Modification of high density lipoproteins: Effects on OxLDL cytotoxicity and on cholesterol efflux

Monday June 26, 2000: Poster Abstracts P:W29 Oxidation and Atherogenesis benzo-furan (BO-653), probucol and butylated hydroquinone (BHQ) were added as a DMSO solution. After 6 hr treatment, the cells were scraped and the mRNA was recovered. The expression of 6,416 genes was analyzed using a set of oligo nucleotide array (DNA chip Affymetrix). Results: Among 6,416 genes, 21 genes including genes encording mitochondrial proteins and proteins related to oxidative stress response were induced more than 3 folds by BO-653, probucol and BHQ. A gene of cytochrome P-450 1A1 isozyme which is one of drug-metabolizing phase 1 enzymes was expressed only by BHQ treatment. For other genes, BO-653 showed a similar induction pattern to BHQ rather than probucol.

MoP8:W29

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I S e r u m bilirubin a n d albumin levels are markers of the extent of atherosclerosls in dyslipidaemic patients

J

J.A. Papadakis 1, E.S. Ganotakis I , G. Vrentzos 1, D. Emmanouel I , D.P. Mikhailidis2 . I Dept. of Internal Medicine, University Hospital of

Heraklion, Greece; 2Dept. of Molecular Pathology & Clinical Biochemistry, Royal Free and University College Medical School (UCL), London, UK There is evidence that bilirubin (Bil) acts as an antioxidant and that its circulating levels are reduced in patients with extensive coronary disease. Serum albumin (Alb) levels may also act as an antioxidant, possibly in collaboration with Bil. We compared, in a retrospective study, serum Bil concentrations in 443 patients with primary dyslipidaemia; 61 (13.8%) patients had ischaemic heart disease (1HD), 111 (25.1%) had peripheral vascular disease (PVD) and the remaining 271 (61.1%) patients had no clinically evident cardiovascular disease (CVD). Patients with PVD had significantly (p < 0.01) lower serum Bil levels when compared with those with IHD or without CVD. Serum Bil levels correlated significantly (rs = 0.12; p = 0.01) with Alb concentrations, when all patients were considered. Smokers had significantly lower Bil concentrations than non-smokers in both the PVD and without CVD subgroups (p = 0.01 and p = 0.02, respectively). Men with PVD had significantly lower the [Bill × [Alb] product (a proposed combined index of Bil and Alb) when compared with those without CVD, in both non-smoker and smoker subgroups (p = 0.009 and p = 0.04, respectively). Bil, Alb and the [Bill × [Alb] product correlated with lipids and fibrinogen. All the results varied depending on the patient subgroup (e.g. divided by gender or smoking status) evaluated. Conclusion: This study supports the concept that low serum Bil and AIb levels are associated with more extensive CVD.

MoP9:W29 ] Measurement of paraoxonase (PON) concentrations in coronary heart desease (CHD) subjects by sandwich Elba T. Kuiiraoka I , M. Ishihara 1, T. Fujioka 2, E. Saitu 2, S. Saito 2, T. Egashira I , H. Hattori I . l Research Department, R&D Center, BML Inc., Kawagoe;

2Second Department of Internal Medicine, Nihon University, Tokyo, Japan Serum paraoxonase (PON) is associated with high density lipoproteins, and has been shown to prevent the peroxidation of low density lipoprotein phospbolipids. We have developed a sensitive sandwich ELISA, using specific monoclonal antibodies against PON to measure serum PON concentration. We have measured serum PON in healthy Japanese subjects (n = 87) and CHD patients (n = 33) diagnosed by angiography. Serum PON concentrations were 59.3 -4- 12.5 /~g/mL (mean =E SD) and 52.0 =E 13.6 #g/mL in healthy subjects and CHD patients, respectively. Serum PON concentrations in healthy subjects at 192 polymorphism were 69.5 4- 10.3 (QQ), 63.0 -4- 11.0 (QR), 52.8 =t= 10.8 (RR)/~g/mL, and in CHD patients were 56.8 -4- 18.2 (QQ), 56.7 =1= 15.5 (QR), 46.9 -4- 9.44 (R~)/~g/mL. Serum PON concentrations in CHD patients were lower than those in healthy subjects, especially in RR (P < 0.001). Both paraoxonase specific activity and arylesterase specific activity in CHD patients were also lower, especially in RR (P < 0.001). These results suggested that PON in CHD patients must be inactivated due to being exposed to high oxidative stress. The measurement of PON concentration can be useful tool for the detection of oxidative stress and the progress of atherosclerosis. I

MoP10:W29 J Rapid isolation of VLDL subfractions: Assessment of composition and susceptibility to oxidation J. McEneny, E.R. Trimble, I.S. Young. Department of Clinical Biochemistry,

The Queen's University of Belfast, Belfast, UK Objective: To establish a procedure for the subfractionation of VLDL by rapid ultracentrifugation.

