[3H1-unesterified cholesterol are expressed as percent of total medium plus cell H]-cholesterol in cell. Mean ± S.D. of three determinations and representative of 3 experiments. , p < 0.00 1 relative to CD1 (+) ApoA-I control and CD2 (+) ApoA-I control. #, p <0.05 relative to NL1 (+) ApoA-I control and NL2 (+) ApoA-I control. A. Media UC 0 in CO = in 0 F— 50 40 30 20 10 — (-) ApoA-I — (+)ApoM Ad-GFP * ApoA-l Ad-hLAL * ApoA-I LXR Agonist + ApoA-I B. Cellular CE C. Cellular UC 0 in Cl) (U 0 F- 55 0 a) CO = (U 0 I— 55 NL1 NL2 CD1 CD2 NL1 NL2 CD1 CD2 Cell Line Cell Line — (-) ApoA-l — (+) ApoA-l — Ad-GFP * ApoA-I — Ad-hLAL cApoA-l — LXR Agonist * ApoA-l NL1 NL2 CD1 CD2 Cell Line — (-) ApoA-I — (+)ApoA-l — Ad-GFP * ApoA-l — Ad-hLAL+ApoA- LXR Agonist • ApoA-l 95 3.22 Increased Phospholipid Efflux with Delivery of Ad-hLAL in CESD Fibroblasts Lastly, the effect of increasing LAL expression on efflux of radiolabeled phospholipids to apoA-I was determined. As we saw previously, CESD fibroblasts showed reduced efflux of phosphatidyicholine (PC) and sphingomyelin (SM) to apoA-I-containing media (Figure 3.4). The addition of the viral control, Ad-GFP plus apoA-I, did not alter the reduced efflux observed in the mutant cells. In the CESD fibroblasts, increasing hLAL expression was able to increase efflux of PC to apoA-I, increasing from 5.98 ± 0.02% to 9.1 ± 0.38% and from 5.6 ± 0.39% to 7.2 ± 0.43% of total[3H]-SM in CD1 and CD2, respectively (Figure 3.20A). This represents a 1.5 ± 0.05- and 1.3 ±0.12-fold increase in PC efflux following delivery of Ad-hLAL. SM efflux, following delivery of Ad-hLAL, increased from 5.7 ± 0.38% to 8.4 ± 0.24% and from 7.2 ± 0.58% to 9.6 ± 0.91% of total[3H]-SM in CD1 and CD2, an increase of 1.5 ± 0.09-fold and 1.3 ± 0.17-fold, respectively (Figure 3.20B). Addition of TO-901317 was also able to increase PC efflux to apoA-I, to 8.1 ± 0.53% and 9.6 ± 0.78% of total [3H]-PC in CD1 and CD2, a fold increase of 1.4 ± 0.07 and 1.7 ± 0.15, respectively (Figure 3.20A). The LXR agonist also increased SM efflux in CD1 and CD2, increasing release to apoA-I to 11.1 ± 0.47% and 13.9 ± 1.0% of total[3H1-SM, a fold increase of 1.9 ± 0.1 and 1.9 ± 0.15, respectively (Figure 3.20B). Interestingly, increasing LAL expression did not increase efflux of PC or SM to apoA-I from normal cells, while addition of TO-90l317 was able to increase PC efflux from 12.1 ± 0.05% to 15.5 ± 2.1 (1.3 ± 0.14-fold) and from 10.2 ± 0.86% to 11.6 ± 0.41% (1.2 ± 0.12-fold) of total [3H]-PC in NL1 and NL2, respectively (Figure 3.20A). SM was also increased with addition of the agonist, from 12.9 ± 0.31% to 19.1 ± 0.15% (1.5 ± 0.03-fold) and from 13.4 ± 0.52% to 20.2 ± 0.87% (1.5 ± 0.08-fold) of total[3H]-SM in NL1 and NL2, respectively (Figure 3.20B). These results indicate that in normal cells efflux of PC and SM, similar to UC efflux in Figure 3.19, is possibly already at maximum capacity, such that increasing LAL expression does not increase UC or phospholipids release. In CESD cells, increasing LAL expression is capable of increasing efflux by releasing more PC and SM, indicating that the LAL is active and is localizing in the lysosomal compartment. Furthermore, direct activation of ABCA1 by addition of TO-90 1317 was able to induce PC and SM efflux to levels equal to or greater than those observed with Ad-hLAL infection. Cellular PC and SM levels were also measured in normal and CESD fibroblasts. Normal cells did not exhibit any significant differences in cellular PC and SM levels following delivery of Ad-hLAL (Figure 3.20C,D). Exposure to TO-901317 did result in a reduction in cellular PC and SM in normal fibroblasts. CESD cells exhibited minor, but significant, reductions in cellular PC and SM levels after delivery of Ad-hLAL (Figure 3.20C,D). 96 Similar to efflux of PC and SM to apoA-I, cellular PC and SM levels were depleted to the same level or greater than that caused by infection with Ad-hLAL. Overall, these results indicate that increasing LAL expression in CESD cells produced significant increases in ABCA 1 protein levels, which leads to a near normalization of PC and SM efflux to apoA-I and a reduction in cellular PC and SM levels. However, increasing LAL expression is unable to significantly increase efflux of phospholipids to apoA-I in normal cells, suggesting these cells already have maximal phospholipid efflux as well as LAL capacity. 