HDL Cholesterol - Metabolic Pathways and Drug Targets

Venue: Royal Sonesta Hotel

Location: Cambridge, Massachusetts,

Event Date/Time: Feb 25, 2002 End Date/Time: Feb 26, 2002
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Monday, February 25, 2001
8:00 Registration, Poster/Exhibit Set-Up, Coffee and Pastries

8:50 Chairperson's Opening Remarks
John F. Oram, Ph.D., Research Professor of Medicine, University of Washington

9:00 Genetic Disorders of High Density Lipoproteins
Jacques Genest, M.D., FRCP(C), Professor, Faculty of Medicine, McGill University, Director, Division of Cardiology, McGill University Health Center/Royal Victoria Hospital, Montreal
Abstract not available at time of print.

9:35 The Molecular Basis of Common HDL Deficiency States
Margaret E. Brousseau, Ph.D., Scientist II & Assistant Professor of Medicine and Nutrition Science and Policy, Lipid Metabolism Laboratory, Tufts University
HDL cholesterol (HDL-C) deficiency is the most common lipid abnormality observed in patients with premature coronary heart disease (CHD). Genetic factors account for approximately 50% of the variation in HDL-C levels in the general population, with several common mutations identified in genes involved in the regulation of plasma HDL-C concentrations and, in some cases, the development of CHD. To further explore the molecular basis of common HDL deficiency states, we have determined the frequencies of polymorphisms in genes encoding for ATP-binding cassette transporter 1 (ABCA1), cholesteryl ester transfer protein (CETP), and lipoprotein lipase (LPL) in men participating in the Veterans Affairs HDL-C Intervention Trial (VA-HIT), a study designed to examine the potential benefits of HDL raising in men having CHD and low HDL-C (<40 mg/dL). Our data suggest that each of these common variants contributes to the pathogenesis of HDL deficiency in men with CHD, with the CETP TaqIB variant having the greatest effect on HDL-C levels in this population.

ABCA1 Transporter Protein
10:10 ABCA1: The Gatekeeper For Eliminating Excess Cholesterol from Tissues
John F. Oram, Ph.D., Research Professor of Medicine, University of Washington
HDL apolipoproteins remove cholesterol from macrophages by an active transport pathway control by an ATP-binding cassette transporter called ABCA1. ABCA1 plays a critical role in transporting cholesterol from tissues into the lipoprotein metabolic pathway and is likely to be a major anti-atherogenic molecule. ABCA1 expression is highly regulated by diverse metabolic factors. Thus, ABCA1 has become an important therapeutic target for preventing cardiovascular disease.

10:45 Refreshment Break and Poster/Exhibit Viewing

11:15 ABCA1: An Exit Strategy for Cholesterol Excess
Mason W. Freeman, M.D., Chief, Lipid Metabolism Unit, Massachusetts General Hospital, Harvard Medical School
The regulation of cellular cholesterol content is tightly regulated by most cells. Professional phagocytes defend the body against pathogens but the innate immune receptors that facilitate this process also make them vulnerable to cholesterol overload. One strategy they can deploy to protect against this overload is to up-regulate an ABC transporter that mediates cholesterol efflux to lipid acceptor proteins found in HDL. Studies of the cell biology of this receptor and its structure/function relationships will be presented.

11:50 ABCA1 Transgenic Mice: Recent Insights into the Role of ABCA1 in Reverse Cholesterol
H. Bryan Brewer, Jr., M.D., Chief, Molecular Disease Branch, National Heart, Lung and Blood Institute, NIH
The effect of overexpression of ABCA1 on the plasma lipids, lipoproteins and reverse cholesterol transport in ABCA1 transgenic mice will be discussed.

12:25 Role of ABCA1 in HDL Formation and Atherosclerosis: Studies in ABCA1-Deficient Mice
Robert J. Aiello Ph.D., Senior Research Investigator, Pfizer Global Research
Targeted disruption of the ABCA1 in mice has a major impact on HDL metabolism and cholesterol efflux from cells. The absence of ABCA1 in mice leads to a marked decrease in HDL and a large accumulation of sterols in some tissues. In hypercholesterolemic apolipoprotein E deficient mice with ABCA1 deficiency, the accumulation of cholesterol in tissue macrophages is more pronounced, but only minor effects on atherosclerotic lesion development.

1:00 Luncheon, Sponsored by The Knowledge Foundation, Inc.