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Methods: Four subfractions of VLDL (A, B, C & D; where A denotes the largest and most buoyant) were isolated by sequential ultracentrifugation requiring a total preparation time of less than 3.5 hours, compared to 18 hours reported by other procedures. Each subfraction was assessed for; lipid composition, preformed hydroperoxides (HPOs; a marker of in viva peroxidation) and conjugated diene production (a marker of in vitro peroxidation). Results: As the subfractions decreased in size and increased in density (A-+D) the percent triglyceride decreased 86-+62% while cholesterol increased 14---,38%. With increasing density of the subfraction the distribution of fatty acid changed; SFA decreased 39.3--~37.1%, MUFA remained unchanged 38.4-+37.6% and PUFA increased 23.2--+27.0%. HPOs were not different between the subfractions 7.4--> 6.4 nmol/mg protein. Lag time results demonstrated that the subfractions became more susceptible to oxidation { 162-+96" min (p < 0.05)} as they decreased in size and increased in density. Conclusions: Four VLDL subfractions (A->D) were isolated by sequential density gradient ultracentrifugation requiring 1/5th of the time of previously reported procedures. For the first time we have shown how, like LDL, VLDL subfractions become more susceptible to oxidation with decreasing size and increasing density. This method may be applied to patient groups for the detection of abnormalities within their VLDL subfractions that may not be detectable when examining whole VLDL.

I MoP11 :W29 I Evidence of ATP and glucose depleted areas within the atherosclerotic plaque in viva M. Levin, M. Evaldsson, O. Wiklund, G. Bondjers, T. Bjtmheden. The

Wallenberg Laboratory for Cardiovascular Research, GSteborg, Sweden According to the Anoxemia theory ofatherosclerosis, an imbalance between the demand for and supply of oxygen and nutrients in the arterial wall is a key factor in the development and progression of atherosclerotic lesions. However, the energymetabolic situation of the arterial wall in viva is largely unknown. At our lab the prescence of hypoxic areas at depth in the atherosclerotic plaque in viva has been demonstrated in lesions > 4 - 5 0 0 / z m thick. Objective: The aim of the present study was to determine local concentrations of ATP (adenosine triphosphate), glucose and lactate within the atherosclerotic arterial wall in viva at high spatial resolution. Methods: The bioluminescence method metabolic imaging was used to study local concentrations of ATP, glucose and lactate. To get a reflection of the in viva situation, aortas from rabbits (n = 9) with experimental atherosclerosis were snap-frozen in situ in the anestethized animal and consecutive cryosections were used for the different analyses. Results: In plaques exceeding a certain thickness, ATP (>4-500/~m) and glucose depleted (>5-600/~m) areas were demonstrated in the central parts of the plaque. Lactate concentrations were homogenous in the plaque. Conclusions: ATP and glucose depleted areas were demonstrated at depth in the athemsclerotic plaque. We believe that this is a result of an insufficient diffusion of glucose and oxygen due to the thickness of the lesion, maybe in combination with an increased local metabolic demand within the plaque. These results lend support to the Anoxemia theory ofatherosclerosis.

I MOP12:W29

J Modification at" high density lipoproteins: Effects on OxLDL cytotoxicity and on cholesterol effiux

A. Pirillo 1, P. Uboldi I , S. Marcovina 3 , A.L. Catapano 1,2. llnstitute of

Pharmacological Sciences, Milan; 2Centro per It Studio, la Prevenzione e la Terapia delle Vasculopatie Aterosclerotiche, Ospedale Bassini, Milan, Italy; 3Department of Medicine, University of Washington, Seattle, USA Elevated plasma levels of high density lipoproteins (HDL) are believed to be antiatherogenic. Among the mechanisms by which HDL might exert their effects, attention has been paid to the role of HDL in reverse cholesterol transport. Furthermore, HDL may be antiatherogenic by preventing the cytotoxicity of oxidized low density lipoproteins (OxLDL). HDL also undergo modifications that may affect their biological activities. Lipoxygenases (LOX) belong to a family of enzymes which may play a role in atherogenesis because of their ability to oxidize lipoproteins. In this study we investigated the effect of lipoxygenase-mediated modification of HDL (LOX-HDL) on their ability to prevent the cytotoxicity of OxLDL and on cholesterol removal from ceils and aimed at relating these effects to changes in apo AI structure. OxLDL are cytotoxic to endothelial cells; this effect could be reverted by the presence of increasing concentrations of HDL (70.83% -t- 2.3 of control in the presence of OxLDL versus 141.33% :i: 2.43 in the presence of OxLDL + HDL 400 /zg protein/ml). When LOX-HDL were added to cells incubated with OxLDL, the improvement of cell viability was lower than in presence of native HDL

