PO12-317 ACTIVATION STATE OF NEUTROPHILS IN PATIENTS WITH STABLE CORONARY ARTERY DISEASE

Poster Sessions PO12 Retention and modification of atherogenic lipoproteins in arterial wall Conclusion: These results showed that: a) 15-PGDH downregulation in vulnerable plaques may increase PGE2 formation leading in turn to MMP-mediated plaque rupture; b) 15-PGDH and COX-2 are inversely regulated in high-risk plaques, and this can contribute to elevated levels of PGE2 promoting plaque instability.

PO12 RETENTION AND MODIFICATION OF ATHEROGENIC LIPOPROTEINS IN ARTERIAL WALL PO12-314

STEROL REGULATORY ELEMENT BINDING PROTEIN DOWNREGULATES LRP1 EXPRESSION AND LRP1-MEDIATED AGGREGATED LDL UPTAKE BY HUMAN MACROPHAGES

V. Llorente Cortes 1 , T. Royo 2 , M. Otero-Vinas 1 , M. Berrrozpe 1 , L. Badimon 1 . 1 Cardiovascular Research Center, CSIC-ICCC. Hospital de La Santa Creu I Sant Pau, Barcelona, Spain; 2 Departamento de Biologia Celular. School of Medicine. UB, Barcelona, Spain

PO12-315

GAMMA-GLUTAMYLTRANSFERASE ACTIVITY IN HUMAN ATHEROSCLEROTIC PLAQUES: ORIGIN, PROOXIDANT EFFECTS AND POTENTIAL ROLES IN PROGRESSION OF DISEASE

A. Pompella 1 , M. Franzini 1 , M. Emdin 2 , C. Passino 2 , A. Paolicchi 1 . of Experimental Pathology, University of Pisa Medical School, Pisa, Italy; 2 Inst. of Clinical Physiology, National Research Council, Pisa, Italy 1 Dept.

Serum gamma-glutamyltransferase (GGT) activity has been identified as a predictor of complications of coronary atherosclerosis and stroke (1, 2). GGT activity can give rise to prooxidant molecular species, and promotes LDL oxidation in vitro. As oxidative processes are involved at various levels in atherogenesis, and human atherosclerotic lesions do contain variable levels of active GGT, it is conceivable that the enzyme can directly contribute to pathogenesis and progression of atherosclerosis (3). The biochemical characteristics of GGT protein were compared in plaque tissue (endoarteriectomy) and in serum and platelets obtained from healthy donors and cardiovascular patients. Electrostatic charge of GGT, its association with serum macromolecules, and the molecular weight of its large subunit were analyzed. Both in serum and plaque extracts, two distinct GGT complexes with lipoproteins were found, corresponding to the m.w. of HDL and VLDL/LDL. Further analysis showed the expression in plaques of GGT-I gene, coding for the complete and active enzyme, as well as its product cysteinyl-glycine, both in free and protein bound forms. Our observations suggest that the predictive value of serum GGT for

cardiovascular death is due to a direct contribution of serum GGT to GGT activity within the plaque, and to prooxidant GGT-driven reactions. References: [1] Paolicchi et al. Circulation 109: 1440, 2004. [2] Pompella et al. Clin. Chem. Lab. Med. 42: 1085-1091, 2004. [3] Emdin et al. Circulation 112: 2078-80, 2005. Supported by Italian Ministry for University and Research, FIRB and PRIN Funds. PO12-316

