- Email: [email protected]
Tu-P7:204 Nutrition, inflammation and atherosclerosis
Tues&ty, June 20, 2006: Poster Session P7 Basic science ( lst parO
ITu-P7:200
I
Ii G R O U P I V C P H O S P H O L I P A S E A 2 S U P P L I E S FATTY ACIDS UTILIZED FOR THE FORMATION OF CHOLESTERYL ESTER AND TRIACYLGLYCEROL IN M A C R O P H A G E S
H. Ii, M. Oka, S. Akiba, T. Sato. Department of Pathological Biochemistty, Kyoto Pla3rna3ceutical University, Kyoto, Japan Objective: A key event in the progression of atherosclerosis is the transformation of macrophages into lipid-laden foam cells, of which formation is associated with the uptake of oxidized low-density lipoprotein (oxLDL) and subsequent overproduction of cholesteryl ester (CE) and triacylglycerol (FG), which are components of intracellulax lipid droplets. However, little is known about the pathways involved in the supply of fatty acids, precursors for acyl-CoA required for the formation of CE and TG. We examined the possible involvement of two group IV phospholipase A2 (PLA2) subtypes, group IVA and IVC PLA2s, in the supply of fatty acids in oxLDL-stimulated mouse peritoneal macrophages ( M P M ) a n d phorbol 12-myristate 13-acetatedifferentiated THP-1 human macrophages (THP-M). Results: In [3H]oleic acid-labeled MPM and THP-M, oxLDL induced the formation of [3H]CE and [3H]TG. These responses were suppressed by methyl arachidonyl fluorophosphonate, an inhibitor of both group IVA and IVC PLA2s. However, increases in [3H]CE and CE mass induced by oxLDL were not different between the MPM from group IVA PLA2-knockout and wild-type mice. Similar results were observed in group IVA PLA2knockdown THP-M. Interestingly, in group IVC PLA2-knockdown THP-M the oxLDL-induced formation of [3H]CE and [3H]TG was suppressed. Conclusion: Our findings suggest that group IVC PLA2 but not group IVA PLA2 is mainly involved in the formation of CE and TG in oxLDL-stimulated macrophages. Therefore, group IVC PLA2 may be a novel therapeutic target in atherosclerosis.
ITu-P7:2011 OP HXOI DSIPZHEODL ILPDI DL ( FORXALCDTLI) OANN DIN TNHEEUI TRR O P H I L MONOCYTES
AND
CULTURE CELLS
M.C. Jurado, S. Jancar, S.L. G6mez, M. Gidlund. Institute of Biomedical Sciences, Universi~ of Scio Paulo, Scio Paulo, Brazil oxLDL is involved in atherosclerosis. Phospholipids fractions (PLox) could exert atherogenic effects in the same way as PAF. Here we assessed the effect of LDL, oxLDL and their phospholipids fractions in neutrophils by hydrogen peroxide production and release of MPO and by monocytes by TNFalpha and IL-10 production (ELISA).LDL was obtained by ultracentrifugation and oxidized with 20uM Cu 2+ for 18h at RT. The phospholipid fraction was obtained by chloroform:methanol extraction and applied to a HPLC system with methanol:acetonitrile. Neutrophils and monocytes were obtained by gradient.Cell cultures were stimulated with LDL, oxLDL and phospholipids fractions in presence or absence of WEB2170 for 2 hours and 24 hours respectively. In neutrophils culture there was no significant difference between LDL and oxLDL in hydrogen peroxide production and MPO released, but the PLox could produce 100 nmoles of H 2 0 2 against 5 nmoles in control. When monocytes were stimulated there was no difference in TNF production between PLox and PL but W E B was able to inhibit the effect of PLox. The 3 components of oxLDL had the same effect on TNF production (250 pg/mL) but the W E B 2170 was able to inhibit only the first peak. oxLDL and LDL was able to induce IL-10 production in monocytes in the same way that LDL and W E B 2170 was able to inhibit the PLox effect.We characterized the phospholipids fractions in oxLDL and observed that its fractions could increase the hydrogen peroxide when compared with the total oxLDL (100 vs 5nmoles) in neutrophils. The PLox fractions could induce the production of TNF as the same way that PAF. Funding: FAPESR CNPq.