Xllth International Symposium on Atherosclerosis, Stockholm, Sweden, June 25-29, 2000

Monday June 26, 2000: Poster Abstracts P:W29 Oxidation and Atherogenesis

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(83.31% + 0.8 of control), suggesting that the oxidative modification impairs their antiatherogenic role. We also investigated the ability of LOX-HDL to stimulate cholesterol efflux from cultured cells. Compared with native HDL, LOX-HDL showed a reduced ability to stimulate cholesterol efflux from preloaded macrophages (at 200 /zg protein/ml: 83% of control with LOX-HDL). Changes in apo AI conformation were evaluated by using a panel of murine monoclonal antibodies. The oxidative modification of HDL (both Cu ++- or LOX-mediated) dramatically increased the expression of epitopes for monoclonals mAb8, mAb9, mAbl 1 and mAbl9, while decreased that for mAb5, suggesting profound changes of apo AI structure that may affect its biological properties.

ity (by 79%). Native AAT only showed concentration and time dependent stimulatory effects on MCP-1 protein expression, and these appear to be independent of oxidative stress. Conclusion: Multiple activities of AAT identify it as a critical agent in orchestrating the inflammatory response in diverse processes including atherogenesis.

MoP15:W29 [ Oxidized LDL triggers the lysis of human macrophages concurrently to but independently of the induction of apoptosis by depleting intracellular ATP i

R. Asmis, J. Jelk, E.S. Wintergerst, Ch. Stark, F. Brawand. Institute of

MOP13:W29 ] Oxidized LDL induce the release of thioredoxin, a chemoattractant, in human endothelial cells. Antagonistic effect of HDL

Biochemistry, University of Basel, Switzerland

i

G.D. Norata 1, A.L. Catapan L2. llnstitute of Pharmacological Sciences, Milan; 2Centro per lo Studio, la Prevenzione e la Terapia delle Vasculopatie Aterosclerotiche, Ospedale Bassini, Milano, Italy Thioredoxin (Trx) is an ubiquitos intracelhilar protein disulfide oxidoreductase with intracelhilar antioxidant activity. Trx regenerates proteins inactivated by oxidative stresses and controls the DNA binding activity of NF-kB. Trx is secreted by monocytes or lymphocytes stimulated with ILl-beta exhibiting several cytokine-like activities such as chemoattractant activity for neutrophils, monocytes and T cells. Trx is locally elevated in cerebral ischemia, brain injury, open-heart surgery and during neo intima formation in rat arteries suggesting a possible role in atherogenesis. Oxidative stress plays a key role in the regulation of gene expression during atherogenesis; one of the earliest events in atherosclerosis is the oxidative modification of lipoproteins (in particular LDL) that generates oxidized LDL (Ox-LDL) whose role in the pathogenesis of atherosclerosis has been well established. Aim of our study was to investigate whether Ox-LDL could modulate the release of Trx in human umbelical cord endothelial cells. Cells were incubated with Ox-LDL 5 to 80/zg/mL for 18 hours, a concentration dependent release of Trx was observed from 1.8 fold at 5 /~g/mL to 7 fold at 80 /zg/mL, native LDL were uneffective while HDL decreased the release of Trx (up to 76% at 400 /~g/mL). When HDL were added to Ox-LDL the release of Trx was reduced toward basal secretion. As oxysterols could regulate also cytokines production we studied the effects of 7K-cholesterol, 7t-OH cholesterol and 5-6 epoxcholesterol (all 2 to 10 #g/mL) on Trx release. No relevant changes in Trx secretion was observed. Since Trx could act as a signal of oxidative stress that amplifies the cellular response at a site of inflammation, our data suggest another pro-inflammatory mechanism of Ox-LDL that involves the release of Trx which promote the recruitment of inflammatory cells in the vascular wall, moreover the observation that HDL alone or when coincubated with Ox-LDL decrease the secretion of Trx suggest another possible antiatherosclerotic role for HDL.

