- Email: [email protected]
WITHOUT ENHANCING PPAR-GAMA EXPRESSION IN 3T3-L1 ADIPOCYTES
Poster Sessions PO4 Receptors and signaling pathways in lipid metabolism separated by SDS-PAGE (10%) and immunoblotted with a polyclonal antibody against pERK 1/2 (1:1000). Aldosterone was measured by RIA and cortisol release was determined by competitive luminometric assay. Results: Incubation of adrenocortical cells with 10 to 100 μg/mL native LDL (natLDL) or oxidized LDL (oxLDL) for 24 h stimulated aldosterone release dose-dependently up to 3-fold. Subsequent stimulation of NCI-H295R cells with angiotensin II induced an additional aldosterone secretion up to 2.9-fold in LDL-pretreated samples. Similar effects were observed for cortisol release. Compared to natLDL, oxLDL induced a smaller hormone release. The ability of oxLDL to induce hormone release decreased with increasing degree of LDL oxidation. NatLDL and oxLDL induced ERK phosphorylation with a maximum at 5 min poststimulation. NatLDL and oxLDL-induced aldosterone synthesis was markedly inhibited by MEK 1/2 inhibitor U0126, but not by p38 MAPK inhibitor SB 203580. Conclusion: Oxidative modification attenuated LDL-mediated steroidogenesis. The ERK signalling cascade is critically involved in LDL-induced adrenal aldosterone and cortisol release.
wild type mice but not in FXRKO mice. In dyslipidemic male LDLRKO mice consuming a Western diet, FXR-450 decreased serum triglyceride and VLDL, LDL and HDL cholesterol levels. The Western diet elevations of hepatic total cholesterol, free cholesterol, or triglyceride content along with the repressions of cholesterol-regulated genes (HMG-CoA synthase and PCSK9) were all attenuated by FXR-450. In dyslipidemic rats consuming a high fructose/cholesterol diet, FXR-450 decreased triglyceride and VLDL cholesterol levels but significantly increased HDL cholesterol levels, in contrast to mice. Although FXR has been suggested to modulate apoAI expression, FXR-450 did not repress apoA gene expression in LDLRKO mice, human apoAI transgenic mice, or rats. Gene expression profiling studies indicated that FXR-450 treatment induced endothelial lipase expression in mice but not in rats, suggesting a possible basis for the differing effects on HDL cholesterol in the two species. Together these results suggest that synthetic FXR agonists may have clinical utility in the treatment of dyslipidemia. PO4-98
ABSTRACT WITHDRAWN
PO4-96
UPREGULATION OF CD36 EXPRESSION MEDIATED BY OXLDL IN MURINE DIFFERENTIATING PREADIPOCYTES INVOLVES NUCLEAR FACTOR ERYTHROID 2 (NF-E2)-RELATED FACTOR 2 (NRF2)
M. D’Archivio, C. Santangelo, R. Vari, C. Filesi, B. Scazzocchio, R. Masella. National Centre for Food Quality and Risk Assessment Istituto Superiore Di Sanita, Roma, Italy The scavenger receptor CD36 has been implicated in many biological processes. It acts as receptor for oxidized LDL (oxLDL) and apoptotic cells on macrophages and as fatty acid translocase in adipocytes. Overexpression of CD36, recently demonstrated in adipocytes of patients with type 2 diabetes and hyperlipidemia, has been associated with insulin resistance. OxLDL, found in the plasma of obese patients, are able to modulate intracellular factors and cell activities. We evaluated oxLDL effects, and the related molecular mechanisms, on CD36 expression in differentiating preadipocytes. 3T3-L1 preadipocytes, induced to differentiate, were treated with oxLDL (50 mg protein/l). CD36 and PPARgamma gene expression was detected by quantitative RT-PCR. Protein expression of CD36, PPARgamma, Nrf2, PKC and phospho-PKC was evaluated by Western blotting. Our findings demonstrated that oxLDL induced CD36 upregulation. This increase seemed not to be mediated by PPARgamma, the key regulator of adipocyte differentiation, but rather by an increased Nrf2 nuclear translocation. The involvement of Nrf2 was confirmed by the rapid upregulation of phosphorylated-PKC protein, the major kinase involved in Nrf2 activation. Moreover, by incubating the cells with the PKC inhibitor staurosporine, the oxLDL-induced upregulation of Nrf2 and, worth of note, the subsequent CD36 increase were significantly counteracted. Our results pointed out Nrf2 activation as an alternative CD36-induction pathway. Since PKC is activated by lipid peroxidation products, we hypothesized that oxLDL activated PKC that, in turn, led to Nrf2 nuclear translocation and CD36 overexpression. These findings suggest that oxLDL could contribute to induce insulin resistance by modulating adipose tissue functions. PO4-97