I Tu-P7:202 I A C O M P R E H E N S I V E
PLATFORM OF CELL-BASED A S S A Y S F O R T A R G E T S R E L E V A N T IN ATHEROS CLEROSIS
C. Caserini, A. Della Bella, M.G. Giribaldi, G. Piazza, M. Bruno, M. De Silvestris, L. Redaelli, A. Rossignoli, L. Scaxabottolo. Axxam Srl, Milan, Italy Objective: Atherosclerosis, which represents the primary cause of heart failure, stroke and peripheral vascular disease, is a progressive disease characterized by the accumulation of lipids and fibrous elements in the large arteries. Atherosclerosis can be considered a form of chronic inflammation
229
resulting from interaction between vascular circulating elements and cellular components of the arterial wall. Several genes influencing the development of the pathology have been recently identified. The goal of our work was the generation of a comprehensive platform of assays for these potential targets, suitable for the high-throughput identification of novel therapeutics. M e t h o d s : Cell based assays suitable for studying the functionality of different targets: chemokine receptors (CCR2b, CXCR2, CX3CR1) involved in monocyte recruitment and infiltration, nuclear hormone receptors (LXR, ER, PPAR, RXR) involved in lipid metabolism, vasodilation modulators (IP, AGTR, NOS) and prothrombotic factors (collagen receptor) have been generated using cell lines expressing luminescence based reporter systems and an innovative calcium sensing photoprotein. Results: All the cell lines obtained produced strong and stable luminescence signals upon stimulation with the specific receptor ligands. Conclusions: We have implemented a platform of cell based assays for several molecular targets involved in different stages of atherosclerotic plaque progression. All these assays showed highly sensitive and reproducible signals and axe particularly suitable for the high-throughput screening of chemical compound libraries.
"I-u-P7:203 ] D I S T U R B E D C A 2 + T R A N S P O R T IN N E U T R O P H I L S OF OBESE PATIENTS I. Seres, G. Paragh, B. Kosztficzky, T. Kalmfir, H.Z. Mirdamadi, A. Kassad, G. Fdris. First Department of Medicine, Unh,ersi~ of Debrecen, Debrecen,
Hungary Objectives: To investigate [Ca2+]i homeostasis in neutrophils of obese (Ob) patients after angiotensin II (Ang II)-stimulation, in comparison with cells of healthy subjects (C) and there of hypercholesterolemic (HC) patients. Design: Studies were carried out on neutrophils of 12 control, 14 Ob and 14 HC patients. M e t h o d s : The Ang II-induced superoxide anion, inositoltrisphosphate (IP3) and Ca 2+ signals were studied. The Ca 2+ signals were determined over 6 rain after stimuli, and the areas under curves (AUC) were calculated, too. The in vitro treatment of cells, with pertussis toxin (PTX), Ca2+-free medium, containing verapamil (V), and fluvastatin (Flu) presents an opportunity to evaluate Ca 2+ balance in Ob-neutrophils. Results: 1.) In neutrophils of Ob patients, similarly to HC-neutrophils, the Ang II-induced Ca 2+ signal originated from the extracellular medium. The delayed return of [Ca2+]i to the basal level after Ang II-stimulus is related to the Ang II-triggered enhancement of superoxide anion generation both in Ob and HC neutrophils. 2.) Evaluating the areas under time-curves (AUC) of Ca 2+ signals, the AUC values of both the Ob and HC neutrophils were significantly higher than those of the control cells. Conclusion: In obesity, similarly to HC, the Ca 2+ signal in neutrophils after Ang II stimulation originated from the extracellulax Ca 2+, and the subsequent normalization of [Ca2+]i was delayed, as a consequence of increased amounts of stimulus-triggered, statin-inhibitable superoxide anion generation.
Tu-P7:204]
NUTRITION, INFLAMMATION AND ATHEROSCLEROSIS
R. Sin~h I , M. Saxena 2, J. Sharma 3. 1Halberg Hospital arm Research bzstitute, Morwdabcud, bMia: "~MetroPathology, Mora&tbad, bMia: 3Sharna3 Matemi~ Nursbtg Home, Morwdabcud, bMia Objective: To evaluate that total fat (TF) saturated fat(SF),and high w-6/w-3 fatty acid may enhance tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6). M e t h o d : Review of studies by Paoletti,Kelly, Rallidis and others that TNFalpha,IL-6,are risk factors of atherosclerosis.Case study among 54 patients with myocardial infarction (MI) diagnosed by W H O criteria. Dietary intakes by, dietary diaries and TNF-alpha and IL-6 were obtained by immulite assay using chemical kit from LA,(USA). Results: Singh observed (2001) in 18 patient of AMI that fish oil caused significant reduction in TNF, IL-6 and oxidative stress, compared to control subject.In a recent study, half of the A M I cases(n=27) were consuming high TFA, diet and half large meals (> 1000 kcal/meal). A large break fast (> 1000 kcal) was consumed by 40.7%(n=22) of which contained excess of total and TFA and a high w-6/w-3 fatty acid ratio diet.Plasma levels of IL-6 (32.6-t-6.5 vs. 27.5+5.2pg/ml,P<0.05) and TNF, (42.5-t-8.8 vs. 38.2-t-10.6 pg/ml,P<0.05) and TBARS were significantly greater and plasma CoQ significantly lower (0.21-t-0.02 vs 0.23-t-0.03 ug/mb, p=0.02) among patients taking large breakfast compared to those eating small breakfast respectively.