97 Figure 3.20 Increased phospholipid efflux in normal and CESD fibroblasts following delivery of Ad-hLAL. Confluent normal (NL 1, NL2) and CESD (CD 1, CD2) fibroblasts were incubated with Ad-hLAL or Ad-GFP with an MOl of 600 and incubated in media containing 1.4% Lipofectamine for 4hr. Non-infected control cells ((-) and (+) ApoA-I) were incubated in media containing 1.4% Lipofectamine for 4 hours without adenovirus. Subsequently, all cells were incubated with 5 p.CiIml [3H) choline and 50 .tg/ml LDL cholesterol for 24hr, followed by a 24 hr incubation in the presence ((+) ApoA-I) or absence ((-) ApoA-I) of 10 .tg/ml ApoA-I. Results are expressed as percent of total cell plus medium[3H]-PC or -SM in the medium (A and B, respectively), cell PC (C), and cell SM (D). Mean ± S.D. of three determinations and representative of 3 experiments. ‘, p <0.05 relative to CD1 and CD2 (+) ApoA-I control. 18 16 o 14 1. — 12 :i: 10 Cu 0 I 6 4 2 0 ,15 :i: 0 I U, I CO 13 I— A. Media PC 20 C. Cellular PC 105 100 () 0 — 95 I Cu 90 NL1 NL2 CD1 CD2 Cell Line iii flu80 NL1 NL2 — (-) ApoA-I — (.)ApoA-I Ad-GFP - ApoA-t Ad-1iLAL + ApoA-I LXR Agonist • ApoA-I B. Media SM 25 20 cell Line — (-I ApoA-I — (.)ApoA-l Ad-GFP + ApoA-l Ad-hLAL + ApoA-I — LXR AgoniSt * ApoA-I 10 NL1 NL2 CD1 CD2 Cell Line I-) ApoA-l — (.lApoA-I Ad-GFP . ApoA-t Ad-CtLAL * ApoA-I — LXR Agonist * ApoA-I Cell Line — (-lApoAl — (.lApoA — Ad-GFP * ApoA-l Ad-IiLAL.ApoA-l — LXR Agonist • ApoA-I 98 66 MISSf13SIU:J71dVIJ3 Our laboratory had previously provided evidence, using fibroblasts from patients with the lysosomal cholesterol storage disorder Niemann-Pick Type C (NPC) disease, that lysosomally derived cholesterol may play a key role in stimulating the expression of ATP-Binding Cassette Transporter Al (ABCA 1)140 In the studies presented here, we have extended these findings to show that the regulation of ABCA1 is also impaired in another lysosomal cholesterol storage disorder, cholesteryl ester storage disease (CESD), due to deficiency of lysosomal acid lipase (LAL). The key findings of these studies are that: 1) fibroblasts from CESD patients exhibit reduced ABCA 1 mRNA and protein levels relative to normal fibroblasts following low density lipoprotein (LDL)-loading; 2) CESD fibroblasts display reduced release of cholesterol and phospholipids to apoA-I, and a massive accumulation of cholesteryl esters (CE), relative to normal fibroblasts; 3) following delivery of an adenovirus expressing full-length human lysosomal acid lipase (Ad-hLAL), ABCA1 protein levels are increased in normal and CESD fibroblasts, ABCA 1-dependent cholesterol release to apoA-I is normalized, and intracellular CE levels as determined by incorporation of LDL-derived[3H]-cholesterol are reduced to levels similar to those observed in normal fibroblasts. In addition, we also observed an increase in phospholipid efflux in the CESD fibroblasts. These studies provide further evidence that lysosomally-derived unesterified cholesterol (UC) plays a major role in the regulation of ABCA 1 expression, and that reduced flux of cholesterol out of lysosomes leading to impaired regulation of ABCA1 is a, or the, likely cause of the low HDL-C levels observed in CESD patients’24. We initially determined that CESD fibroblasts exhibited impaired regulation of ABCA 1 mRNA, which translated into reduced ABCA1 protein levels, following LDL-loading (Figure 3.1, 3.2). These results suggested that the known mutation in lysosomal acid lipase (LAL) in CESD fibroblasts, which manifests as a significant reduction in release of UC from the lysosomal compartment, may be the cause of ABCA1 dysregulation at a transcriptional and translational level. To investigate the functional repercussions of ABCA 1 mRNA and protein dysregulation, we measured release of [3H1-cholesteryl linoleate-labelled LDL-derived cholesterol to apoA-I. The CESD fibroblasts showed significantly less efflux of LDL-derived [3H]-cholesterol to apoA-I, and also exhibited massive accumulation of radiolabeled cellular LDL-derived CE and reduced amounts of labeled cellular LDL-derived UC, compared to normal fibroblasts (Figure 3.3A,B,D,E). These results support previous studies that had observed dysregulation of sterol metabolism in fibroblasts from CESD patients’37. Importantly, these results also indicate that in addition to reduced upregulation of ABCA 1 protein with LDL loading in CESD fibroblasts, there is also dysregulation of ABCA1 function. 