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Conference Topics 1:15 Chairperson’s Opening Remarks John F. Oram, Ph.D., Department of Medicine, University of Washington Raising HDL-C 1:20 Developing the Next Drugs for Atherosclerosis: the Promise of HDL-Raising Drugs Donald M. Black, M.D., Vice President, Global Strategic Development, Merck and Company The development of drugs for reducing cardiovascular disease have been primarily focused on reducing LDL-cholesterol. New research has lead to the development of compounds that can reliably increase HDL-cholesterol. The approach to the clinical testing of these drugs will be challenging, but will provide opportunities to assess the best approach to addressing a key medical need. 1:50 Paraoxonase 1: A Multifunctional Enzyme With Anti-Atherosclerotic Properties Trudy M. Forte, Life Sciences Division, Lawrence Berkeley National Laboratory Paraoxonase 1 (PON1) is transported on HDL and is believed to protect against atherosclerosis. In mice overexpressing mouse PON1, the enzyme is increased in HDL and the particles appear to have increased protective functions against oxidation events. Studies with recombinant PON1 (rPON1) suggest that the enzyme has a non-catalytic antioxidative function associated with its free thiol at residue C284. During oxidation by lipoxygenase, wt rPON1, but not the C284A rPON1 mutant, maximally protects against oxidation. This protective activity of rPON depends upon association of the enzyme with a lipid surface. The rPON1 also possesses a catalytic activity similar to platelet activating-factor acetylhydrolase that is not dependent on C284 or calcium. Together, these two activities, one non-catalytic and the other catalytic may be functionally important in protection against atherosclerosis. 2:20 Discovery of HDL376, a Novel Thiourea-Based HDL-Elevating Agent Gary M. Coppola, Senior Scientist II, Diabetes and Metabolism Disease Area, Chemistry, Novartis Institutes for Biomedical Research One of the risk factors of coronary heart disease is reduced HDL cholesterol levels. Our goal was to develop a new chemical entity which raised HDL cholesterol at least twice that of gemfibrozil. This presentation will describe the identification of the thiourea SDZ 45-904 as an HDL-elevating agent and subsequent optimization both chemically and analytically to the development compound HDL376. Nuclear Receptors 2:50 The Role of Nuclear Hormone Receptors in the Regulation of HDL Metabolism During Infection and Inflammation Kenneth Feingold M.D., Professor of Medicine, University of California at San Francisco The effect of inflammation and infection on HDL metabolism and the link with nuclear hormone receptors will be discussed. 3:35 Chairperson’s Remarks Alan T. Remaley, M.D., Ph.D., Senior Staff, National Institutes of Health 3:40 Second Generation RXR Agonists as Regulators of Dietary Cholesterol Magnus Pfahl, Ph.D., President and CEO, MaxoCore Pharmaceuticals, Inc. The nuclear retinoid X receptors (RXRs) play key roles in regulating fat metabolism by serving as obligatory co-receptors for PPARs, FXR, and LXR. In vitro and in vivo data have shown that RXR ligands can affect critical pathways affecting cholesterol uptake and secretion. However, first generation RXR agonists, similar to LXR agonists, lead to unacceptable increases in triglyceride levels in animal models. We will describe second generation RXR agonists that effectively control dietary cholesterol, increasing HDL and decreasing LDL in the absence of triglyceride increases. Their mechanism of action will be discussed. 4:10 LXRs, ABCs and Cholesterol Balance Joyce J. Repa, Ph.D., Assistant Professor, Departments of Physiology and Internal Medicine, Touchstone Center for Diabetes Research, UT Southwestern Medical Center 4:40 Role of LXR in Lipid Metabolism and Cardiovascular Disease Brandee Wagner, X-Ceptor Therapeutics, Inc. LXR modulators regulate key genes in macrophages that control cholesterol transport, including members of the ABC transporters, as well as genes involved in inflammation. Loss of LXR expression or ligand activation of the receptor both lead to increase macrophage ABCA1 expression and serum HDL levels suggesting that LXR can both positively and negatively regulate target gene activation. In rodent models of atherosclerosis, LXR modulators not only reduce the progression of atherosclerosis but cause regression of atherosclerotic plaques. Bone marrow transplant studies demonstrate that the macrophage LXR is necessary and sufficient for the anti-atherogenic effect of the ligand. These data suggest that regulation of cholesterol transport and inflammatory signaling in the macrophage contributes to the anti atherogenic activity of LXR. 5:10 Target Gene Regulation and HDL Induction in Mice by Liver X Receptors: Implications for Atherosclerosis and Potentially Other Diseases Guoqing Cao, Ph.D., Cardiovascular Discovery, Lilly Research Laboratories The