XIV bztetTtational Symposium on Atherosclerosis, Rome, Italy, June 18-22, 2006
230
Tues&ty, June 20, 2006: Poster Session P7 Basic science ( lst part)
Conclusions: The findings indicate that increased intake of w-3 fatty acids can decrease inflammation whereas excess intake of total and SF and L A may have adverse effects. Funding: Center of Nutrition Res. and Metro Patho,India. Tishcon Corporation,USA,
I Tu-P7:207 I R E S I S T A N C E
TO DIABETIC VASCULAR DISEASE A N D T H E H A P T O T G L O B I N 1-1 G E N O T Y P E : T H E ROLE OF OXIDATIVE STRESS AND THE INDUCTION OF INTERLEUKIN-10
J. Guetta, N. Levy, A.P. Levy. Rappaport Facul~ of Medicine, Technion,
Haifit, Israel Tu-P7:205
CHARACTERIZATION OF INTRACELLULAR LOOPS O F P R O S T A C Y C L I N R E C E P T O R C O U P L E D T O GS PROTEIN USING MINIGENE TECHNIQUE
K.-H. Ruan, L. Zhang. Universi~ of Texas Health Science Center At Houston,
Houston, USA The C-terminal domain of the human alpha-subunit of Galpha s protein (Galpha s Ct) and the first intracellular loop OLP1) of human prostacyclin receptor (IP), a prostanoid receptor (PR), involved in the receptor signaling mediated by the Gs protein coupling have been predicted by our previous NMR studies using synthetic peptides (Zhang et al., Biochemistry 44, 2005). To test whether the results of the NMR/peptide studies can be applied to the protein properties in cells, a minigene technique was used to construct cDNAs that encoded either the amino acid sequences of the Galpha s Ct or that of the intracellulax loops of the IE The effects of the minigene-expressed protein fragments on cAMP production mediated by the IP/Gs coupling were evaluated through experiments that co-expressed either the Galpha s Ct or the individual IP intracellular loop with the IP in HEK293 cells. The iLP1 and third OLP3) IP intracellular loops, as well as the Galpha s Ct, which are important to the receptor/Gs coupling-mediated signaling, were identified by the significant reduction of the cAMP production when the corresponding peptides expressed in the cells. Furthermore, the cAMP productions were significantly impaired in the Galpha s C-terminal mutants, E392A, L393A and L394A co-expressing with the IP in a Galpha s-knockout cell line. These results revealed that the three residues (E392 ~ L394) of the GTs protein predicted from NMR peptide studies play a key role in the Gs-mediated IP signaling that mediates vasodilatation and inhibition of platelet aggregation, and may be a general binding site for other PRs that couple to the Gs protein. The studies have also provided clues for drug designs to regulate the receptor signaling targeted the IP and other PRs using minigenes encoding the intracellulax loop domains of the receptors, and/or the Galpha s Ct domain.
Tu-P7:206 ] M O L E C U L A R S I G N A T U R E S D E T E R M I N I N G CORONARY ARTERY AND SAPHENOUS VEIN SMOOTH MUSCLE CELL PHENOTYPES: DISTINCT RESPONSES TO STIMULI D.X. Deng 1 , J.M. Spin 2, A. Tsalenko 1 , A. Vadlaya 1 , A. Ben-Dor 1 , Z. Yakhini ] , E Tsao-, L. Bruhn ] , T. Quertermous-. Agtlent Technologws,
Palo Alto, USA." 2Stcmford Universi~ School of Medicine, Stcmford, USA Objectives: We compared gene expression profiles and functional responses to oxidized LDL (OxLDL) and platelet-derived growth factor (PDGF) between cultured SMCs from human coronary artery (CASM) and saphenous vein (SVSM) to better understand the molecular mechanisms underlying functional differences among distinct SMC subtypes. M e t h o d s a n d Results: OxLDL and PDGF elicited markedly different functional responses and expression profiles between the two SMC subtypes. In CASM, OxLDL inhibited cell proliferation and migration, and modified gene expression of some chemokines, proinflammatory cytokines, insulin-like growth factor binding proteins (IGFBPs), and endothelial and smooth muscle marker genes. In SVSM, OxLDL promoted proliferation partially via IGF1 signaling, activated NF-kappaB and phosphatidylinositol signaling pathways, and upregulated prostaglandin receptors and synthases. In untreated cells, alpha-chemokines, proinflammatory cytokines and genes associated with lipid biosynthesis were higher in CASM, while some beta-chemokines, metalloproteinase inhibitors and IGFBPs were higher in SVSM. The expression levels of subset of genes distinguishing the two SMC subtypes were consistent with relative expression levels in in vivo tissues between human arteries (radial and internal mammary arteries) and saphenous veins. Interestingly, the basal expression levels of these genes seemed closely related to their responses to OxLDL and PDGF. Conclusions: Our results reveal molecular signatures that define the distinct phenotypes characteristics of coronary artery and saphenous vein SMC subtypes.