OXIDATION OF HUMAN LOW-DENSITY LIPOPROTEIN CHOLESTEROL TAKEN UP BY HUMAN ENDOTHELIAL CELLS IN TISSUE CULTURE

L.H. Krut 1,2 . 1 Department of Internal Medicine, St. Mary’s Health Center, St. Louis, MO, USA; 2 Department of Internal Medicine, St. Louis University, St. Louis, MO, USA The only tissue cells ordinarily exposed to low-density lipoprotein (LDL) cholesterol at its concentration in plasma are hepatocytes and endothelial cells. Hepatocytes do oxidize cholesterol. We know little on cholesterol oxidation by endothelial cells. To examine this, human umbilical vein endothelial cells (HUVEC) grown in tissue culture were exposed for 15, 60, 180 and 300 minutes to 3 H-cholesterol incorporated into human LDL. The uptake of 3 H from medium by cells at the above times ranged between 0.8 and 1.9%, 1.1 and 4.8%, 2.9 and 7.1% and 4.5 and 10.7% respectively. Lipids extracted from media and cells were chromatographed on thin layer silica gel (TLC) and the TLC plates subjected to autoradiography. The media extracts showed a band in the position of cholesterol, and a trace amount in the position of cholesterol esters. The cell extracts showed the above plus at least six additional constituents more polar than cholesterol. The latter are believed to be oxysterols. Some of these migrate as do oxysterols generated by oxidizing cholesterol in vitro. The distribution of radioactivity was quantified by scraping the TLC plates and counting activity in the sterol esters, cholesterol and the total oxysterols. The distribution was similar for all incubation periods, respectively 1.7-4.7%, 71.5-76.0% and 19.1-24.2%. Cholesterol entering HUVEC in human LDL is oxidized rapidly, substantially and consistently. Oxidation of LDL cholesterol in endothelial cells is clearly part of the normal metabolism of cholesterol in these cells. These findings have implications for postulates on atherogenesis. PO12-317

ACTIVATION STATE OF NEUTROPHILS IN PATIENTS WITH STABLE CORONARY ARTERY DISEASE

E. Sarndahl 1 , I. Bergstrom 1 , V. Patcha 2 , J. Nijm 3 , H. Lundqvist Setterud 1 , L. Jonasson 3 . 1 Division of Medical Microbiology, IMK, Faculty of Health Sciences, Linkoping University, Linkoping, Sweden; 2 Division of Cell Biology, IBK, Faculty of Health Sciences, Linkoping University, Linkoping, Sweden; 3 Division of Cardiology, IMV, Faculty of Health Sciences, Linkoping University, Linkoping, Sweden Background: High levels of circulating neutrophils are observed in patients with stable coronary artery disease (CAD) and a primed state of the neutrophils has been proposed. We investigated neutrophil activation during basal and stimulatory conditions in patients with stable CAD. Methods: We included 30 patients with stable CAD and 30 individually matched healthy controls. Five patients with acute coronary syndrome (ACS) served as “positive” controls. Whole blood or isolated neutrophils were stimulated with IL-8 and LTB4. The expression of CD18 and high-affinity state of CD11b was analysed by flow cytometry before and after stimulation. The production of reactive oxygen species (ROS) was also determined. Results: During basal conditions, the expression of CD18 and the high-affinity state of CD11b did not differ between stable CAD patients and controls. IL-8 and LTB4 up-regulated the expression of CD18 to a similar extent in patients and controls. IL-8 and LTB4 induced an increase in beta2-integrin affinity that did not differ between patients and controls (137±9 vs 145±12% and 293±44 vs 235±18%, respectively). The ROS production was similar in the 2 groups. On the other hand, the neutrophil activation state in the ACS group was markedly increased during both basal and stimulatory conditions. Conclusions: Neutrophils from stable CAD patients were not more activated than cells from healthy controls, neither were the neutrophils from patients more prone to activation than cells from controls. The data

76th Congress of the European Atherosclerosis Society, June 10–13, 2007, Helsinki, Finland

POSTER SESSIONS

Background and Aims: In the extracellular intima, extracellular matrix proteoglycans favour LDL retention and aggregation (agLDL). Contrarily to native LDL (nLDL), agLDL is a potent inducer of massive intracellular cholesteryl ester (CE) accumulation in macrophages. It has been suggested that low density lipoprotein receptor related protein (LRP1) is involved in agLDL binding and internalization by macrophages. The aim of this work was to analyze whether sterol regulatory element binding protein (SREBP) modulates LRP1 expression and LRP1-mediated agLDL uptake in human monocyte-derived macrophages (HMDM). Methods and Results: The treatment of HMDM with small anti-LRP1 interfering RNA (siRNA-LRP1) led to the specific inhibition of LRP1 mRNA expression and also to the inhibition of LRP1 protein expression in these cells. In siRNA-LRP1-treated HMDM, CE accumulation from agLDL uptake (84.66±5 μg CE/mg protein) was reduced by 95.76±5.22%. This suggests that LRP1 plays a pivotal role in agLDL uptake by HMDM. N-acetyl-leucyl-leucyl-norleucinal (ALLN), an inhibitor of SREBP catabolism, maintained high levels of active SREBP-2 and SREBP-1a even in the presence of nLDL and agLDL. Therefore, ALLN induced LDL receptor (LDLR) upregulation. Concomitantly, a strong downregulation of LRP1 mRNA and LRP1 protein was observed in ALLN-treated macrophages. By decreasing LRP1 expression levels, ALLN reduced CE accumulation from agLDL at all tested concentrations. Conclusions: These results suggest that high levels of active SREBP downregulate LRP1 expression and intracellular CE accumulation in HMDM.

