Gladstone Institute of Cardiovascular Disease -

Cardiovascular disease continues to be the leading cause of death in the U.S., claiming more than 900,000 American lives annually. High blood pressure, coronary heart disease (heart attack and angina), congestive heart failure, and stroke account for more deaths than all other major causes combined. Cardiovascular disease is a complicated, multifactorial disease with atherosclerosis at its base. Therefore, GICD emphasizes a multidisciplinary research approach to understand risk factors for atherosclerosis, with studies in the areas of vascular biology, inflammation, fat and cholesterol metabolism, obesity, and clinical molecular genetics. The investigators committed to these areas of research within GICD are Israel F. Charo, M.D., Ph.D.; Robert V. Farese, Jr., M.D.; Robert W. Mahley, M.D., Ph.D.; and Tom Bersot, M.D.
Laboratory of Israel F. Charo
Atherosclerosis begins early in life with the formation of fatty streak deposits of lipid laden macrophages in arteries. The Charo laboratory seeks to understand why monocytes leave the bloodstream and enter the artery wall and what cues induce lipid accumulation in macrophages, thereby contributing to fatty streak formation.
Fatty streak formation in atherosclerosis. The processes leading to atherosclerotic plaque formation are depicted. Most recently, the Charo laboratory has been focusing on the step of monocyte recruitment to the vessel wall and the role that various chemokines and their receptors, such as MCP-1 and CCR2, play in this process.
Cellular interactions in the initiation of atherosclerosis. An important step in formation of athersclerotic plaques is the recruitment of monocytes (inflammatory cells) to the vasular wall. Monocytes are recruited by a cell-surface receptor, CCR2, which recognizes MCP-1, a chemokine secreted from numerous cell types at sites of plaque formation. The Charo laboratory discovered CCR2 and uncovered its role in recruiting monocytes and macrophages.
	Decreased macrophage recruitment in CCR2-deficient mice. When fed a high-fat diet, vessels (green) of mice deficient in apolipoprotein E (apo^{E^–/–) develop atherosclerotic plaques containing large numbers of macrophages (orange). However, without the CCR2 gene, the number of macrophages and degree of atherosclerosis within the vessel wall is decreased, demonstrating the requirement of CCR2 for macrophage recruitment and plaque formation.}
Laboratory of Robert V. Farese, Jr.
The ability to utilize energy in a regulated manner is fundamental to life. However, abnormalities in energy metabolism and in fat synthesis and storage play a central role in diseases such as obesity, type 2 diabetes, fatty liver disease, and atherosclerosis. Nearly all cells synthesize and store hydrophobic fats (e.g., triglycerides and cholesterol esters) in organelles called lipid droplets. These droplets serve as storage depots, providing reservoirs of lipids for fuel and other cellular needs such as membrane synthesis. The Farese laboratory cloned many of the genes that encode enzymes that synthesize hydrophobic lipids and elucidated their biochemical and physiological functions. These enzymes include those that catalyze the synthesis of triglycerides (DGAT), cholesterol esters (ACAT), diacylglycerols (MGAT), retinol esters (ARAT), and waxes (wax synthase). By studying mice that lack these enzymes, the Farese laboratory identified several new drug targets. For example, the knockout of DGAT1 results in mice that are resistant to obesity, diabetes, and fatty liver, providing the biological rationale for developing DGAT1 inhibitors.
Model of diacylglycerol acyltransferase (Dgat) enzymes and triglyceride synthesis. The membrane bound Dgat enzymes, Dgat1 and Dgat2, convert fatty acyl CoA and diacylglycerol into triacylglycerol. These triglycerides are subsequently packaged into lipid droplets. The Farese laboratory uses approaches ranging from basic biochemical and cellular studies to those involving physiology and disease in genetically modified mice to better understand the mechanisms of lipid synthesis and storage.
	Genetic knockout of the gene encoding the enzyme, Dgat1, in mice. Dgat1-deficient mice are leaner than their wild-type littermates (photo, left) due to perturbed triglyceride synthesis in the absence of Dgat1. Dgat1-deficient mice also tend to outlive their wild-type littermates (graph, below left) and studies are under way in the Farese laboratory to determine the mechanisms underlying the increased longevity of Dgat1-deficient mice.

	A screen to identify genes important for lipid storage in cells. To identify novel genes involved in fat storage, the Farese laboratory individually inactivated every gene in yeast cells carrying a green fluorescent protein to mark lipid droplets, enabling them to visualize cells with altered lipid droplet formation after gene inactivation. From this screen, they identified genes whose absence positively or negatively influenced lipid droplet formation. They are currently advancing the most interesting genes into functional studies in mice. These new studies promise two major outcomes: significant advances in understanding the processes that regulate fat metabolism in cells and novel therapeutic targets for treating diseases such as obesity and diabetes.
Laboratory of Robert W. Mahley
The Mahley laboratory has contributed greatly to our understanding of how cholesterol homeostasis contributes to atherosclerosis and coronary artery disease through extensive studies of apolipoprotein E (apoE). They described apoE’s ligand function, determined its protein and gene sequences, mapped the amino acid residues involved in receptor binding, defined the three-dimensional structure of the ligand-binding domain, and identified mutations that established its role in the pathogenesis of type III hyperlipoproteinemia. Dr. Mahley also directs an epidemiological study that identified the major risk factor for heart disease in Turkey: genetically determined low levels of plasma high density lipoproteins. The genetic nature of this disorder is under investigation.
The Turkish Heart Study. Since 1988, the Mahley lab has conducted a large-scale epidemiological study of risk factors for heart disease in six regions of Turkey (red symbols). The Turkish Heart Study has shown that high density lipoprotein cholesterol (HDL-C) levels are 10–15 mg/dl lower in Turks than in western Europeans or Americans and are associated with a 25–30% increase in hepatic lipase (HL) activity. The decrease in HDL-C is primarily genetic in origin, as it was even observed in Turks living in Germany or San Francisco.

	Genome scanning of Turkish families. Very large Turkish families offer an unusual opportunity to identify genes related to lipid disorders. For example, in this family, 171 members were analyzed by genome scanning across all chromosomes.
Genome scan of chromosome 15 identified linkage of HDL-C level to chromosome 15q22 in Turkish families. As part of a large international study (Genetic Epidemiology of the Metabolic Syndrome), Dr. Mahley conducted genome-wide scans for quantitative trait loci affecting triglyceride and HDL-C levels in 40 Turkish families with atherogenic dyslipidemia. These studies showed a significant linkage to triglycerides at chromosome 11q22 (LOD = 3.34), near the apoAI/AIV/CIII/A5 locus (not pictured), and a linkage to HDL-C at chromosome 15q22 (LOD = 3.05), near the HL locus (right).