Objectives: We have demonstrated in longitudinal and cross sectional studies that haptoglobin (Hp) genotype is a major determinant of susceptibility to diabetic vascular complications. Hp binds to hemoglobin (Hb) and Hp-Hb complexes bind to the CD163 receptor on macrophages. We proposed that haptoglobin protein products differ in their ability to protect against hemoglobin driven oxidative stress and in modulating macrophage activation. M e t h o d s : Oxidative stress in tissue culture cells was mesured using DCFH. Peritoneal macrophages(MPM) were taken from wild type mice (Hp 1) or transgenic (Hp 2) mice with or without streptozotocin induced diabetes (DM). Human monocyte - derived macrophages were isolated using Histipaque-1077. Cytokine concentrations were measured by ELISA. Results: We found that oxidative stress was significantly increased in vitro in Chinese Hamster Cells (CHO) by Hp 2-2-Hb protein complex under hyperglycemia compared to Hp 1-1-Hb. DFO, an iron chelator, inhibited the increased oxidation. In addition we found that oxidative stress was significantly increased in the MPMs from DM Hp 2 mice. We found that Hp 1-1-Hb complexes stimulated production of IL-10 by human monocyte - derived macrophages more than Hp 2-2-Hb complexes. Antibody to CD163 receptor abolished Hp 1-1-Hb increases in IL-10. Conclusions: The protection provided by the Hp 1-1 genotype against the development of diabetic vascular disease may be mediated by its antioxidant capacity and its ability to induce the anti-oxidant anti-inflammatory cytokine IL-10. Funding: D Cure Diabetes Care in Israel and the Russell Berrie Foundation.
I Tu-P7:208 I A T H E R O S C L E R O T I C
LESION PROGRESSION CHANGES LYSOPHOSPHATIDIC ACID H O M E O S T A S I S T O FAVOR ITS A C C U M U L A T I O N
M. Bot, I. Bot, T.J.C. Van Berkel, E.A.L. Biessen. Division of Biopharntaceutics, LACDR, Leiden Universi~, Leiden, The Netherlands Objective: Lysophosphatidic acid (LPA) accumulates in the lipid core of human atherosclerotic plaques and is the primary platelet-activating lipid. Plaque rupture will expose entrapped LPA, which may trigger thrombus formation. Here, we aimed to temporally profile the metabolic regulation of LPA homeostasis and LPA accumulation during lesion progression. M e t h o d s : Atherosclerosis was induced in LDLr-/- mice by placing carotid artery collars. At 0, 2, 4, 6 and 8 weeks after surgery, RNA and lipids were extracted from the plaques. Gene expression of enzymes involved in LPA homeostasis was analyzed by RT-PCR, while lipid extracts were evaluated for LPA content. Results: Phospholipase D3, which generates phosphatidic acid (PA), showed a 4-fold increase in expression. Cytoplasmic phospholipase A2IVA, which converts PA to LPA, was 8-fold increased. Conversely, a 50% reduction was seen for LPA acyltransferase, which hydrolyzes LPA. LPA receptor 2 and 3 showed respectively a 12 and 5-fold increase. Plaque lipid analysis revealed a significant increase in LPA content of advanced lesions (0.9 to 37.6 pmol LPA/mg tissue, P<0.05) similar to human advanced plaques. Conclusions: Atherosclerosis progression results in a changed expression of proteins involved in LPA homeostasis reflecting a strongly induced intracellulax LPA production and an increased signal transduction. Lipid composition analysis confirmed accumulation of LPA in atherosderotic tissue. We propose that intervention in the LPA metabolism could be a promising therapeutic entry in reducing atherogenesis. Funding: Dutch Thrombosis Foundation grant 2004.01
I Tu-P7:209
I
Ii T H E R E D U C T I O N O F S E R U M A T H E R O G E N I C I T Y BY NATURAL ANTI-INFLAMMATORY DRUG
T.V. Gorchakova I , V.A. M,/asoedova 1, I.A. Sobenin 1 , A.N. Orekhov 1'2 .
l b~stitute of General Pathology arm Pathophysiology, Moscow, Russia: 2bzstitute for Atherosclerosis Research, Moscow, Russia Objective: The aim of this investigation was to study antiatherosclerotic effects of natural anti-inflammatory drug inflaminat, based on black elder berries, calendula flowers and violet flowers, in vitro and ex vivo cell culture-based models.
