W10-P-002 Ageing and cardiovascular diseases,implication of HDL and paraoxonase in reverse cholesterol transport

Workshops WIO Regulation of plasma HDL levels and metabolism Method: To clarify the effect of EL-inactivation in the development of atberosclerosis, EL knockout mice (EL-KO) were bred with the apoE knockout mice (apoE-KO), and the aortic lesion and lipid profile of homozygous double knockout mice (apoE/EL-KO) were characterized. EL expression in atherosclerotic lesion was evaluated by immunohistochemisty and immunoblotting. Ex vivo adhesion assays were performed using cultured monocyte/macrophages and aortic strips from apoE-KO and apoE/EL-KO. Results: Both apeE-KO and apoE/EL-KO had low HDL cholesterol levels compared to wild-type mice, but the HDL levels of apoE/EL-KO was higher than that of apoE-KO (21.4-1-1.9 vs. 11.1-1-2.2 mg/dl, p<0.05). VLDL and IDL/LDL cholesterol levels of apoE/EL-KO were also greater than those of apoE-KO. Despite this increase in plasma cholesterol lipid, aortic atberosclerotic area in apoE/EL-KO was a significantly decreased by 71% compared with that in apoE-KO. Also, macrophage infiltration in the atherosclerotic lesion was significantly decreased in apoE/EL-KO. Smooth muscle and T-cell contents in the lesion were not affected by the EL deficiency. EL expression is increased in the atberosclerotic aortas of the apoE-KO as well as early coronary lesions in humans, suggesting a local effect of EL in the diseased vessel wall. Ex vivo adhesion assays revealed that the number of adherent monocyte to the aoaic strips was significantly lower in apoE/EL-KO than in apoE-KO. Conclusions: These data suggest that EL has atherogenic actions in vivo by modulating circulating HDL cholesterol, vascular wall lipoprotein uptake, and monocyte recruitment.

47

3H-cholesterol or 3H-choline, equilibrated in presence or absence of 22OH 51xg/ml-cRA 101xM or cAMP 0.3mM and exposed to apoA-I 201xg/ml. To analyze mRNA and protein content of ABCA1, RT-PCR and western blot were performed. Results: Pitavastatin dose-dependently increased cholesterol efflux from Fu5AH both in basal condition and when ABCA1 was stimulated by 22OH/cRA. This effect was fully recovered by mevalonate and geranyl geraniol. In J774 macrophages pitavastatin significantly reduced cAMP ability to stimulate cholesterol and phospholipid efflux and to up-regulate ABCA1 mRNA and protein. Mevalonate, but not geranyl geraniol reversed these inhibitions. No effect of pitavastatin was detected when ABCA1 was induced by 22OH/cRA. Importantly, pitavastatin up to 50$tM did not affect lipid ettlux or ABCA1 expression in cholesterol-loaded macrophages, a cellular model of foam cells. Conclusions: Pitavastatin promotes ABCAl-mediated efflux from hepatic cens; this effect could partially explain the improvement in HDL plasma profile observed in patients treated with statins. Pitavastatin inhibits ABCAl-mediated efflux in macrophages only when the protein expression is induced by cAMP, suggesting that depletion of cellular sterols is potentially involved in cAMP-mediated activation of ABCA1. The lack of influence in foam cells is likely to exclude a potential negative pleiotropic effect by statins.

I W10-P-001 / COMPARISON O F EFFECTS OF FISH OILS AND ATORVASTATIN ON HDL METABOLISM IN OBESITY J

W l 0 - 0 - 0 0 2 I CATALYTICALLLY ACTIVE PLTP P R O M O T E S HI)L-FACILITATED C H O L E S T E R O L EFFLUX F R O M M A C R O P H A G E FOAM CELLS R. Vikstedt, J. Metso, C. Elmholm, M. Jauhiainen. Molecular Medicine,

National Public Health Institute, Helsinlt, Finland Objective: Plasma phospholipid transfer protein (PLTP) is important in lipoprotein metabolism. PLTP is present in two distinct forms, one with high and the other with low phospholipid transfer activity. PLTP modulates cholesterol effiux from macrophages to HDL via the ATP-binding cassette transporter A1 (ABCA1) pathway. The aim of this study was to clarify the role of catalytically active and inactive forms of PLTP in reverse cholesterol transport. Methods: Active and inactive PLTP were isolated from plasma. Active recombinant PLTP (rPLTP) was produced in the baculoviras expression system. THP-1 macrophages were loaded with [3H]cholesteryl linoleate containing acetyl-LDL and cholesterol efltux to human HDL3 and apoA-I was investigated. Cyclic AMP was used to increase ABCA1 expression level. Results: Inactive PLTP did not have any effect on cholesterol efflux to HDL3, while catalytically active PLTP promoted efltux to HDL3. This increase in efflux was more pronounced when HDL3 and active PLTP were co-incubated for 20 h at +37 °C prior to their addition to macrophages. Similar results were obtained with catalytically active recombinant PLTP. Neither the active PLTP nor the inactive PLTP affected the high specificity and low capacity ettlux to lipid free apoA-I. 8-Br-cAMP increased cholesterol efflux to HDL3. Conclusions: PLTP has an effect on cholesterol effux from macrophage foam cells. However, only PLTP with phospholipid transfer activity is able to promote reverse cholesterol transport.