ACTIVATION OF FARNESOID X RECEPTOR (FXR) PROTECTS AGAINST DIET-INDUCED DYSLIPIDEMIA
M.J. Evans 1 , P.E. Mahaney 2 , H.B. Hartman 1 , L. Borges-Marcucci 1 , C. Huard 3 , K. Lai 1 , R. Martinez 3 , S. Wang 1 , D.C. Harnish 1 . 1 Cardiovascular & Metabolic Diseases Research, Wyeth, Collegeville, PA, USA; 2 Chemical & Screening Sciences, Wyeth, Collegeville, PA, USA; 3 Biological Technologies, Wyeth, Cambridge, MA, USA The nuclear hormone receptor farnesoid X Receptor (FXR) plays a critical role in the regulation of bile acid synthesis and cholesterol homeostasis. Here we demonstrate the synthetic ligand FXR-450 is a potent FXR agonist using both in vitro and cell based coactivator recruitment assays. FXR-450 activated known FXR target genes following treatment of human HepG2 or mouse AML12 hepatocyte cell lines, and induced expression of FXR target genes in both liver (SHP, BSEP, apoC2) and ileum (SHP, FGF15) in
PERFLUOROHEXANESULFONATE AND PERFLUOROOCTANESULFONATE DECREASE PLASMA CHOLESTEROL AND TRIGLYCERIDES IN APOE*3LEIDEN TRANSGENIC MICE. INDICATION FOR A PPARα AGONIST MECHANISM
E. Pieterman 1 , A.M. van den Hoek 1 , L.M. Havekes 1 , D.J. Ehresman 2 , S. Chang 2 , J.L. Butenhoff 2 , H.M. Princen 1 , L.H. Cohen 1 . 1 TNO BioSciences, Leiden, The Netherlands; 2 3M Company, Medical Department, St. Paul, MN, USA Objective: Perfluorinated alkyl sulphonates are fully fluorinated amphiphilic organic molecules with strong surface-tension reducing properties. They are stable to environmental and metabolic degradation. Perfluorooctanesulfonate (PFOS) is widely dispersed in humans, fish-eating wildlife, and surface waters. Toxicological studies in rats and monkeys have shown a reduction in serum cholesterol after treatment with PFOS; however, such reductions have not been observed among exposed workers. In the present study we investigated the mechanistical background of the effect of perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS) and PFOS on cholesterol and triglyceride metabolism in APOE*3Leiden transgenic mice (a mouse model with a human-like lipoprotein profile). Methods and Results: Whereas PFBS treatment (30 mg/kg/day) had no effect, both PFHS (6 mg/kg/day) and PFOS (3 mg/kg/day) reduced plasma cholesterol (-36% and -31%) and triglycerides (-50% and -22%) in APOE*3Leiden mice, by a decrease in VLDL/IDL, and caused a concomitant shift of HDL towards larger particles. Mice treated for 10 weeks with PFHS and PFOS had increased ALAT levels (2-5 fold), enlarged livers (2-3 fold) and reduced cholesterol 7-α-hydroxylase activities (-60% and -77%) with a decreased fecal excretion of bile acids (-25% and -48%). Furthermore, PFHS and PFOS showed elevated plasma ketone bodies (2-3 fold), decreased respiratory exchange ratios, increased amounts of liver mitochondria and loss of perigonadal fat, all indicative of an increased fatty acid oxidation. Conclusions: Treatment of APOE*3Leiden mice with PFHS and PFOS results in reduced plasma cholesterol and triglycerides, increased liver size, decreased cholesterol 7-α-hydroxylase and increased fatty acid oxidation, suggesting a PPARα agonist activity. PO4-99
HDL AND ATORVASTATIN COUNTERBALANCE PROINFLAMMATORY EFFECT OF OXIDIZED LDL BY INHIBITING NF-KAPPAB ACTIVATION WITH/WITHOUT ENHANCING PPAR-GAMA EXPRESSION IN 3T3-L1 ADIPOCYTES
X. XZ, Z. SP, Y. BL. Department of Cardiology,The Second Xiangya Hospital of Central South University, Changsha, China Background and Aims: Adipose tissue is an important source of endogenous tumor necrosis factor α (TNFα) production, which is an inflammatory cytokine involved in atherogensis. This work aimed to investigate how high-density lipoprotein (HDL) and atorvastatin may prevent TNFα expression in oxidized low-density lipoprotein (oxLDL)-stimulated 3T3-L1 adipocytes. Methods: Fully differentiated 3T3-L1 adipocytes were incubated in the medium containing various concentrations of HDL (10 - 100 μg/ml) or atorvastatin (0.1 -10 μM) with oxLDL (50 μg/ml) stimulated, with/without protein kinase A (PKA) inhibitor H-89 (10 μM) preincubated. TNFα and
76th Congress of the European Atherosclerosis Society, June 10–13, 2007, Helsinki, Finland
POSTER SESSIONS
PO4-95
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Poster Sessions PO4 Receptors and signaling pathways in lipid metabolism