MoP14:W29 ] Fibrillar C-terminal fragment of alpha-l-antitrypsin activates human monocytes via oxidative mechanisms S. Janciauskiene, S. Lindgren. Department of Medicine, University Hospital MAS, MalmO S-20502, Sweden Objective: Monocytes increase expression of serine proteinase inhibitor, alpha-l-antitrypsin (AAT) in response to inflammatory mediators. Proteolytic inactivation of AAT with subsequent generation of cleaved fibrillogenic form of AAT, C-terminal fragment (C-36) may amplify the inflammatory response. Fibril cytotoxicity is suggested to be mediated via a common oxidative stress mechanism. We undertook to determine whether fibrillar C-36 effects also involve this common pathway. Methods: Monocytes were isolated from buffy coats by the Ficoll-Hypaque procedure. Chemoattractant protein-1 (MCP-I) expression was assayed by immunoassay, caspase activity determined by the spectrofluorometry. Superoxide produced from the NADPH oxidase was monitored by the superoxide dismutase-inhibitable rate of cytochrome c reduction. Mitochondrial membrane potential was measured from the intracelhilar distribution of dye, JC-1. Measurement of malonaldehyde and 4-hydroxy-alkenals was used as an indicator of lipid peroxidation. Results: Monocytes stimulated with C-36 fibrils (10/zM) for 1 h showed elevation in MCP-1 by 36 times, induced NADPH oxidase activity by 6.5-fold, increased lipid peroxidation by 25-fold, alteration in mitochondria membrane potential and increased caspase-3 activity by 24%. Treatment of monocytes with C-36 fibrils for 24 h resulted in increased cytosolic cathepsin D activ-

In previous studies, we showed that programmed cell death in mature human macrophages induced by oxidized LDL (OxLDL) involves oxidized thiols on apoB100 and requires CD36. Here we tested the hypothesis that OxLDL concurrently triggers apoptosis and cell lysis in human macrophages. We found that compounds inhibiting OxLDL-induced apoptosis did not prevent cell lysis Apoptosis was detected with the monoclonal antibody apo2.7 directed at the mitochondrial antigen 7A2. Cell lysis was measured as the release of radiolabel from [3H]adenine-loaded macrophages. Our studies with an oxidation-sensitive fluorescent probe suggest that OxLDL stimulated peroxide formation in macrophages. Furthermore, the radical scavenger Trolox inhibited OxLDL-induced cell lysis but did not prevent apoptosis. Depletion of cellular glutathione (GSH) with diethylmaleate and inhibition of GSH synthesis with buthionine sulphoximine dramatically increased the cell lyric effect of OxLDL. Conversely, loading macrophages with ascorbic acid and concurrently increasing cellular GSH with L-oxothiazolidine-4-carboxylic acid reduced the susceptibility of macrophages to cell lysis. Finally, incubation with OxLDL resulted in the total depletion of ATP from macrophages. This loss of ATP was prevented by Trolox but not by dehydroascorbic acid, an inhibitor of OxLDL-induced apoptosis. We conclude that in addition to the induction of apoptosis, OxLDL triggers the loss of membrane integrity in human macrophages. This concurrent but independent process appears to involve the initiation of radical-mediated membrane (lipid) oxidation, possibly by direct diffusion of lipid-centered radicals from OxLDL to the macrophage membrane. The expansion of the oxidative reactions into the cell interior results in the depletion of cellular ATP which in turn leads to the loss of membrane integrity.

I MoP16:W29 ] Foam cell formation reduces cellular vitamin E and I

increases the susceptibility of human macrophages to lysis by oxidized LDL

R. Asmis, J. Jell(. Institute of Biochemistry, University of Basel, Switzerland Several studies in macrophage cell lines, rodent macrophages and animal models of atherosclerosis suggest that vitamin E may prevent the formation of foam cells. We tested this hypothesis in macrophages from 8 individuals in a fully autologous in vitro model of human foam cell formation. Foam cell formation was stimulated in mature unsupplemented and vitamin E-loaded macrophages for one week with 50 tzg autologous aggregated LDL in the presence of unsupplemenwxl and vitamin E-loaded serum, respectively. Vitamin E-loading had no effect on the lipid composition of macrophages and did not affect their growth. We observed no effect of vitamin E supplementation on the formation of foam cells. However, foam cell formation resulted in a 36% and 44% reduction in the cellular a-tocopherol/total cholesterol ratio in unsupplemented and vitamin E-supplemented foam cells, respectively. The loss of vitamin E was accelerated with increasing concentrations of aggregated LDL and was accompanied by an increase in the susceptibility of these foam cells to succumb to the cell lytic effects of oxidized LDL. However, vitamin E-supplementation did not protect macrophages or foam cells from OxLDL-mediated cell lysis, indicating that vitamin E loss in foam cells is not the cause of their increased susceptibility to cell lysis. Our results suggest that the beneficial effects of vitamin E on cardiovascular disease observed in humans are neither due to a reduction in the propensity of macrophages to form foam cells nor to an increased resistance of these cells to cytolytic OxLDL.

Xllth International Symposium on Atherosclerosis, Stockholm, Sweden, June 25-29, 2000