W l 0-O-003 I PITAVASTATIN DIFFERENTLY M O D U L A T E S ABCA1-MEDIATED LIPID EFFLUX IN MACROPHAGES AND HEPATIC CELLS I. Zanotti, E. Favari, M. Adorni, E Zimetti, E Bemini. Dept. of

Pharmacological and Biol. Sciences and Appl. Chemistries, University of Parma, Parma, Italy The beneficial effects of statins on the treatment of cardiovascular disease are in part related to pleiotropic effects, whose underlying mechanisms are not fully understood. ABCA1 exerts antiatherosclerotic properties by promoting the formation of HDL in the liver and by inducing lipid ettlux from macrophages in the arterial wall. Objective: We investigated the ability of pitavastatin to modulate ABCAl-mediated effux from hepatic cells and macrophages. Methods: Cholesterol and phospholipid efflux were quantified in Fu5AH rat hepatoma cells and J774 macrophages. Cells were radiolabeled with

H. Barrett, M. Nguyen, D. Chan, G. Watts. School of Medicine and

Pharmacology, University of Western Australia, Perth, Australia Objective: In obesity, alterations in HDL metabolism may partly account for the increased risk of cardiovascular disease (CVD). Statins and fish oils (FO) are known to reduce CVD risk, but their effects on HDL metabolism have not been fully elucidated. We studied the effect of Atorvastatin (AT), FO and AT plus FO (ATFO) on HDL metabolism in obese men. Method: In a placebo-controned trim we examined the independent and combined effects of AT (40 mg/day) and FO (4 g/day) on HDL metabolism in 38 viscerally obese men (age 53.6-t-9.1 yr, BMI 33.65:4.5; TG 1.954-0.79 mmol/L; total cholesterol (TC) 5.834-0.54 mmol/L; HDL-C 0.99+0.14 mmol/L). HDL apoAI and apoAII kinetics were measured using d3-1eucine and GCMS. Kinetic parameters for HDL apoAI, apoAII, LpAI and LpAI:AII were derived by compartmental modelling. Results: FO significantly (main effect) reduced TG (-0.354-0.09 mmol/L, p<0.01) and increased HDL-C (0.09+0.04 mmol/L, p<0.05). AT significantly (main effect, p<0.001) reduced TG (-0.384-0.09 mmol/L), TC (-1.90+0.18 mmol/L) and LDL-C (-1.81-t-0.16 mmol/L). In addition, FO significantly (p<0.05) reduced the fractional catabolic rate (FCR) and production rate (PR) of HDL apoAI (-0.04-t-0.02 pools/d; -2.33-t-0.93 mg/kg/d), HDL apoAII (-0.044-0.02 pools/d; -0.574-0.24 mg/kg/d), and LpAI:AII (-0.044-0.02 pools/d; -1.474-0.72 mg/kg/d) with no significant change in concentrations. FO also increased HDL-C to apoA protein ratio (0.054-0.03, p=0.051), reflecting an increase in HDL particle size. Conclusion: In obese men, FO but not AT significantly infuences HDL metabolism. FO increases HDL-C concentration and HDL particle size; HDL apoAI, apoAII and LpAI:AII concentrations, however, are not altered by FO, and this is related to commensurate reductions in the corresponding rates of catabolism and synthesis. These kinetic effects of FO may relate to HDL particle remodelling and down-regulation of expression of both apoAI and apoAII. AND CARDIOVASCULAR DISEASES, IWlO-P-OO2JAGEING IMPLICATION OF HDL AND PARAOXONASE IN REVERSE C H O L E S T E R O L T R A N S P O R T H. Berrougui, M. Cloutier, A. Khalil. Research Centre on Aging,

Sherbrooke, Canada During ageing, several changes in the composition of HDL may occur and then affect their function. In this study, we investigate: 1- Whether ageing can affect the ability of HDL, (HDL2 & HDL3) to take up cellular free cholesterol (FC) execs and their susceptibility to oxidation. 2- the role of paraoxonase-1 (PON-1) in the RCT. HDL cholesterol effiux (CE) was carried out using human THP-1 macrophage incubated in the presence of: A - Native HDL, HDL2 or HDL3; B - Oxidised HDL; C - Oxidised HDL in the presence of increased concentrations of PON-1. HDL oxidation