Transfer Proteins/ Lipase
2:25 Chairperson's Remarks
Silvia Santamarina-Fojo, M.D., Ph.D., Chief, Section of Molecular Biology, Molecular Disease Branch, National Heart, Lung, and Blood Institute, NIH

2:30 An Immunotherapeutic Approach for the Treatment of Low Plasma HDL Cholesterol: Phase 1 Clinical Studies of CETi-1
Charles W. Rittershaus, Director, Discovery Research, AVANT Immunotherapeutics, Inc.
The inhibition of cholesteryl ester transfer protein (CETP) activity has been proposed as a therapeutic strategy to raise the plasma concentration of HDL-cholesterol. CETi-1 vaccine has been designed to induce the production of antibodies that specifically bind to and inhibit the function of endogenous CETP and thereby elevate plasma HDL-cholesterol concentration, which could be of significant clinical value in the treatment of atherosclerosis.

3:05 Lipases and HDL: Lessons Learned from Induced Mutant Mouse Lines
Rudolf Zechner, Ph.D., Institute of Molecular Biology, Biochemistry, and Microbiology University of Graz, Austria
The generation of induced mutant mouse lines that lack or overexpress lipoprotein lipase in various tissues permitted extensive studies and revealed important insights on the role of the enzyme for the maturation of HDL. Additionally, recent studies demonstrated that the expression levels of other lipases such as hormone-sensitive lipase and endothelial lipase also affects the metabolism of HDL by various mechanisms. Taken together lipases and lipolysis are important factors in the regulation of HDL cholesterol levels.

3:40 New Insights into the Role of HL in Lipoprotein Metabolism and Atherosclerosis
Silvia Santamarina-Fojo, M.D., Ph.D., Chief, Section of Molecular Biology, Molecular Disease Branch, National Heart, Lung, and Blood Institute, NIH
Abstract not available at time of print.

4:15 Refreshment Break and Poster/Exhibit Viewing

Scavenger Receptor B
4:45 Scavenger receptor class B type I (SR-BI) and alpha-tocopherol (alpha-T) metabolism
Attilio Rigotti, Assistant Professor, Gastroenterology Department, Catholic University School of Medicine, Santiago, Chile
Recent studies have suggested that selective lipid uptake is important for alpha-T delivery to cells. We have found that SR-BI, which mediates selective lipid uptake, facilitates efficient transfer of alpha-T from HDL to cultured cells. In SR-BI-deficient mice, relative to wild-type animals, there was a significant increase in plasma alpha-T levels that was mostly due to elevated alpha-T content in abnormally large plasma HDL particles. This finding was associated with a significant decrease in alpha-T content of both the bile and several tissues, including ovary, testis, lung and brain of SR-BI knockout mice. Defective tissue uptake of lipoprotein alpha-T in SR-BI-deficient mice may contribute to the reproductive and cardiovascular abnormalities found in these animals.

5:20 Scavenger Receptor BI: Recent Advances
David L. Williams, Ph.D., Professor and Interim Chair, Pharmacological Sciences, University Medical Center, SUNY at Stony Brook
Scavenger receptor BI (SR-BI) is an HDL receptor that mediates the selective uptake of cholesteryl ester into the liver and steroidogenic cells where it is expressed at high levels. SR-BI is also expressed at lower levels in macrophages in atherosclerotic lesions and in endothelial cells of the vasculature. In addition to its activity in mediating the selective uptake of cholesteryl ester from HDL, SR-BI enhances the flux of free cholesterol between cells and lipoproteins and alters the distribution of cholesterol in plasma membrane domains. Plasma membrane morphology is altered in cells overexpressing SR-BI in culture and in adrenal cells from the SR-BI knockout mouse, suggesting that SR-BI plays a role in the organization of plasma membrane domains that may be involved in cholesterol flux. Thus, SR-BI influences the uptake and distribution of both HDL cholesteryl ester and free cholesterol and may play a key role in reverse cholesterol transport.

5:55 The Role of Apolipoprotein A-II in HDL Metabolism and Insulin Resistance
Lawrence W. Castellani, Ph.D., Associate Research Cardiologist, Department of Medicine/Cardiology, University of California at Los Angeles
The function of apolipoprotein A-II in HDL metabolism is unknown. ApoA-II does not appear to be important for the anti-atherogenic properties of HDL. Studies with both apoA-II transgenic and apoA-II knockout mice have demonstrated a role in insulin resistance. Data on the altered metabolism in the apoA-II transgenic mice will be presented, as well as recent findings suggesting that apoA-II can cause insulin resistance in humans.