XIV bztentational Symposium on Atherosclerosis, Rome, Italy, June 18-22, 2006
ITu-P7:200
I
Ii G R O U P I V C P H O S P H O L I P A S E A 2 S U P P L I E S FATTY ACIDS UTILIZED FOR THE FORMATION OF CHOLESTERYL ESTER AND TRIACYLGLYCEROL IN M A C R O P H A G E S
H. Ii, M. Oka, S. Akiba, T. Sato. Department of Pathological Biochemistty, Kyoto Pla3rna3ceutical University, Kyoto, Japan Objective: A key event in the progression of atherosclerosis is the transformation of macrophages into lipid-laden foam cells, of which formation is associated with the uptake of oxidized low-density lipoprotein (oxLDL) and subsequent overproduction of cholesteryl ester (CE) and triacylglycerol (FG), which are components of intracellulax lipid droplets. However, little is known about the pathways involved in the supply of fatty acids, precursors for acyl-CoA required for the formation of CE and TG. We examined the possible involvement of two group IV phospholipase A2 (PLA2) subtypes, group IVA and IVC PLA2s, in the supply of fatty acids in oxLDL-stimulated mouse peritoneal macrophages ( M P M ) a n d phorbol 12-myristate 13-acetatedifferentiated THP-1 human macrophages (THP-M). Results: In [3H]oleic acid-labeled MPM and THP-M, oxLDL induced the formation of [3H]CE and [3H]TG. These responses were suppressed by methyl arachidonyl fluorophosphonate, an inhibitor of both group IVA and IVC PLA2s. However, increases in [3H]CE and CE mass induced by oxLDL were not different between the MPM from group IVA PLA2-knockout and wild-type mice. Similar results were observed in group IVA PLA2knockdown THP-M. Interestingly, in group IVC PLA2-knockdown THP-M the oxLDL-induced formation of [3H]CE and [3H]TG was suppressed. Conclusion: Our findings suggest that group IVC PLA2 but not group IVA PLA2 is mainly involved in the formation of CE and TG in oxLDL-stimulated macrophages. Therefore, group IVC PLA2 may be a novel therapeutic target in atherosclerosis.
ITu-P7:2011 OP HXOI DSIPZHEODL ILPDI DL ( FORXALCDTLI) OANN DIN TNHEEUI TRR O P H I L MONOCYTES
AND
CULTURE CELLS
M.C. Jurado, S. Jancar, S.L. G6mez, M. Gidlund. Institute of Biomedical Sciences, Universi~ of Scio Paulo, Scio Paulo, Brazil oxLDL is involved in atherosclerosis. Phospholipids fractions (PLox) could exert atherogenic effects in the same way as PAF. Here we assessed the effect of LDL, oxLDL and their phospholipids fractions in neutrophils by hydrogen peroxide production and release of MPO and by monocytes by TNFalpha and IL-10 production (ELISA).LDL was obtained by ultracentrifugation and oxidized with 20uM Cu 2+ for 18h at RT. The phospholipid fraction was obtained by chloroform:methanol extraction and applied to a HPLC system with methanol:acetonitrile. Neutrophils and monocytes were obtained by gradient.Cell cultures were stimulated with LDL, oxLDL and phospholipids fractions in presence or absence of WEB2170 for 2 hours and 24 hours respectively. In neutrophils culture there was no significant difference between LDL and oxLDL in hydrogen peroxide production and MPO released, but the PLox could produce 100 nmoles of H 2 0 2 against 5 nmoles in control. When monocytes were stimulated there was no difference in TNF production between PLox and PL but W E B was able to inhibit the effect of PLox. The 3 components of oxLDL had the same effect on TNF production (250 pg/mL) but the W E B 2170 was able to inhibit only the first peak. oxLDL and LDL was able to induce IL-10 production in monocytes in the same way that LDL and W E B 2170 was able to inhibit the PLox effect.We characterized the phospholipids fractions in oxLDL and observed that its fractions could increase the hydrogen peroxide when compared with the total oxLDL (100 vs 5nmoles) in neutrophils. The PLox fractions could induce the production of TNF as the same way that PAF. Funding: FAPESR CNPq.