75th EAS Congress, 23-26 April 2005, Prague, Czech Republic

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I 0 "D GO

Workshops WIO Regulation of plasma HDL levels and metabolism

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was evaluated by measuring CD and MDA formation and vitamin E disappearance as well as apoA1 degradation. Our results show that HDL and HDL3 induces mobilisation of cellular FC which was significantly higher in younger than for older subjects. In the younger patients, CE was more important for HDL3 than HDL2 (P=0.0186). Oxidation of HDL induces an increase in the CD, MDA production and the rate of vit-E disappearance, which was high in young than older subjects. Negative correlation was observed between oxidised HDL and CE from THP-1 (P<0.001). However, the mean of CE was significantly higher with oxidised-HDL from young than from elderly. The decrease of cholesterol removing from macrophage in the HDL oxidised transports was abolished in the presence of increased concentrations of PON- 1. These data indicate that ageing can affect negatively the ability of HDL to mobilise FC from cells and that PON-1 protecting HDL towards oxidation may restore and enhance RCT.

I W l 0 - P - 0 0 3 I REDUCED HDL AND INFLAMMATION C O R R E L A T E S O F MICROVASCULAR DYSFUNCTION IN IDIOPATHIC DILATED CARDIOMIOPATHY F. Bigazzi 1, B. Dal Pint 1, M. Puntoni I , A. Morales 1, F. MinichiUi 1, S. Lusse 2, F. Sbrana 2, A. Bionda 2, D. Neglia 1, T. Sampietro 1. ]CNR

Institute of Clinical Physiology, Pisa, Italy; 2Department of lnternal Medicine, University of Pisa, Italy

Combination therapy with T/A may provide additive benefits when treating various dyslipidemias that may slow the progression of atherosclerosis and reduce cardiovascular risk. Rationale and objective: Measurement of CIMT is a well-validated technique for evaluating atherosclerotic progression. In clinical trials, treatment-related changes in CIMT have been associated with reductions in cardiovascular events. The objective of this study is to show that the combination of T/A can slow atherosclerotic progression, as measured by the change in CIMT, more than A alone in subjects with mixed hyperlipidemia. Design: This Phase 3, multi-center, double-blind, randomized, parallelgroup study is enrolling ~840 men and women at 64 sites in North America and Europe. Subjects are aged 18 to 70 years with mixed hypedipidemia (type I/b). Prior to screening, subjects undergo a 4-week wash-out period when lipid-lowering therapy is discontinued and counseling on lifestyle changes is given. Eligible subjects then begin an A only run-in period (starting dose 10 nag once daily) and are titrated to a target LDL-C level defined by NCEP ATP III guidelines. Subjects reaching target are randomized to (i) fixed combination T/A (T 60 nag combined with A once daily) or (ii) A alone. In both treatment arms, the dose of A (10, 20, 40 or 80 nag) is that established during the run-in period. B-mode ultrasonography is performed to measure CIMT at baseline and eve*y 6 months for 24 months of treatment. The primary efficacy variable is the annualized rate of change in maximum CIMT of 12 pre-defined carotid segments. Clinical safety and/or lipid efficacy assessments are performed at each visit. Enrollment has now completed and results are expected in 2007.

Objective. Coronary microcixculation is altered in idiopathic dilated cardiomyopathy (IDC) possibly due to endothelial dysfunction. High density lipoproteins (HDLs) have the potential to regulate endothelial function. We investigated whether IDC is associated with reduced HDLs and whether HDL values correlate with corenaty microvascular function. Methods. Fifty-five IDC patients, without evidence of other organ or systemic diseases, were compared with 55 healthy controls for HDLs, lipid profile, C3 and C4 complement fractions, C Reactive Protein, and adhesion molecules sICAM-1 and sELAM-1. MBF Resting/dipyridamole myocardial blood flow (MBFb/MBFd) was assessed by Positron Emission Tomography and 13N-NH3. Remits. IDC patients differed from controls because of lower HDL (43.8 4- 12.6 vs 55.4 4- 13.8 mg/dl, p<0.0001), ApoA-I (134.6 4- 22 vs 154.8 5= 26.4 mg/dl, p<0.0001), ApoA-II (29.1 5= 4.7 vs 33.1 5= 3.9 mg/dl, p<0.001) and higher triglycerides (127.5 4- 98.6 vs 72.1 4- 44.6, p<0.001), but not for total and LDL cholesterol, ApoB or Lp(a). Inflammation and endothelial dysfunction markers (C3, C4, CRP, sICAM-1 and sELAM-1) were significantly higher in IDC patients in respect to controls and were negatively correlated with HDLs. HDL values positively correlated with MBFb and MBFd (r 0.54, p<0.005 and r 0.46, p<0.05). MBFd negatively correlated with age (r -0.46, p<0.05). Categorical HI)L, lower or higher than 40 mg/dl, was strongly and independently associated with IDC (odds ratio 0.10,p 0.0003). Conclusions. Reduced HDL and high inflammation markers, like in the Familial Hypoalphalipoproteinemia condition, were found in IDC. The linear positive correlation between HDL and MBF suggests a role for HDLs in microvascular dysfunction in IDC.