100 To further investigate impaired ABCA1 function in CESD fibroblasts, we measured the delivery of [3H] phosphatidyicholine (PC) and {3H] sphingomyelin derived from [3H] choline to apoA-I-containing media. Following a 241w incubation in the presence of apoA-I, CESD fibroblasts demonstrated a significant reduction in the release of[3H]-PC and[3HJ-SM (Figure 3.4A,C), as well as an accumulation of cellular[3H]-PC and[3H]-SM (Figure 3.4B,D) compared to normal fibroblasts. These results support the findings from the[3H]-cholesterol efflux assay, which suggested that ABCA1 function is impaired in CESD fibroblasts. In addition to the radiolabeled cholesterol and phospholipid efflux studies, we were able to further study the dysregulation in CESD sterol metabolism by utilizing gas chromatography (GC) analysis to measure whole cell CE and UC mass, as well as media UC mass, following LDL-loading. Similar to the results obtained from the radiolabeled cholesterol efflux study, we observed a significant reduction in the mass of UC released to apoA-I in CESD fibroblasts compared to normals, which correlated with the significant accumulation of cellular CE mass in the CESD fibroblasts (Figure 3.5A,B). The results accumulated thus far support previous findings, which demonstrated a gross accumulation of cholesteryl esters within the lysosomal compartment of CESD fibroblasts. However, our finding that CESD fibroblasts exhibit impaired ABCA1 mRNA and protein levels, as well as impaired ABCA1 function, are novel findings. In addition, these results further support a key regulatory role for lysosomal cholesterol with regards to ABCA1 expression. In addition, these findings also suggest that the increased de novo synthesis of cholesterol in CESD cells, that is increased due to upregulation of HMG-CoA reductase64,is unable to compensate and upregulate ABCA1. This de novo synthesized cholesterol therefore does not appear to join the pooi of cholesterol that regulates both oxysterol formation and ABCA1 regulation. In order to determine whether ABCA1 can be upregulated in CESD cells, we incubated the cells with a non-oxysterol LXR agonist (TO-9013 17), which has been shown in previous studies to upregulate ABCA1 expression and activity in human NPCI’ fibroblasts, including normalizing efflux of radiolabeled PC and cholesterol mass to apoA-I’41. However, we also hypothesized that addition of TO-901317 would not correct the sterol accumulation in CESD, as we had not corrected CE hydrolysis. We initially measured ABCA1 mRNA levels, following incubation with TO-901317, and found that both normal and CESD fibroblasts exhibited upregulation of ABCA1 mRNA (Figure 3.6A). In CESD fibroblasts, the normalization of ABCA1 mRNA levels indicated that ABCA1 was transcriptionally responsive. Additon of TO 901317 also increased ABCA1 protein levels, indicating that ABCA1 translation is not disturbed 101 in CESD if directly activated (Figure 3.6B). Importantly, these results also suggest that following correction of the reduced UC flux from the lysosome, it would be possible to ameliorate impaired ABCA1 mRNA and protein expression via increased oxysterol formation. Functionally, addition of TO-90 1317 to CD 1 and CD2 was able to increase the release of total[3H1-PC to apoA-I from 5.7 ± 0.52% to 8.1 ± 0.58% (1.4 ± 0.16-fold increase) and from 5.4 ± 0.29% to 9.1 ± 0.61% (1.7 ± 0.10-fold increase), respectively (Figure 3.7A). Similarly, TO- 9013 17 was also able to increase release of total[3H]-SM to apoA-I from 5.6 ± 0.38% to 11.4 ± 0.47% (2.0 ± 0.10-fold increase) and from 7.3 ± 0.58% to 13.9 ± 1.1% (1.9 ± 0.16-fold increase) in CD1 and CD2, respectively (Figure 3.7B). We also investigated release of[3H]-LDL-derived UC to apoA-I following activation by TO-901317 and found that it was increased from 7.3 ± 0.34% to 14.1 ± 0.73% (1.9 ± 0.09-fold increase) and from 10.3 ± 0.25% to 20.8 ± 0.62% (2.0 ± 0.05-fold increase) in CD1 and CD2, respectively (Figure 3.8). Importantly, although these results represent relatively large increases in phospholipid and cholesterol efflux in the CESD fibroblasts compared to their respective basal levels, the percentage total[3H]-PC,[3H]-SM and [3H]-UC was still significantly less than that observed in normal fibroblasts under the same conditions. However, these results demonstrate that under basal conditions ABCA1 function is impaired in CESD fibroblasts, but that ABCA1 activation is capable of improving function in the form of increased phospholipid and cholesterol efflux. Having demonstrated impaired expression and function of ABCA1 in CESD fibroblasts, the second stage of the project was to determine the effect of delivering Ad-hLAL to both normal and CESD fibroblasts, with the intent of increasing LAL activity and correcting/increasing the release of UC from the lysosomes. We utilized an adenoviral delivery protocol previously developed by a research associate from our lab, which involved incubating the fibroblasts with a cationic lipid, Lipofectamine 2000, during incubation with the adenovirus. As previously described, for an adenovirus to efficiently enter a cell, it must bind via a coxsackievirus and adenovirus receptor (CAR), which allows interaction between the virus and host cell surface integrins, af33 and aV1352292 1. As human skin fibroblasts have limited or no CAR receptor expression231,we relied on the use of Lipofectamine, which complexes with the adenovirus and allows cell fusion and virus internalization232.Previous studies have shown that Lipofectamine allows effective infection of cells such as macrophages and adipocytes233’234 however there are limited references describing adenoviral infection of human skin fibroblasts using Lipofectamine. Over the course of optimizing the protocol for delivery of Ad-hLAL, we discovered that at elevated concentrations Lipofectamine has adverse, function-altering effects 102 on human skin fibroblasts, a potential result of permeabilization of the cell membrane by the detergent activity of Lipofectamine. Importantly, studies using Lipofectamine in HeLa cells have shown that at higher concentrations the cationic lipid can induce cytotoxicity 235 It was therefore critical to optimize the delivery conditions to limit cell toxicity as well as effects on our key assays of ABCA1 function. Following our optimization studies, we concluded that 1.4% Lipofectamine and a multiplicity of infection (MOl) of 600 allowed effective LAL expression while preventing adverse alterations in cell function (Section 3.9 to 3.18). After successfully optimizing the delivery protocol to maintain cell confluency, and to maximize hLAL and ABCA1 expression, we determined the LAL activity following infection with Ad-hLAL. As previously described, cells were provided with 4-methylumbelliferyl oleate (4-MUO), which upon enzymatic cleavage produces a fluorescent by-product which can be quantitated. Following incubation with Ad-hLAL, NL1 and NL2 exhibited a 1.6 ± 0.09-fold and 2.0 ± 0.43-fold increase in LAL activity, respectively, while CD 1 and CD2 LAL activities were brought up to levels observed in normal control fibroblasts, increasing 14 ± 0.72-fold and 3.8 ± 0.06-fold, respectively (Figure 3.17). This experiment demonstrated two critical points, first that we were adequately infecting the fibroblasts to observe changes in enzyme activity, and second that following Ad-hLAL delivery the expressed product was enzymatically active at a lysosomal pHof5.5. Having found that the expressed hLAL was functionally active, we determined whether increasing LAL activity in normal and CESD fibroblasts was capable of increasing ABCA 1 