talk will discuss the factors involved in LXR mediated HDL metabolism, new LXR target genes and the implication for multiple diseases. Tuesday, March 9, 2004 8:25 Chairperson’s Remarks Norman E. Miller, Professor, London, UK CETP/ HL 8:30 Role of Hepatic Lipase as a Multifunctional Protein that Modulates Lipoprotein Metabolism and Atherogenic Risk Santamarina-Fojo, Silvia, MDB, NHLBI, National Institutes of Health Hepatic lipase (HL) is a multifunctional protein that plays a major role in lipoprotein metabolism as both, a lipolytic enzyme that hydrolyzes triglycerides and phospholipids present in circulating plasma lipoproteins and as a ligand that facilitates the uptake of lipoproteins and/or lipoprotein lipids by cell surface receptors and proteoglycans. Recently, we have identified an alternative pathway by which HL may modulate atherogenic risk, separate from HL-mediated changes in plasma lipid and lipoprotein concentrations. Bone marrow transplantation studies demonstrate that production of hepatic lipase by mouse macrophages present in aortic lesions markedly alters aortic lesion formation, even in the absence of changes in plasma lipids. The concept of hepatic lipase as mainly a lipolytic enzyme that reduces atherogenic risk has evolved into that of a complex protein with multiple functions which, depending on genetic background and sites of expression, can have a variable effect on atherosclerosis. In collaboration with: Gonzalez, Herminia, MDB, NHLBI, NIH, USA; Nong, Zengxuan, MDB, NHLBI, NIH, USA; Paigen, Beverly, Bar Harbour, Maine, USA; Najib-Fruchart, Jamila, Pasteur Institute, Lille, France; and Freeman, Lita, MDB,NHLBI,NIH, USA 9:00 Natural Genetic Variation and Its Use as a Tool in Understanding the Regulation of HDL Cholesterol John F. Thompson, Cardiovascular and Metabolic Diseases, Pfizer Global Research and Development Cholesterol ester transfer protein (CETP) has been the subject of much study for its role in affecting HDL levels in humans. Resequencing the gene from multiple individuals and association of the polymorphisms with CETP mass and HDL cholesterol has allowed a better understanding of CETP’s role. The CETP ass ociation studies will be contrasted with the results from other genes linked to HDL cholesterol including endothelial lipase, LXR alpha, and LXR beta. Other genetic approaches directed at better understanding the regulation of HDL cholesterol will also be presented. 9:30 Phase 2 Results with CETi-1, an Immunotherapeutic Vaccine to Raise HDL-C Charles W. Rittershaus, Director, Discovery Research, AVANT Immunotherapeutics, Inc. CETi-1 is a vaccine designed to elicit antibodies that bind to and inhibit endogenous CETP function in order to raise plasma HDL-C concentrations. In a placebo-controlled, dose-escalating Phase 2 clinical trial, CETi-1 was shown to be well-tolerated and Immunogenic, eliciting anti-CETP antibodies in > 90% of those vaccinated in the high dose group. In the overall treated population, CETi-1 raised plasma HBL-C significantly from baseline, but not compared to placebo. However, in a pre-defined sub-group analysis, CETi-1 raised plasma HDL-C significantly compared to both baseline and placebo. Details of the Phase 2 study will be presented. Scavenger Receptor B 10:30 Analysis of the HDL Receptor SR-BI Monty Krieger, Thomas D. & Virginia W. Cabot Professor, Biology Department, Massachusetts Institute of Technology 11:00 SCARB1: Beyond HDL-C. On the Crossroad of Diabetes and Clinical Disease Jose M. Ordovas, PhD, Director, Nutrition and Genomics, Professor Nutrition and Genetics, JM-USDA-HNRCA at Tufts University The scavenger receptor class B type I (SCARB1) is a key component in the reverse cholesterol transport pathway. We have reported associations between common polymorphisms at this locus and plasma lipids and body mass index. Moreover, we have shown that SCARB1 gene variation modulates the lipid profile, particularly in type 2 diabetes, contributing to the metabolic abnormalities in these subjects. Finally, our data provide substantial evidence for a QTL on chromosome 12 influencing ICA IMT, and association of a rare variant of SCARB1, or a nearby locus, with ICA IMT. Therefore, this locus plays an important role on the risk of metabolic syndrome and cardiovascular disease. 