Selective oxidation of methionine residues enhances rather than decreases potential anti-atherogenic properties of apolipoprotein ai

Thursday 27 May 1999 Workshop: Modi[ied lipoptrJteins, their receptors and potential roles in atherosclemsis NEW INSIGHTS INTO THE ROLE OF HDL IN REVERSE C H O L E S T E R O L TRANSPORT AND ATHEROSCLEROSIS H.B. Brewer, Jr.. National Heart. Lung. and Blood Institute, Molecular Disease Branch. Bethesda. MD, USA Epidemiological evidence has clearly established that increased and decreased plasma high density lipoprotein (HDL) levels are associated with decreased and increased risk of cardiovascular disease (CVD), respectively. The current practice o f routine HDL screening in clinical medicine has identify a large cohort of individuals with low HDL levels some of which have associated elevated plasma triglycerides and dense low density lipoproteins (LDL). HDL has been proposed to protect against the development of CVD by facilitating the transport of cholesterol in peripheral cells back to the liver for removal from the body. a process termed reverse cholesterol transport. There is increasing evidence primarily from transgenic animal studies that raising HDL by increasing apoA-I results in protection against diet induced atherosclerosis. Initial studies in LCAT transgenic rabbits established that overexpression of LCAT results in markedly increased plasma HDL cholesterol (C) levels, reduced LDL-C levels and significant protection against diet induced atherosclerosis. Kinetic studies established that the elevated HDL and reduced LDL levels were due to decreased catabolism and increased catabolism, respectively. The role of LCAT in modulating HDL levels has recently been extended to the non-human primate. Adenovirusmediated expression of LCAT in monkeys resulted in a marked increase in plasma HDL-C and decreased LDL-C levels similar to results observed in LCAT transgenic rabbits. In contrast to the kinetic studies in the rabbit, HDL kinetic studies in monkeys established that the increased HDL-C was due to a combination of increased synthesis and increased catabolism of apoAI. These combined results indicate that apoA-I and LCAT represent ideal candidate genes for gene therapy as well as a target for drug development for the treatment of atherosclerosis in man.

Workshop: Modified lipoproteins, their receptors and potential roles in atherosclerosis IMPLICATION OF ENZYMATIC LIPID PEROXIDATION IN EARLY ATHEROGENESIS. PRO- AND/OR ANTI-ATHEROGENIC ACTIVITY? H. Kfihn. Institute of BiochemisOy. Unit~ersity Clinics (CharitL Humboldt

UniuersiO, Hessische Str. 3~1. 10115 Berlin. Gelw~any Oxidative modification of low density lipoprotein [LDL) converts the LDL particle to an atherogenic form which is rapidly taken up by macrophages and/or smooth muscle cells via scavenger receptor mediated pathways. Since these pathways are not feedback controlled the internalized lipids are not completely metabolized but may be deposited inside the cells. Accumulation of such lipid-laden foam cells in the subendothelial space of the arteries leads to the formation of fatty streak which may represent early atherosclerotic lesions. Although the detailed in vivo mechanism of oxidative LDL modification is still unknown structural studies on the composition of oxidized lipids in atherosclerotic lesions of different stages suggested the involvement of enzymatic and non-enzymatic lipid peroxidation, tn early atherogenesis the enzymatic processes appear to be more important than in older lesions. Over the last 3-4 years a substantial body of experimental evidence has been accumulated which implicate the lipid peroxidizing enzyme 12/15LOX in early atherogenesis. Because of its capability to create an oxidizing environment the enzyme is commonly regarded as pro-atherogenic stimulus and the majority of the experimental data published so far support this hypothesis. However, there is also a report on a potential anti-atherogenic activity of the 12/15-LOX in two rabbit atherosclerosis models. Here the most recent experimental findings on the potential role of 12/15LOX in atherogenesis will be summarized and novel data suggesting an anti-atherogenic activity of the 12/15-LOX will be presented.