6:30 End of Day One

Tuesday, February 26, 2002
8:00 Poster/Exhibit Viewing, Coffee and Pastries

Scavenger Receptor B
8:20 Chairperson's Opening Remarks
Attilio Rigotti, Assistant Professor, Catholic University School of Medicine, Santiago, Chile

8:25 SR-BI Action in Endothelial Cells
Philip W. Shaul, M.D., Department of Pediatrics, University of Texas Southwestern Medical Center
SR-BI expression has recently been described in endothelial cells, where the receptor mediates potent effects of HDL on nitric oxide (NO) production. The capacity for endothelial SR-BI to modify endothelial NO synthase (eNOS) subcellular localization will be considered in relation to the adverse effects of oxidized LDL on the enzyme. The mechanisms by which HDL binding to SR-BI stimulates nitric oxide production will also be discussed. Gaining further insight into SR-BI action in endothelial cells will enhance our understanding of the anti-atherogenic properties of HDL.

9:00 Mechanisms of Polar HDL Transport and Sorting
David Silver, Ph.D., Associate Research Scientist, Columbia University
The mechanism by which HDL donates cholesterol for polar secretion into bile by the liver has not been known. Here evidence will be presented showing that HDL cholesterol is independently sorted from HDL apoprotein leading to the secretion of HDL cholesterol from the apical membrane. SR-BI is expressed in a polar fashion in MDCK cells that leads to the polar secretion of HDL cholesterol from basolateral to apical membrane. In addition, an analysis of SR-BI structure/function and its interaction with a PDZ domain containing protein, PDZK1, in relation to polar sorting will be presented.

Nuclear Receptors
9:35 Transcriptional Regulatory Mechanisms of the Human ApoA-I/ApoCIII Gene Cluster in vitro and in Transgenic Mice: The Role of Nuclear Receptors
Vassilis I. Zannis, Ph.D., Professor, Medicine and Biochemistry, Director, Section of Molecular Genetics, Boston University School of Medicine
The human apoA-I and apoCIII genes utilize a common enhancer and their activity is modulated by nuclear receptors and Smad proteins. The JNK NFkB and TGFb signal transduction pathways affect the activity of the apoCIII promoter and enhancer. Studies in transgenic mice established that the hormone response element (HRE) and the Sp1 sites of the apoCIII enhancer are required for the intestinal expression and contribute to the hepatic expression of the two genes. The proximal apoA-I promoter or the apoCIII enhancer can independently drive the hepatic transcription of the apoA-I gene. Combination of both results in transcriptional synergism in vivo.

10:10 LXR is an Endogenous Inhibitor of Atherosclerosis
Richard A. Heyman, Ph.D., Vice President of Research, X-Ceptor Therapeutics

The orphan nuclear receptors, LXRa and LXRb (Liver X Receptors), function as ligand dependent transcription factors that serve as important regulators of cholesterol metabolism and transport. Ligand activation of the LXRs controls the transcription of genes critical to a broad range of biological functions including increasing reverse cholesterol transport in peripheral cells, cholesterol catabolism in hepatocytes and inhibition of intestinal sterol absorption. We have used bone marrow transplantation as an experimental model to selectively knockout LXRs in macrophages in murine models of atherosclerosis. The results demonstrate that LXRs are potent endogenous inhibitors of atherogenesis. Selective elimination of LXR activity in macrophages mimics many aspects of Tangier disease, including lipid accumulation in macrophages, increased atherosclerosis and splenomegaly. These results suggest that LXR is a novel target for intervention in cardiovascular disease.

10:45 Refreshment Break and Poster/Exhibit Viewing

11:15 Agonists of Orphan Nuclear Receptors: Relationship between Induction of Apo E Synthesis in Cultured Cells and HDL Levels in Animal Models
Eric J. Niesor, Ph.D., Director of Biosciences and Pharmacology, ILEX Research-Europe, Geneva, Switzerland
The orphan nuclear receptors FXR and LXRa have been shown to control a number of genes involved in cholesterol and HDL metabolism. Natural and synthetic modulators of these receptors can be tested in vitro in the monocyte-macrophage (THP-1) and in the hepatoma (HepG2) cell lines to determine their effect on Apo E mRNA expression and secretion in culture medium. Selected compounds where synthesized in sufficient quantity to be administered orally to rodent and non-rodent species. The levels of plasma HDL cholesterol and apoproteins were measured in an attempt to correlate the results obtained in vitro with in vivo activities.