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Poster Sessions PO12 Retention and modification of atherogenic lipoproteins in arterial wall

contradict the theory that priming of circulating neutrophils in stable CAD is of importance in the progress of disease. PO12-318

ACIDIC PH ENHANCES LIPOLYSIS OF SMALL VLDL, IDL, AND LDL BY SPLA2-V AND ENHANCES THEIR BINDING TO HUMAN AORTIC PROTEOGLYCANS

K. Lahdesmaki 1 , E. Hurt-Camejo 2 , P.T. Kovanen 1 , K. Oorni 1 . 1 Wihuri Research Institute, Helsinki, Finland; 2 AstraZeneca, R&D, Molecular Pharmacology, Molndal, Sweden Binding of lipoproteins to intimal proteoglycans and enzymatic modifications of lipoproteins are key processes in atherogenesis. During atherogenesis the extracellular pH of the atherosclerotic lesions decreases. The objective of this study was to examine how pH influences lipolysis of small very low density lipoprotein (sVLDL), intermediate density lipoprotein (IDL), and low density lipoprotein (LDL) particles by recombinant human group V secretory phospholipase A2 (sPLA2-V), and to test the effect of pH on the binding of native and sPLA2-V-treated sVLDL, IDL, and LDL particles to human aortic proteoglycans. To determine the degree of lipolysis at different pH values, lipoproteins were incubated with sPLA2-V at pH 5.5-7.5 for 24h. The binding of native or sPLA2-V-treated lipoprotein particles to proteoglycans was measured in microtiter well assays at pH 5.5 and pH 7.5. Although pH optimum of sPLA2-V is slightly basic, the ability of the enzyme to lipolyze sVLDL, IDL, and LDL was increased at acidic pH values. At the lowest pH tested (pH 5.5), the degree of lipolysis of sVLDL, IDL and LDL were 1.4-2-fold higher than at pH 7.5. Also, at acidic pH, the binding of these lipoproteins to proteoglycans did increase dramatically, and sPLA2-treatment further increased the amounts of proteoglycan-bound lipoproteins. Taken together, our results suggest that in areas of atherosclerotic arterial intima, where the extracellular pH decreases and sPLA2-V is present, retention of sVLDL, IDL and LDL by the intimal proteoglycans is enhanced, and may lead to extracellular lipid accumulation and progression of the disease. PO12-319

BINDING OF LIPOPROTEINS TO PROTEOGLYCANS AND AGGREGATION OF MODIFIED LIPOPROTEINS IS ENHANCED AT ACIDIC PH

K. Oorni, M. Sneck, K. Lahdesmaki, P.T. Kovanen. Wihuri Research Institute, Helsinki, Finland Binding of apolipoprotein B-100 –containing lipoproteins (VLDL, IDL, and LDL) to proteoglycans and modifications of the lipoproteins, whether bound or unbound, are key processes in atherogenesis. The complex interplay between binding and modification has traditionally been studied at conditions of neutral pH. Recently, it has been demonstrated that during atherogenesis the extracellular pH of the lesions decreases. Many of the enzymes that are found in the arterial intima, such as secretory sphingomyelinase and cathepsins, are able to hydrolyze lipoproteins in vitro. Since these enzymes have acidic pH optima (pH 5.5-6.5), they act on lipoproteins particularly at acidic pH. Interestingly, aggregation of modified lipoproteins is strongly enhanced at acidic pH. Furthermore, the ability of human aortic proteoglycans to bind native VLDL, IDL, and LDL is dramatically increased at acidic pH, and this binding can be further increased if these apoB-100-containing particles are hydrolytically modified. Our present findings suggest that in areas of atherosclerotic arterial intima where the extracellular pH is decreased, both binding of apoB-100-containing lipoproteins to proteoglycans and modification of the lipoproteins by acidic enzymes are enhanced. The pH-induced amplification of these processes will lead to enhanced extracellular accumulation of lipoproteins and accelerated progression of the disease. PO12-320