peroxisome proliferator- activated receptor γ (PPARγ) mRNA expression, nuclear factor-κB (NF-κB) activity and IκB protein level were evaluated. Results: OxLDL stimulation induced a significant increase of TNFα expression and NF-κB activity. Both atorvastatin and HDL showed antiinflammatory effects in a dose-dependent manner. Compared with that of oxLDL stimulation, treatment with atorvastatin at 10 μM significantly suppressed TNFα expression by 56.5%, inhibited NF-κB activation by 41.2%, and increased PPARγ expression to 2.83-fold; and treatment with HDL at 100 μg/ml suppressed TNFα expression by 64.5%, inhibited NF-κB activation by 49%, and stabilized IκB significantly, but had no effect on PPARγ expression. The anti-inflammatory effect of HDL could be abolished by H-89 partially. Conclusions: HDL could suppress TNFα expression in oxLDLstimulated 3T3-L1 adipocytes by PKA- IκBα- NF-κB signaling pathway, without any contact between HDL and oxLDL. Atorvastatin inhibited TNFα expression in oxLDL-stimulated 3T3-L1 adipocytes by NF-κB and PPARγ pathways. The anti-inflammatory intensity of HDL was similar with or even more powerful than that of atorvastatin. PO4-100
CONJUGATED LINOLEIC ACID ALTERS THE PRO-ATHEROSCLEROTIC PHENOTYPE OF THE MONOCYTE/MACROPHAGE – EVIDENCE FOR THE INVOLVEMENT OF A NOVEL SIGNALLING PATHWAY
S.E. McClelland, R. O’Connor, P.E. Buckmaster, D.J. Fitzgerald, O.A. Belton. Department of Medicine and Medical Therapeutics, Conway Institute, University College Dublin, Dublin, Ireland Conjugated linoleic acid (CLA), a peroxisome-proliferator-activator receptor (PPAR) agonist, denotes a group of naturally occurring isomers of linoleic acid. We have shown that CLA induces regression of pre-established atherosclerosis, however the mechanism by which CLA mediates this effect is unknown. Monocyte-derived macrophages, critical in atherosclerosis development, are believed to be an important target. Here we describe a novel pathway, induced by CLA, which is associated with an altered macrophage phenotype. CLA treatment of THP-1 macrophages induced expression of PPARtarget genes, as expected. Interestingly, CLA also induced expression of Retinoic-acid-related-Orphan Receptor-α (RORα), a nuclear transcription factor (endogenous ligand cholesterol), involved in inflammation and lipid metabolism. Importantly, RORα-signalling has not previously been associated with CLA. In addition, CLA induced expression of the RORα-target gene, PPARγ-coactivator-1(PGC-1), providing an important link between RORα and PPAR-signalling pathways. Many PPAR target genes are also targets for RORα. CLA induced expression of several PPAR/RORα targets such as CD-36 (the oxLDL scavenger receptor) and ABCA-1 (a cholesterol-efflux mediator) demonstrating a potential vascular-protective effect by coupling LDL-uptake with its efflux to HDL. Monocyte transendothelial migration, an atherosclerosis-initiating event, is preceded by platelet accumulation. We showed that activated-plateletreleasate induces THP-1 migration, linking platelet accumulation with monocyte-migration. CLA inhibited both platelet-releasate and monocyte chemoattractant protein-1(MCP-1)-induced migration. Furthermore, CLA decreased cyclooxygenase-2 expression, prostaglandinE2 and MMP-9 production, and altered PPARγ-dependent monocyte/macrophage differentiation; demonstrating that CLA is associated with a “less-atherogenic” macrophage phenotype. CLA induces RORα and PGC-1 expression and alters PPAR/RORα target genes that regulate cholesterol, fatty acid metabolism, inflammation and monocyte-migration. PO4-101
EFFECT OF ENDOTHELIN RECEPTOR ANTAGONISTS ON SYMPATHETIC INNERVATION IN HYPERLIPIDEMIC RABBITS
N.C. Chang 1 , M.S. Lin 2 , T.M. Lee 3 . 1 Cardiology Section, Department of Internal Medicine, Taipei Medical University and Hospital, Taipei, Taiwan; 2 Department of Pharmacy, National Taiwan University Hospital, Taipei, Taiwan; 3 Cardiology Section, Department of Internal Medicine, Taipei Medical University and Chi-Mei Medical Center, Tainan, Taiwan Background: Activation of the endothelin (ET) receptor exerts an arrhythmogenic effect of ET-1; however, the mechanisms remain unclear. We investigated whether selective ETA or nonselective ETA/ETB recep-