11:50 Regulation of Lipid Metabolism by the Nuclear Receptor PPARd
David G. Hassall, GlaxoSmithKline
The Peroxisome Proliferator Activated Receptors (PPARs) play important roles in the regulation of lipid and glucose metabolism. GW501516 is a potent and selective PPARd agonist, which was developed with the aid of structure-based drug design. The pharmacology of GW501516 has established PPARd as a key regulator of macrophage lipid metabolism and HDL-cholesterol levels in non-human primates.

12:25 Lunch on Your Own

1:45 Selective Modulation of RXR and Its Heterodimeric Partners in the Treatment of Obesity and Dyslipidemia
Magnus Pfahl, Ph.D. Executive Chairman, Chief Scientific Officer, MAXIA Pharmaceuticals, Inc.
¥ Recognizing the varied disease targets of obesity and dyslipidemia ¥ The limitations of our current therapeutics, and the challenges for the next generation of anti-dyslipidemic drugs ¥ Nuclear receptors as targets for the treatment of obesity and dyslipidemia ¥ Selective modulation of nuclear receptors with novel small molecule therapeutics ¥ Proof of Principle ¥ Future challenges.

New Drug Targets
2:20 Chairperson's Remarks
Daniel J. Rader, M.D., Associate Professor of Medicine, University of Pennsylvania Medical Center

2:25 Pharmacological Profile of Novel Lipid Synthesis Inhibitors in Models of Dyslipidemia, Diabetes and Obesity
Michael E. Pape, Ph.D., Senior Director, Cellular and Molecular Biology, Esperion Therapeutics
The statin class of drugs inhibits cholesterol biosynthesis resulting in marked reduction of plasma LDL-C and triglycerides in humans and relevant animal models. Although statins are potent hypolipidemic agents they do not significantly raise HDL-C or affect obesity and diabetes parameters. In light of this we have developed dual inhibitors of cholesterol and fatty acid synthesis with the hope of identifying compounds that elevate HDL-C and affect aspects of obesity and diabetes. We will describe the effects of these dual inhibitors on lipoprotein metabolism, nuclear hormone receptors, and relevant animal models of metabolic syndrome and atherosclerosis.

3:00 HDL Remodelling: Target for Therapeutic Intervention?
Daniel J. Rader, M.D., Associate Professor of Medicine, University of Pennsylvania Medical Center
HDL is extensively remodelled in the plasma compartment by lipases and lipid transfer proteins. This remodelling process has substantial effects on the rate of HDL turnover and HDL cholesterol and apoA-I levels. Targeting components of the remodelling process to delay the catabolism of HDL is an attractive strategy for raising plasma HDL cholesterol and apoA-I levels.

3:35 Refreshment Break and Poster/Exhibit Viewing

4:00 What is the Utility of an HDL Mimetic in the Treatment of Cardiovascular Disease?
Charles L. Bisgaier, Ph.D., Vice-President, Discovery Pharmacology, Esperion Therapeutics
Acute coronary syndromes are the result of plaque rupture in the coronary arteries. Lesions within these vessels contain lipid-rich macrophage-foam cells, thin fibrous caps and are rupture-prone. We are investigating the use of various preparations of synthetic HDL to enhance reverse lipid transport systemically with potential utility to acutely reduce lipid and macrophage content of diseased blood vessels. We are investigating additional therapeutic applications for these preparations.

4:35 Crystal Structures Of Human Squalene Synthase Enzyme: Inhibitor Complexes
Jay Pandit, Senior Research Investigator, Pfizer
Global Research and Development
We have determined the crystal structure of recombinant human squalene synthase (rhSQS) to assist in the discovery of selective, potent inhibitors of SQS which could be developed as potential anti-hypocholesterolemic drugs. Squalene synthase catalyzes the biosynthesis of squalene, a key cholesterol precursor, through a reductive dimerization of two C15 farnesyl diphosphate (FPP) molecules. Since FPP is located at the final branchpoint in the isoprenoid biosynthesis pathway, its conversion to squalene through the action of SQS represents the first committed step in the formation of cholesterol. The structures of rhSQS complexed with different inhibitors, in combination with biochemical and mutagenesis data have also provided insights into critical features underlying the unique two-step reaction mechanism of this enzyme.

5:15 End of Conference