mRNA levels. The results, shown as fold increase over non-LDL-loaded cells, indicated no major differences in ABCA1 mRNA levels between the LDL control, the Ad-GFP virus control and the Ad-hLAL infected cells in normal and CESD fibroblasts (Figure 3.18A). However, incubation with TO-9013l7 did increase ABCA1 mRNA levels in both normal and mutant fibroblasts. Previous results indicated that hLAL is both highly expressed and active at lysosomal pH, but we did not yet know that the enzyme was localized in lysosomes and capable of hydrolyzing LDL-derived CE. This was indicated by the large drop in radiolabeled CE and increase in UC in CESD cells following rescue of LAL activity with the adenovirus. However, although increased ABCA1 mRNA levels were not observed following delivery of Ad-hLAL, the Western blot analysis showed increased ABCA1 protein in the NL2, CD1 and CD2 cell lines when compared to non-infected cells. Upregulation of ABCA 1 protein expression was also observed in NL1, however a similar increase was also observed in the Ad-GFP condition. The increased ABCA1 protein levels following Ad-hLAL delivery, but absence of increase in 103 ABCA1 mRNA, indicated a potential methodological issue with quantitation of ABCA1 mRNA. Upon examination of the results, we observed a much larger increase in the upregulation of ABCA1 mRNA with LDL-loading under non-Lipofectamine conditions (-8-fold increase) (Figure 3.1) compared to the same conditions with Lipofectamine (-.2-fold increase)(Figure 3.18A). These results suggest that Lipofectamine may be causing adverse effects on RNA stability, potentially directly or indirectly inducing RNA degradation. However, as we observed increased ABCA1 protein expression with Ad-hLAL, it is likely that although Lipofectamine obscures changes in mRNA levels, there is sufficient translation to visualize increases in ABCA1 protein. Importantly, Lipofectamine may also have an effect on protein stability as ABCA1 protein levels did not increase with LDL loading in normal and CESD fibroblasts in the presence of Lipofectamine. Previous Western blot analyses had shown that following addition of LDL, ABCA1 expression was upregulated in normal fibroblasts (Figure 3.2). The inability to distinguish increased ABCA1 protein levels in LDL-loaded conditions relative to non-cholesterol loaded conditions further indicates potential adverse effects of Lipofectamine which prevents us from visualizing the expected increase in ABCA 1 mRNA, as well as protein, once generation of UC from CE is corrected in lysosomes with expression of higher levels of LAL in the cells. In order to confirm whether increased ABCA1 protein levels in response to increased LAL acitivity translated to a functional improvement in normal and CESD fibroblasts, we repeated the radiolabeled cholesterol efflux studies. We confirmed our previous findings that CESD fibroblasts exhibit reduced cellular LDL-derived UC levels and release of UC to apoA-I, and accumulation of LDL-derived CE compared to normal fibroblasts (Figure 3. 19A-C). Following delivery of Ad-hLAL to the CESD fibroblasts, release of LDL-derived UC to apoA-I was normalized, the accumulation in cell CE were reduced to near normal levels and cellular LDL derived UC levels were increased to similar levels observed in the normal fibroblasts (Figure 3. 19A-C). Interestingly, delivery of Ad-hLAL did not significantly alter cellular or media UC levels in normal cells, however did cause a small, but significant, decrease in cellular CE levels. As previously discussed, these results suggest that in normal fibroblasts there may already be maximal LAL activity. In addition, increasing LAL activity in CESD led to significant increases in [3H]-PC and [3H]-SM release to apoA-I (Figure 3.20A,B). Interestingly, in CESD fibroblasts addition of an LXR agonist was able to correct[3H]-PC and[3H]-SM efflux to apoA-I to levels equivalent to, or greater than, those observed following Ad-hLAL delivery. As the mutation in LAL had not been corrected in the LXR agonist condition, a possible explanation is that sufficient hydrolysis 104 of LDL-derived