DECREASE IN PH STRONGLY ENHANCES AGGREGATION OF SPHINGOMYELINASE-TREATED LDL PARTICLES

M. Sneck, P.T. Kovanen, K. Oorni. Wihuri Research Institute, Helsinki, Finland The key processes in the development of atherosclerotic lesions include binding of LDL to intimal proteoglycans and modification of LDL, leading to aggregation of the LDL particles. Recently, it has been demonstrated

that during atherogenesis, the extracellular pH of the atherosclerotic lesions decreases. We have now examined whether the decreased pH has an effect on aggregation of sphingomyelinase (SMase) or phospholipase A2 (PLA2)-treated LDL particles and on the binding of LDL to proteoglycans. Aggregation of the lipolyzed LDL particles was followed at pH 5.5-7.5. We found that the lower the pH of the incubation medium was, the higher was the SMase-induced aggregation of LDL particles, while the pH had no effect on PLA2-induced LDL aggregation. At pH 5.5, the SMase-treated LDL particles formed large aggregates having diameters up to 1 μm. The lipid aggregates became dissolved at high ionic strength indicating that the aggregated LDL particles are attached to each other via ionic interactions. Interestingly, emulsion particles lacking any protein failed to form such large aggregates at low pH revealing that the protein moieties of the LDL particles were necessary for the formation of large aggregates. These results suggest that in the areas of the arteries, where the extracellular pH decreases, modification of LDL by SMase leads to formation of large extracellular LDL aggregates. PO12-321

DIFFERENTIAL SUSCEPTIBILITY OF HUMAN GRANULOCYTES AND MACROPHAGES TO ENZYMATICALLY MODIFIED LDL

C.A. Lux 1 , K. Dersch 1 , A. Koschinski 2 , M. Husmann 1 , S. Bhakdi 1 . for Medical Microbiology and Hygiene, Johannes Gutenberg-University Mainz, Mainz, Germany; 2 Rudolf-Buchheim-Institute for Pharmacology, Justus-Liebig-University Gießen, Gießen, Germany 1 Institute

Background: We have previously demonstrated that LDL entrapped in the arterial intima is cleaved by proteases and cholesterin esterase. The resulting enzymatically modified LDL (E-LDL) is rich in free fatty acids and free cholesterol. Methods: The susceptibility of human granulocytes and monocytederived macrophages towards enzymatically altered LDL was assayed by measurement of LDH release and ATP loss. Respiratory burst, release of myeloperoxidase and calcium influx were determined as markers for PMN activation. Results: E-LDL displayed toxicity towards PMN at lower concentrations than towards MDM. Oxidized or unmodified LDL had no effect. Free fatty acids are known to cause damage and death of different cells and proved to be the cause of the observed toxicity. E-LDL activated PMN and caused calcium influx. Inhibition of either oxidative burst or calcium influx did not affect toxicity, nor did depletion of ATP. Evidence is presented that cytotoxicity of E-LDL towards PMN is derived from detergent-like effects of the free fatty acids contained in the modified lipoprotein. Conclusions: The free fatty acids contained in E-LDL, by virtue of their capacity to directly disrupt cell membranes, exert potent cytotoxic effects on PMN, whereby these cells simultaneously display characteristics of apoptotic and necrotic cell death. Differences in the membrane composition of granulocytes and macrophages may account for the different sensitivity of these cells, constituting a possible reason for the absence of granulocytes from the atherosclerotic lesion. PO12-322

EFFECTS OF PHOSPHATIDYLETHANOL ON ENDOSOMES IN CULTURED HUMAN ENDOTHELIAL CELLS

M.K. Liisanantti 1 , T. Lehto 1 , T. Huusko 1 , S. Eskelinen 2 , R. Sormunen 2 , J. Vuoristo 2 , M.J. Savolainen 1 . 1 Clinical Research Center, Department of Internal Medicine, University of Oulu, Oulu, Finland; 2 Biocenter Oulu, University of Oulu, Oulu, Finland Background and Aims: Phosphatidylethanol (PEth) is an aberrant phospholipid formed only in the presence of ethanol that is shown to be beneficial against coronary heart disease. Our recent studies have shown that lipoprotein-associated PEth is transferred from lipoproteins to vascular wall cells and increases vascular endothelial growth factor secretion from these cells by protein kinase c and mitogen-activated protein kinase mediated pathways. In this study we wanted to test whether lipoproteinassociated PEth affects the structure and function of endosomes in vascular endothelial cells. Methods: Lipoproteins were isolated from the plasma of healthy volunteers. In this study, cultured human EA.hy 926 endothelial cells were used to study the effects of PEth on the structure and function of endothelial cells. Affymetrix microarray and semi-quantitative RT-PCR were used to measure mRNA expression of endothelial cells treated by PEth. Western

76th Congress of the European Atherosclerosis Society, June 10–13, 2007, Helsinki, Finland