11:30 PDZK1, a Novel PDZ Domain-Containing Protein Involved in Lipoprotein Metabolism Olivier Kocher, M.D., Ph.D., Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School 1:25 Chairpersons Remarks Jose M. Ordovas, PhD, Director, Nutrition and Genomics, Professor Nutrition and Genetics, JM-USDA-HNRCA at Tufts University ABCA1 1:30 Expression and Activity Modulators of the Cell Cholesterol Transporter ABCA1 John F. Oram, Ph.D., Department of Medicine, University of Washington Population studies have shown an inverse relationship between plasma HDL levels and risk for cardiovascular disease, implying that factors associated with HDL metabolism are atheroprotective. One of these factors is a cell membrane ATP-binding cassette (ABC) transporter called ABCA1, which mediates the transport of excess cholesterol from cells to HDL apolipoproteins. ABCA1 mutations cause a severe HDL deficiency syndrome characterized by cholesterol deposition in tissue macrophages and prevalent atherosclerosis. Genetic manipulations of ABCA1 expression in mice also affect plasma HDL levels and atherogenesis. The cellular content and lipid transport function of ABCA1 are highly regulated. Cellular sterols induce ABCA1 gene expression, and apolipoproteins and different metabolic factors modulate ABCA1 protein stability. In addition, distinct signal transduction pathways regulate ABCA1 activity. These studies have identified novel cellular processes that may impair ABCA1-mediated lipid transport in atherogenic metabolic disorders and are candidate therapeutic targets for treating cardiovascular disease. 2:00 Molecular Dynamics of ApoA-I: The Complementary Roles of the N and C-terminal Regions Michael N. Oda, Ph.D., Associate Staff Scientist, Children’s Hospital Oakland Research Institute During lipid association apoA-I undergoes a dramatic conformational transition. A majority of this conformational transition occurs at apoA-I’s C-terminal residues (188 - 210) in a manner analogous to the conformational trigger found in viral fusion proteins. To determine whether the stimulus derived regulation observed in viral fusion proteins is present in apoA-I, we examined its N-terminal residues (1 - 81) through a combination of electron paramagnetic resonance spectroscopy and fluorescence resonance energy transfer studies. Our results suggest that this region of apoA-I may stabilize the lipid-free conformation of the apoA-I C-terminus and participate in the regulation of apoA-I’s conformational transition. 3:00 Metabolic Effects of Intravenous Infusion of Apolipoprotein A-I / Phosphatidylcholine (apo a-I/PC) Discs in Humans Norman E. Miller, Professor, London, UK To gain insight into the anti-atherogenic action of HDLs, apo A-I/PC discs (ZLB, Swiss Red Cross, Bern) were infused into healthy males (40 mg apo A-I/kg over 4 h) under metabolic ward conditions. Changes in peripheral tissue fluid were followed by continuous collection of prenodal lymph from a vessel in the leg. Increases were observed in (1) the production of pre-beta HDLs in plasma, (2) pre-beta HDL conc in tissue fluid, (3) efflux of unesterified cholesterol (UC) from tissues into tissue fluid HDLs, (4) esterification of cholesterol in plasma, (5) plasma HDL cholesterol concentration, and (6) synthesis and fecal excretion of bile acids. CETP concentration and PLTP specific activity also increased in plasma. In a single subject with familial LCAT deficiency, plasma pre-beta apo A-I and HDL-UC concentrations increased (no lymph data). In normal subjects plasma LDLs became depleted of minimally modified LDLs, as assessed in human arterial endothelium-smooth muscle cell co-cultures. Plasma PON concentration was unchanged. PAF-AH redistributed from LDLs to HDLs in plasma. These findings suggest that elevation of plasma HDLs protects against atherosclerosis both via increased reverse cholesterol transport, and diminished oxidation of LDLs. [Parts of this work were done in collaboration with UCLA Department of Cardiology, Polish Academy of Sciences, and BML Inc, Japan.] 3:30 Interaction of Apolipoproteins with the ABCA1 Transporter and Their Structural Motifs that Promote Lipid Efflux. Alan T. Remaley, M.D., Ph.D., Senior Staff, National Institutes of Health ABCA1, an ABC transporter that is defective in Tangier disease, mediates the cellular efflux of lipids to extracellular apolipoproteins. The nature of the interaction of ABCA1 with apolipoproteins is not well understood. Results from studying a wide variety of apolipoproteins and synthetic peptides indicate that the type A amphipathic helix is an important structural motif for a protein to efflux lipid by ABCA1 partly because of its ability to microsolubilize membranes. 4:00 The Role of Ceramide in the Modulation of ABCA1-Mediated Cellular Cholesterol Efflux W. Sean Davidson, Ph.D., Associate Professor, Department of Pathology and Laboratory Medicine, University of Cincinnati We have discovered that ceramide stimulates cholesterol efflux from cells in a manner that is specific for lipid-free apolipoproteins and requires preexisting ABCA1 expression. Ceramide increases the cell surface localization of ABCA1, apolipoprotein binding and the cell surface delivery of cholesterol. However, the effect is abolished in cells lacking the Niemann-Pick C1 protein. Our current model is that ceramide enhances the vesicular delivery of either ABCA1 or cholesterol to the membrane for export. As gene induction strategies for enhancing ABCA1 activity are proving to be problematic, it will be important to explore such post-transcriptional mechanisms of ABCA1 regulation for possible therapeutic use.