CE occurred such that formation, and ABCA 1-mediated efflux, of PC and SM were not significantly inhibited. Therefore, activation of ABCA1 was capable of increasing phospholipid efflux to levels equivalent to, or greater than, that caused by Ad-hLAL in the face of the LAL mutation. It is also possible that increasing ABCA1 via an LXR agonist leads to increased phospholipid efflux primarily by mediating efflux of phospholipids that reside in the plasma membrane. These experiments provided convincing results suggesting that, following correction of the LAL mutation in CESD fibroblasts and increasing the flux of UC from the lysosome, we are capable of normalizing ABCA1 function. As discussed in previous sections of this thesis ABCA1 gene transcription is tightly regulated by liver X receptor (LXR), a nuclear hormone receptor that is activated by oxysterol ligands’°°. Therefore, we conclude that the correction in ABCA1-mediated lipidation of apoA-I observed following addition of Ad-hLAL in CESD fibroblasts is a result of the increased flux of UC from the lysosomal compartment, which led to increased oxysterol formation and subsequent activation of ABCA1 expression. The accumulated results obtained over the course of this study have provided new additional evidence that lysosomally-derived cholesterol plays a key role in the regulation of ABCA1 expression. The reduced ABCA1 mRNA and protein levels in CESD fibroblasts and increased ABCA1 protein and function following delivery of Ad-hLAL suggest that lysosome-derived UC has a major role in upregulating ABCA1 expression. However, due to the unexpected mRNA results it will be necessary to repeat the quantitative RT-PCR experiments, investigating the potential adverse affects of Lipofectamine on both mRNA and therefore protein expression. It is quite possible that we would see an even greater response in ABCA1 expression and lipid efflux in both CESD and normal cells following upregulation of LAL expression and activity without the possible reduction in ABCA1 mRNA stability induced by Lipofectamine. Other potential reasons for the reduced ABCA1 mRNA response may need to be explored. The overall results, however, strongly support a role of LAL and lysosomally-derived cholesterol as a major regulator of both ABCA 1 mRNA and protein levels, and HDL formation. Additional future studies should include GC analysis of normal and CESD fibroblasts cellular UC and CE mass, as well as media UC mass, following incubation with Ad-hLAL. We had intended to conduct this experiment, however the expected readiness of our GC mass detector has taken longer to achieve than we originally predicted. In addition, our GC will also be used to measure oxysterol levels in CESD cells compared to normal cells, before and following delivery of Ad-hLAL, to determine whether increasing LAL activity also leads to 105 increased oxysterol formation. As well, it would be of interest to investigate HDL particle formation using 2-dimensional gel electrophoresis to determine the type of particles produced by CESD fibroblasts before and following incubation with Ad-hLAL. Future directions also include infecting normal and CESD fibroblasts with an adenovirus expressing full length ABCA1 cDNA, to assess the effect on ABCA1 expression, lipid efflux and HDL formation by CESD cells. Finally, current studies in the Francis laboratory involve knocking down LAL expression using siRNA in normal fibroblasts and HepG2 cells in an attempt to reproduce the CESD phenotype, which will hopefully provide further insight into a potential role for lysosomal cholesterol in ABCA1 regulation. In conclusion, although the introduction of exogenous hLAL to correct the CE accumulation in the lysosomes and increase ABCA 1 expression has potential therapeutic implications, the purpose of this study was not to develop a mechanism of treating CESD patients, as they can already live extended lives with the appropriate medications. Our purpose was to elucidate further the importance of the lysosomal UC pool as a regulator of ABCA1 expression. 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