Post-congress, Kos Animal models of cardiovascular disease and gene therapy 165

CE-HDL kinetic and liver studies utilizing HDL isolated from all 3 double transgenic lines demonstrated normalization of the dysfunctional properties of LCAT-transgenic mice HDL and restored reverse cholesterol transport. Overexpression of apoA-I and CETP as well as HL-deficiency significantly reduced or abolished diet-induced aortic atherosclerosis emphasizing the importance of functional HDL in the prevention of atherosclerosis in LCAT- transgenic mice. Our studies provide new insights into the coordinate role that apoA-I, HL, CETE and LCAT play in HDL metabolism and function as well as into potential mechanisms that underlie the differences in atherosclerosis susceptibility between LCAT transgenic mice and rabbits.

ANALYSIS OF LIPOPROTEIN REMOVAL PATHWAYS IN TRANSGENIC AND KNOCKOUT MICE

J. Herz. Department of Molecular Genetics, UT Southwestern, Dallas, TX 75235. USA

The complex pathways that regulate cholesterol and triglyceride transport through the circulation converge on the cell surface where proteoglycans, receptors and lipolytic enzymes work together to mediate the uptake of the lipids into the target tissues. The low density lipoprotein (LDL) receptor and the LDL receptor-related protein (LRP), in concert, play pivotal roles in the hepatic removal of apoE containing lipoproteins into the liver. Adenoviral gene transfer of RAP, a universal inhibitor of ligand binding to LRP, completely prevents the removal of chylomicron remnants in LDL receptor deficient mice, causing marked hypertriglyceridemia and hyper- cholesterolemia. In contrast, liver-specific inactivation of the LRP gene in LDL receptor knockout mice also causes hypercholesterolemia, but no significant hypertriglyceridemia, suggesting that RAP also affects other LRP independent pathways that participate in the metabolism of chylomicron remnants. Analysis of these distinct animal models suggests that RAP acts in the periphery by modulating lipoprotein lipase activity.

ANALYSIS OF THE HDL RECEPTOR SR-BI IN MICE USING ADENOVIRUS-MEDIATED HEPATIC OVEREXPRESSION AND TARGETED GENE DISRUPTION

M. Krieger. Department o f Biology; Massachusetts Institute o f Technology. 77 Massachusetts Avenue, Cambridge, MA, 02139, USA

Risk for cardiovascular disease due to atherosclerosis increases with in- creasing concentrations of low density lipoprotein (LDL) cholesterol and is inversely proportional to the levels of high density lipoprotein (HDL) cholesterol. The receptor-mediated control of plasma LDL levels has been well understood for over two decades and has been a focus for the pharmaco- logic treatment of hypercholesterolemia. In contrast, the first identification and characterization of a receptor which mediates cellular metabolism of HDL was only recently reported. This receptor, called scavenger receptor class B type I or SR-BI, is a fatty acylated glycoprotein which can cluster in caveolae-like domains on the surfaces of cultured cells. SR-BI mediates selective lipid uptake from HDL to cells. The mechanism of selective lipid uptake is fundamentally different from that of classic receptor-mediated endocytic uptake via coated pits and vesicles (e.g., the LDL receptor pathway) in that it involves efficient receptor-mediated transfer of the lipids, but not the outer shell proteins, from HDL to cells. In mice, SR-BI plays a key role in determing the levels of plasma HDL cholesterol, in mediating the regulated, selective delivery of HDL-cholesterol to steroidogenic tissues and the liver and in the development of atherosclerosis. SR-BI may play a similar role in humans; thus, modulation of its activity may provide the basis of future approaches to the treatment and prevention of cardiovascular, and possibly other, disorders.

THE ROLE OF APOLIPOPROTEINS, RECEPTORS, AND ENZYMES IN GENETIC MODELS OF HDL METABOLISM AND ATHEROSCLEROSIS

H.B. Brewer Jr.. National Heart, Lung, and Blood Institute, Molecular Disease Branch, Bethesda, MD, USA