I Tu-P7:202 I A C O M P R E H E N S I V E
PLATFORM OF CELL-BASED A S S A Y S F O R T A R G E T S R E L E V A N T IN ATHEROS CLEROSIS
C. Caserini, A. Della Bella, M.G. Giribaldi, G. Piazza, M. Bruno, M. De Silvestris, L. Redaelli, A. Rossignoli, L. Scaxabottolo. Axxam Srl, Milan, Italy Objective: Atherosclerosis, which represents the primary cause of heart failure, stroke and peripheral vascular disease, is a progressive disease characterized by the accumulation of lipids and fibrous elements in the large arteries. Atherosclerosis can be considered a form of chronic inflammation
229
resulting from interaction between vascular circulating elements and cellular components of the arterial wall. Several genes influencing the development of the pathology have been recently identified. The goal of our work was the generation of a comprehensive platform of assays for these potential targets, suitable for the high-throughput identification of novel therapeutics. M e t h o d s : Cell based assays suitable for studying the functionality of different targets: chemokine receptors (CCR2b, CXCR2, CX3CR1) involved in monocyte recruitment and infiltration, nuclear hormone receptors (LXR, ER, PPAR, RXR) involved in lipid metabolism, vasodilation modulators (IP, AGTR, NOS) and prothrombotic factors (collagen receptor) have been generated using cell lines expressing luminescence based reporter systems and an innovative calcium sensing photoprotein. Results: All the cell lines obtained produced strong and stable luminescence signals upon stimulation with the specific receptor ligands. Conclusions: We have implemented a platform of cell based assays for several molecular targets involved in different stages of atherosclerotic plaque progression. All these assays showed highly sensitive and reproducible signals and axe particularly suitable for the high-throughput screening of chemical compound libraries.
"I-u-P7:203 ] D I S T U R B E D C A 2 + T R A N S P O R T IN N E U T R O P H I L S OF OBESE PATIENTS I. Seres, G. Paragh, B. Kosztficzky, T. Kalmfir, H.Z. Mirdamadi, A. Kassad, G. Fdris. First Department of Medicine, Unh,ersi~ of Debrecen, Debrecen,
Hungary Objectives: To investigate [Ca2+]i homeostasis in neutrophils of obese (Ob) patients after angiotensin II (Ang II)-stimulation, in comparison with cells of healthy subjects (C) and there of hypercholesterolemic (HC) patients. Design: Studies were carried out on neutrophils of 12 control, 14 Ob and 14 HC patients. M e t h o d s : The Ang II-induced superoxide anion, inositoltrisphosphate (IP3) and Ca 2+ signals were studied. The Ca 2+ signals were determined over 6 rain after stimuli, and the areas under curves (AUC) were calculated, too. The in vitro treatment of cells, with pertussis toxin (PTX), Ca2+-free medium, containing verapamil (V), and fluvastatin (Flu) presents an opportunity to evaluate Ca 2+ balance in Ob-neutrophils. Results: 1.) In neutrophils of Ob patients, similarly to HC-neutrophils, the Ang II-induced Ca 2+ signal originated from the extracellular medium. The delayed return of [Ca2+]i to the basal level after Ang II-stimulus is related to the Ang II-triggered enhancement of superoxide anion generation both in Ob and HC neutrophils. 2.) Evaluating the areas under time-curves (AUC) of Ca 2+ signals, the AUC values of both the Ob and HC neutrophils were significantly higher than those of the control cells. Conclusion: In obesity, similarly to HC, the Ca 2+ signal in neutrophils after Ang II stimulation originated from the extracellulax Ca 2+, and the subsequent normalization of [Ca2+]i was delayed, as a consequence of increased amounts of stimulus-triggered, statin-inhibitable superoxide anion generation.
Tu-P7:204]
NUTRITION, INFLAMMATION AND ATHEROSCLEROSIS
R. Sin~h I , M. Saxena 2, J. Sharma 3. 1Halberg Hospital arm Research bzstitute, Morwdabcud, bMia: "~MetroPathology, Mora&tbad, bMia: 3Sharna3 Matemi~ Nursbtg Home, Morwdabcud, bMia Objective: To evaluate that total fat (TF) saturated fat(SF),and high w-6/w-3 fatty acid may enhance tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6). M e t h o d : Review of studies by Paoletti,Kelly, Rallidis and others that TNFalpha,IL-6,are risk factors of atherosclerosis.Case study among 54 patients with myocardial infarction (MI) diagnosed by W H O criteria. Dietary intakes by, dietary diaries and TNF-alpha and IL-6 were obtained by immulite assay using chemical kit from LA,(USA). Results: Singh observed (2001) in 18 patient of AMI that fish oil caused significant reduction in TNF, IL-6 and oxidative stress, compared to control subject.In a recent study, half of the A M I cases(n=27) were consuming high TFA, diet and half large meals (> 1000 kcal/meal). A large break fast (> 1000 kcal) was consumed by 40.7%(n=22) of which contained excess of total and TFA and a high w-6/w-3 fatty acid ratio diet.Plasma levels of IL-6 (32.6-t-6.5 vs. 27.5+5.2pg/ml,P<0.05) and TNF, (42.5-t-8.8 vs. 38.2-t-10.6 pg/ml,P<0.05) and TBARS were significantly greater and plasma CoQ significantly lower (0.21-t-0.02 vs 0.23-t-0.03 ug/mb, p=0.02) among patients taking large breakfast compared to those eating small breakfast respectively.