IWl0-P-004

I

I DESIGN O F A STUDY OF THE E F F E C T OF L

TO RC ETRA P I B/ATO RVASTATIN VERSUS ATORVASTATIN A L O N E ON INTIMA-MEDIA THICKNESS IN PATIENTS W I T H MIXED HYPERLIPIDEMIA

M.L. Bots 1, W.A. Riley 2, G.W. Evans 2, J.J.P. Kastelein 3, J.H. Revkin 4, "Ii Thuren 4, C.L. Shear 4, T.T. Nguyen 4 . ~University Medical Center

Utrech~ the Netherlands; 2 Wake Forest University School of Medicine, Winston-Salem, NC, USA; ~Academic Medical Center, University of Amsterdam, the Netherlands; 4Pfizer Global Research and Developmen~ New London, CI:, USA Background: Elevated levels of LDL-C and low levels of HDL-C are strong, independent risk factors for coronary heart disease (CHD). Ultrasound measurements of carotid intima-media thickness (CIMT) have been shown to correlate with CHD risk. Studies show that statins lower LDL-C and reduce cardiovascular risk and that intensive therapy with atorvastatin (A) can slow the progression of atherosclerosis measured by CIMT. Torcetrapib (1") is a novel cholesteryl ester transfer protein (CETP) inhibitor that raises HDL-C, decreases LDL-C, and increases lipid particle size.

I W l 0 - P - 0 0 5 I LOSARTAN INCREASES HDL C H O L E S T E R O L AND EFFECTIVELY REDUCES BLOOD PRESSURE IN Y O U N G HYPERTENSIVE PATIENTS W. Bryl 1, A. Miczke I , M. Cymerys 1, P. Bogdaltski 1, M. Kujawska-Luczak 1, D. Pupek-Musialik 1, O. Trojnarska 2. 1Department

of lnternal Medicine, Metabolic Disorders and Hypertension, Poznad, Poland; 2Department of Cardiology, Poznad, Poland Introduction. Angiotensin receptor antagonists have the clinical effectiveness and metabolic neutrality. There are the goals of hypertensive therapy in young patients. The aim of this work was the assessment of the systolic (SBP) and diastolic blood pressure (DBP) and given metabolic parameters in young hypertensive patients after losartan therapy. Material and methods. There were 16 young men under examination aged 16-19 with mild-to-moderate essential hypertension. They were never treated with antihypertensive drugs. Before enrolling and after 6 month therapy with losartan (50 nag per day) the SBP, DBP, total cholesterol, HDL cholesterol, LDL cholesterol, triglyceride, uric acid were assessed. Results. The average SBP before treatment was 146.3 4- 8.8 mmHg, after 6 month losartan therapy 124.2 4- 7.3 mmHg (p<0.05). The average DBP was 88.6 4- 7.6 mmHg before and 82.3 4- 7.6 mmHg after, respectively (p<0.05). The losartan therapy caused significant increase of HDL cholesterol (1.29 4- 0.27 versus 1.38 4- 0.25 mmol/1, p=0.041). The analyzed hypertensive therapy did not influence others lipid parameters and uric acid concentration. Conclusion. Losartan in dose 50 mg per day caused significant increase of HDL cholesterol and did not influence the other lipid parameters. At the same time losartan taken in monotherapy successfully reduced blood pressure in young hypertensive patients.

I WIO-P-O06 I HTS ASSAY D E V E L O P M E N T

FOR CARDIOVASCULAR-RELEVANT LIPASES

D. Carettoni, P. Arioli, B. Be//anti, L. RedaeUi. AXXAM s.r.l., Via Olgettina

58, 20132 Milan, Italy Objective: Hepatic Lipase (HL) and group-IIa secretory Phospholipase A2 (sPLA2-IIa) are recognized targets for therapeutic intervention of atherosclerotic vascular diseases, playing a key role respectively in either lipid hydrolysis of HDL, or in the production of lipid mediators of inflammatory states occurring during atherosclerosis. Standard lipase activity assays (e.g., HPLC, TLC) have precluded so far the application of parallel screening of large compound libraries in a high-throughput format, hindering the discovery of novel therapeutic drugs directed against HL and sPLA2-IIa. Our experimental approach is intended to fill this gap, by generating homogenous lipase assays based on fluorescent readouts compatible with the high throughput screening (FITS) criteria. Method: We expressed the human HL and sPLA2-IIa genes in insect

75th EAS Congress, 23-26 April 2005, Prague, Czech Republic