Over the last two decades epidemiological studies have established that low plasma high density lipoprotein (HDL) levels are associated with an increased risk of cardiovascular disease (CVD). HDL has been proposed to modulate the development of atherosclerosis by facilitating the transport of cholesterol back from peripheral cells to the liver by reverse cholesterol transport. Increasing plasma HDL has been proposed to decrease the risk

of premature cardiovascular disease. ApoA-l, lecithin cholesterol acyltrans- ferase (LCAT) and the SR-BI receptor, which facilitates the selective uptake of cholesterol esters (CE) without whole particle catabolism, are critical components of the reverse cholesterol pathway. Overexpression of apoA-I in mice and rabbits as well as kindreds with selective increased synthesis of apoA-I is associated with a decreased risk of cardiovascular disease. Re- cently we have established that overexpression of LCAT has been identified using a combination of transgenic mice and rabbits as well as recombinant adenovirus mediated gene transfer as a potential approach to the treatment of low plasma HDL. Studies in LCAT transgenic rabbits established that overexpression of LCAT results in markedly increased plasma HDL levels, reduced LDL levels and significant protection against atheroselerosis. The importance of SR-BI in reverse cholesterol transport and protection has not been definitively established. In order to evaluate the role of SR-BI in protection against cardiovascular disease, we have overexpressed SR-BI using adenovirus vectors in the atherosclerosis prone apoE-knockout mice. Expression of SR-BI decreased total and apoB lipoprotein cholesterol levels without reducing plasma apoB by a selective uptake pathway. Liver uptake of CE was increased in the SR-B! expressing mice when compared to control mice expressing luciferase. These results define a new in vivo pathway for the selective removal of CE from proatherogenic apoB-lipoproteins mediated by SR-BI. These combined results suggest that overexpression of apoA-l, LCAT and SR-B! are potential candidate genes for modulating the development of premature cardiovascular disease.

DISRUPTION OF THE LDL RECEPTOR GENIE IN TRANSGENIC SREBP-la MICE UNMASKS HYPERLIPIDEMIA AND ATHEROSCLEROSIS RESULTING FROM THE PRODUCTION OF LIPID-RICH VLDL

J.D. Horton, H. Shimano, R.L. Hamilton, M.S. Brown, J.L. Goldstein. Affiliation missing, USA

Transgenic mice that overexpress the nuclear form of sterol regulatory element-binding protein- I a (SREBP- I a) in liver (TgBP-I a mice) were shown previously to overproduce cholesterol and fatty acids and to accumulate massive amounts of cholesterol and triglycerides in hepatocytes. Despite the hepatic overproduction of lipids, the plasma levels of cholesterol (% 45 mg/dl) and triglycerides (96055 mg/dl) were not elevated, perhaps owing to degradation of lipid-enriched particles by the LDL receptors. To test this hypothesis, we bred TgBP-la mice to LDL receptor knockout mice. As previously reported, LDLR -/- mice manifested a moderate elevation in plasma cholesterol (%o215 mg/dl) and triglyc~;rides (%0155 mg/dl) on a chow diet. In contrast, the doubly mutant TgBP-Ia; LDLR -/- mice exhibited marked increases in plasma cholesterol (%01050 mg/dl) and triglycerides (960900 mg/dl). These lipids were contained predominantly within large VLDL particles that were relatively enriched in cholesterol and apoprotein E. Isolated hepatocytes from TgBP-Ia and TgBP-la; LDLR -/- mice over- produced cholesterol and fatty acids and secreted increased amounts of these lipids into the medium. Electron micrographs of livers from TgBP-la mice showed large amounts of enlarged lipoproteins within the secretory pathway. We conclude that the TgBP-Ia mice produce large lipid-rich lipoproteins, but these particles do not accumulate in plasma because they are degraded through the action of LDL receptors.

MACROPHAGE SPECIFIC OVER.EXPRESSION OF THE HUMAN MACROPHAGE SCAVENGER RECEPTOR, USING A 180 kb YEAST ARTIFICIAL CHROMOSOME, REDUCES ATHEROSCLEROSIS IN LOW DENSITY LIPOPROTEIN DEFICIENT MICE

M.P.J. De Winther, K. Willems van Dijk, L.M. Havekes, M.H. Hotker. Leiden Univ Med Ctr and TNO-PG Gaubias Laboratory, Leiden, The Netherlands

Macrophage scavenger receptors class A (MSR) are thought to play an important role in atherogenesis by mediating the unrestricted uptake of modified lipoproteins by macrophages in the vessel wall leading to foam cell formation. To investigate the in vivo role of the MSR in this process, a transgenic mouse model expressing both isoforms of the human MSR was generated. A 180 kb yeast artificial chromosome (YAC) containing the human MSR gene (MSRI) with 60 kb and 40 kb flanking sequence at the 5' and 3' end, respectively, was obtained by reducing the size of a 1050 kb YAC by homologous recombination. This 180 kb YAC was microinjected into mouse oocytes. In the resulting transgenic mice, high levels ofmRNA for both type I and type It human MSRl were detected in peritoneal macmphages and trace levels in other organs, known to contain

71st EAS Congress and Satellite Symposia

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