XIV bztetTtational Symposium on Atherosclerosis, Rome, Italy, June 18-22, 2006
230
Tues&ty, June 20, 2006: Poster Session P7 Basic science ( lst part)
Conclusions: The findings indicate that increased intake of w-3 fatty acids can decrease inflammation whereas excess intake of total and SF and L A may have adverse effects. Funding: Center of Nutrition Res. and Metro Patho,India. Tishcon Corporation,USA,
I Tu-P7:207 I R E S I S T A N C E
TO DIABETIC VASCULAR DISEASE A N D T H E H A P T O T G L O B I N 1-1 G E N O T Y P E : T H E ROLE OF OXIDATIVE STRESS AND THE INDUCTION OF INTERLEUKIN-10
J. Guetta, N. Levy, A.P. Levy. Rappaport Facul~ of Medicine, Technion,
Haifit, Israel Tu-P7:205
CHARACTERIZATION OF INTRACELLULAR LOOPS O F P R O S T A C Y C L I N R E C E P T O R C O U P L E D T O GS PROTEIN USING MINIGENE TECHNIQUE
K.-H. Ruan, L. Zhang. Universi~ of Texas Health Science Center At Houston,
Houston, USA The C-terminal domain of the human alpha-subunit of Galpha s protein (Galpha s Ct) and the first intracellular loop OLP1) of human prostacyclin receptor (IP), a prostanoid receptor (PR), involved in the receptor signaling mediated by the Gs protein coupling have been predicted by our previous NMR studies using synthetic peptides (Zhang et al., Biochemistry 44, 2005). To test whether the results of the NMR/peptide studies can be applied to the protein properties in cells, a minigene technique was used to construct cDNAs that encoded either the amino acid sequences of the Galpha s Ct or that of the intracellulax loops of the IE The effects of the minigene-expressed protein fragments on cAMP production mediated by the IP/Gs coupling were evaluated through experiments that co-expressed either the Galpha s Ct or the individual IP intracellular loop with the IP in HEK293 cells. The iLP1 and third OLP3) IP intracellular loops, as well as the Galpha s Ct, which are important to the receptor/Gs coupling-mediated signaling, were identified by the significant reduction of the cAMP production when the corresponding peptides expressed in the cells. Furthermore, the cAMP productions were significantly impaired in the Galpha s C-terminal mutants, E392A, L393A and L394A co-expressing with the IP in a Galpha s-knockout cell line. These results revealed that the three residues (E392 ~ L394) of the GTs protein predicted from NMR peptide studies play a key role in the Gs-mediated IP signaling that mediates vasodilatation and inhibition of platelet aggregation, and may be a general binding site for other PRs that couple to the Gs protein. The studies have also provided clues for drug designs to regulate the receptor signaling targeted the IP and other PRs using minigenes encoding the intracellulax loop domains of the receptors, and/or the Galpha s Ct domain.
Tu-P7:206 ] M O L E C U L A R S I G N A T U R E S D E T E R M I N I N G CORONARY ARTERY AND SAPHENOUS VEIN SMOOTH MUSCLE CELL PHENOTYPES: DISTINCT RESPONSES TO STIMULI D.X. Deng 1 , J.M. Spin 2, A. Tsalenko 1 , A. Vadlaya 1 , A. Ben-Dor 1 , Z. Yakhini ] , E Tsao-, L. Bruhn ] , T. Quertermous-. Agtlent Technologws,
Palo Alto, USA." 2Stcmford Universi~ School of Medicine, Stcmford, USA Objectives: We compared gene expression profiles and functional responses to oxidized LDL (OxLDL) and platelet-derived growth factor (PDGF) between cultured SMCs from human coronary artery (CASM) and saphenous vein (SVSM) to better understand the molecular mechanisms underlying functional differences among distinct SMC subtypes. M e t h o d s a n d Results: OxLDL and PDGF elicited markedly different functional responses and expression profiles between the two SMC subtypes. In CASM, OxLDL inhibited cell proliferation and migration, and modified gene expression of some chemokines, proinflammatory cytokines, insulin-like growth factor binding proteins (IGFBPs), and endothelial and smooth muscle marker genes. In SVSM, OxLDL promoted proliferation partially via IGF1 signaling, activated NF-kappaB and phosphatidylinositol signaling pathways, and upregulated prostaglandin receptors and synthases. In untreated cells, alpha-chemokines, proinflammatory cytokines and genes associated with lipid biosynthesis were higher in CASM, while some beta-chemokines, metalloproteinase inhibitors and IGFBPs were higher in SVSM. The expression levels of subset of genes distinguishing the two SMC subtypes were consistent with relative expression levels in in vivo tissues between human arteries (radial and internal mammary arteries) and saphenous veins. Interestingly, the basal expression levels of these genes seemed closely related to their responses to OxLDL and PDGF. Conclusions: Our results reveal molecular signatures that define the distinct phenotypes characteristics of coronary artery and saphenous vein SMC subtypes.