OXIDATIVE MODIFICATION OF HDL DECREASES SCAVENGER R E C E P T O R BI-MEDIATED C H O L E S T E R O L EFFLUX FROM CELLS M.C. de Beer 1, P.M. Connell 1, C.L. Banka 2, WJ.S. de Villiers, EC. de Beer 1.3 D.R. van der Westhuyzenl. 1Department of Internal Medicine,

Uniuersi O' of Kentuc~'v Medical Center. Lexington. KY 40536: 3Department ~f Veterans A.[faitw Medical Centel: Levington. KY 4051 I: 2Department of lmmunoloKt; Scripl)s Research h~stitute. La Jolla, CA 92037, USA HDL stimulates cellular cholesterol efflux and also acts as a donor of cholesterol ester that is taken up by cells through a selective lipid uptake process. Oxidative modification of HDL has been shown to reduce the efficiency with which HDL stimulates cellular cholesterol effiux. The HDL receptor, scavenger receptor-Bl (SR-BI), mediates selective cholesterol ester uptake from HDL and also facilitates the efflux of cellular cholesterol. The aim of the present study was to investigate the effect of HDL oxidation on SR-BI-mediated cholesterol flux between HDL and cells. SR-Bl-mediated activities were analyzed using human HDLa and transfected CHO cells expressing human SR-BI. Untransfected CHO cells were used as controls. Human HDL was oxidized using 5 [ttM copper acetate at 37 C for different lengths of time. HDL was labeled by iodination with 1251 or by 3Hcholesteryl oleoyl ether. SR-BI-mediated cell association, degradation and cholesterol ester uptake of normal and oxidized HDL were compared in dose and time-course experiments. Both ligands selectively delivered cholesterol ester to cells via SR-BI. Although oxidized HDL showed significantly greater cell-association than normal HDL, the rates of SR-BI-mediated selective lipid uptake were similar for the two ligands (38+5% and 30+5% of HDL added/3 h for normal and oxidized HDL at 10 ttg/ml, respectively). Both control and oxidized HDL stimulated SR-Bl-mediated cholesterol cffiux from CHO cells. However, the rate of SR-Bl-dependent efflux was markedly lower {2-3 fold) for oxidized than for control HDL. These data indicate that oxidative modification of HDL significantly alters its ability to mediate cellular cholesterol effiux through SR-BI and that HDL oxidation may therefore contribute to lipid accumulation in cells. SELECTIVE OXIDATION OF M E T H I O N I N E RESIDUES ENHANCES RATHER THAN DECREASES P O T E N T I A L ANTIA T H E R O G E N I C PROPERTIES OF APOLIPOPROTEIN AI U. Panzenboeck, K.-A. Rye 1, L. Kritharides, R, Stocker. The Heart

Research Institute, 145 Missenden Road. Camperdown. Sydm:v NSW 2050; /Lipid Research Laborutoo,. Royal Adelaide Hospital, Frome Road, Adelaide. 5000, Australia The earliest stages of HDL oxidation are accompanied by the formation of Met sulfoxides (Met(O)) in apoAI and All, due to the reduction and hence detoxification of lipid hydroperoxides by methionine residues (J. Biol. Chem. 1998; 273: 6080; ibid 273: 6088). Here we have investigated the secondary structure, lipid affinity, LCAT activation and cholesterol-effiux promoting properties of native and selectively oxidized apoAl (apoAl+32, containing Met(O) at Met zl2 and Met1481. Circular dichroism studies revealed that selective oxidation of Met residues does not alter alpha helicity of the protein. The lipid affinity was determined as the rate of clearance of DMPC vesicles. ApoAl+32 induced a 2-3 fold faster rate of clearance versus apoAl, suggesting increased protein-lipid interactions due to the presence of Met(O). Kinetic experiments have revealed that the affinity for LCAT is comparable for HDL reconstituted with either apoAl or apoAl+3,. Efflux of [~H]-cholesterol from human macrophage foam cells was enhanced for apoAl+32 compared with apoAl, consistent with the DMPC clearance data, and cholesterol effiux from lipid-laden hMDM was paralleled by the effiux of vitamin E. Together, these findings suggest that selective oxidation of Met residues of apoAl may enhance rather than diminish known anti-atherogenic activities of the apolipoprotein. MODIFIED LIPOPROTEINS, T H E I R R E C E P T O R S P O T E N T I A L ROLES IN ATHEROSCLEROSIS

AND

D. Steinberg. Department o f Medicine. UniuersiO, of California. Son Diego.

9500 Gilman Drive La Jolla. CA 92093-0682. USA It is generally accepted that circulating LDL is the ultimate source of the cholesterol accumulating in fatty streaks, the earliest lesion of atherosclerosis. Yet the cell types that give rise to foam cells - the monocyte/macrophage and the smooth muscle cell - do not take up native LDL at a sufficient rate to increase their cholesterol content significantly under in vitro conditions. This

71st E,4S Congress and Satellite Symposia