Objectives: We have demonstrated in longitudinal and cross sectional studies that haptoglobin (Hp) genotype is a major determinant of susceptibility to diabetic vascular complications. Hp binds to hemoglobin (Hb) and Hp-Hb complexes bind to the CD163 receptor on macrophages. We proposed that haptoglobin protein products differ in their ability to protect against hemoglobin driven oxidative stress and in modulating macrophage activation. M e t h o d s : Oxidative stress in tissue culture cells was mesured using DCFH. Peritoneal macrophages(MPM) were taken from wild type mice (Hp 1) or transgenic (Hp 2) mice with or without streptozotocin induced diabetes (DM). Human monocyte - derived macrophages were isolated using Histipaque-1077. Cytokine concentrations were measured by ELISA. Results: We found that oxidative stress was significantly increased in vitro in Chinese Hamster Cells (CHO) by Hp 2-2-Hb protein complex under hyperglycemia compared to Hp 1-1-Hb. DFO, an iron chelator, inhibited the increased oxidation. In addition we found that oxidative stress was significantly increased in the MPMs from DM Hp 2 mice. We found that Hp 1-1-Hb complexes stimulated production of IL-10 by human monocyte - derived macrophages more than Hp 2-2-Hb complexes. Antibody to CD163 receptor abolished Hp 1-1-Hb increases in IL-10. Conclusions: The protection provided by the Hp 1-1 genotype against the development of diabetic vascular disease may be mediated by its antioxidant capacity and its ability to induce the anti-oxidant anti-inflammatory cytokine IL-10. Funding: D Cure Diabetes Care in Israel and the Russell Berrie Foundation.
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LESION PROGRESSION CHANGES LYSOPHOSPHATIDIC ACID H O M E O S T A S I S T O FAVOR ITS A C C U M U L A T I O N
M. Bot, I. Bot, T.J.C. Van Berkel, E.A.L. Biessen. Division of Biopharntaceutics, LACDR, Leiden Universi~, Leiden, The Netherlands Objective: Lysophosphatidic acid (LPA) accumulates in the lipid core of human atherosclerotic plaques and is the primary platelet-activating lipid. Plaque rupture will expose entrapped LPA, which may trigger thrombus formation. Here, we aimed to temporally profile the metabolic regulation of LPA homeostasis and LPA accumulation during lesion progression. M e t h o d s : Atherosclerosis was induced in LDLr-/- mice by placing carotid artery collars. At 0, 2, 4, 6 and 8 weeks after surgery, RNA and lipids were extracted from the plaques. Gene expression of enzymes involved in LPA homeostasis was analyzed by RT-PCR, while lipid extracts were evaluated for LPA content. Results: Phospholipase D3, which generates phosphatidic acid (PA), showed a 4-fold increase in expression. Cytoplasmic phospholipase A2IVA, which converts PA to LPA, was 8-fold increased. Conversely, a 50% reduction was seen for LPA acyltransferase, which hydrolyzes LPA. LPA receptor 2 and 3 showed respectively a 12 and 5-fold increase. Plaque lipid analysis revealed a significant increase in LPA content of advanced lesions (0.9 to 37.6 pmol LPA/mg tissue, P<0.05) similar to human advanced plaques. Conclusions: Atherosclerosis progression results in a changed expression of proteins involved in LPA homeostasis reflecting a strongly induced intracellulax LPA production and an increased signal transduction. Lipid composition analysis confirmed accumulation of LPA in atherosderotic tissue. We propose that intervention in the LPA metabolism could be a promising therapeutic entry in reducing atherogenesis. Funding: Dutch Thrombosis Foundation grant 2004.01
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Ii T H E R E D U C T I O N O F S E R U M A T H E R O G E N I C I T Y BY NATURAL ANTI-INFLAMMATORY DRUG
T.V. Gorchakova I , V.A. M,/asoedova 1, I.A. Sobenin 1 , A.N. Orekhov 1'2 .
l b~stitute of General Pathology arm Pathophysiology, Moscow, Russia: 2bzstitute for Atherosclerosis Research, Moscow, Russia Objective: The aim of this investigation was to study antiatherosclerotic effects of natural anti-inflammatory drug inflaminat, based on black elder berries, calendula flowers and violet flowers, in vitro and ex vivo cell culture-based models.
XIV bztentational Symposium on Atherosclerosis, Rome, Italy, June 18-22, 2006