tor blockade exerts antiarrhythmic effects through attenuated sympathetic reinnervation in hyperlipidemic rabbits. Methods: We evaluated the hemodynamic, biochemical, morphological, and electrophysiological responses to endothelin receptor blockers in cholesterol-fed (1%) rabbits. We randomized male New Zealand White rabbits to either vehicle, hydralazine, ABT-627 (selective ETA receptor antagonist), or bosentan (nonselective ETA/ETB receptor antagonist) for 8 weeks. Results: Myocardial ET-1 levels revealed a significant elevation after cholesterol-feeding in comparison to that in normocholesterolemic rabbits, consistent with increased activities of ET-1 after hyperlipidemia. Sympathetic reinnervation and nerve function were parallel to ET-1 levels. Sympathetic hyperinnervation was blunted after administering either ETA or ETA/ETB blockade to a similar extent, assessed by immunohistochemical analysis of tyrosine hydroxylase, growth associated protein 43 and neurofilament, and Western blot and real-time quantitative RT-PCR of nerve growth factor. Dissociation between the effects of blood pressure and sympathetic innervation was noted, because ET receptor antagonists and hydralazine reduced arterial pressure similarly. Arrhythmic scores during programmed stimulation in ET receptor antagonists-treated rats were significantly lower than those treated with vehicle. Conclusions: Our data indicate that the ET system, especially via ETA receptors, plays an important role in the sympathetic innervation in hyperlipidemic rabbits. Independent of their hemodynamic effects, chronic use of either ETA or ETA/ETB antagonists may modify the arrhythmogenic response to programmed electrical stimulation (corresponding author: Tsung-Ming Lee). PO4-102
ISOFORM-SPECIFIC INTERFERENCE OF APOE WITH THE REELIN SIGNALING PATHWAY
N. Andrade 1 , M. Butterweck 1 , J. Owen 2 , W.J. Schneider 1 , J. Nimpf 1 . of Medical Biochemistry, Max F. Perutz Laboratories, Medical University of Vienna, Vienna, Austria; 2 Department of Medicine, University College London, London, UK 1 Department
Background and Aims: The Reelin signaling pathway plays a key role in neuronal migration and proper layer formation during embryonic brain development. ApoER2 and VLDL receptor are integral parts of this pathway, inducing phosphorylation of Dab1 upon reelin-induced receptor clustering. Subsequently a complex downstream signaling cascade which involves activation of PI3-kinase and Akt is triggered. Since ApoER2 and VLDL receptor bind apoE we investigated whether apoE containing lipoproteins interfere with the Reelin signaling pathway. Results: ApoE2-, apoE3-, and apoE4-containing lipoprotein particles were prepared using stable transfected CHO cells. Stimulation of primary rat neurons with the apoE-containing particles induced an isoform-specific activation of the Reelin-signaling pathway with E4 having the strongest effect. The signaling effect of apoE was confirmed using subventricular zone explants cultivated in Matrigel. These explants develop chains of migrating neurons which disintegrate upon Reelin stimulation. Addition of apoE4 dissociated the chains equally well as Reelin. The effect of apoE3 was significantly weaker and that of apoE2 was only slightly above background. Conclusion: These results demonstrated that there is an isoform-specific interference of apoE with the Reelin signaling pathway which might have an implication for the development of Alzheimer’s disease which is prevalent in carriers of the ε4 allele. PO4-103
INHIBITION OF CHOLESTEROL BIOSYNTHESIS AFFECTS LIPID RAFT/CAVEOLAE AND SIGNAL TRANSDUCTION IN ADIPOGENESIS
R. Busto 1 , J. Sanchez-Wandelmer 1 , A. Davalos 1 , O. Pastor 1 , M.A. Lasuncion 1,2 . 1 Servicio Bioquimica-Investigacion, Hospital Ramon y Cajal, Madrid, Spain; 2 Departamento de Bioquimica y Biologia Molecular, Universidad de Alcala, Madrid, Spain Background and Aims: Acute cell cholesterol extraction by cyclodextrins results in the reversible disruption and flattering of lipid raft domains, which subsequently alters the interaction between lipid-raft associated proteins, the mediated signalling and cellular function. Adipogenesis has been shown to be inhibited by lipid raft disruption. Adipocyte differentiation is regulated by several signalling pathways such as AKT and mitogen-activated protein kinase (MAPK). The aim of the study was to elucidate whether cholesterol biosynthesis inhibitors disrupt lipid raft domains and signal transduction in 3T3-L1 cells.
76th Congress of the European Atherosclerosis Society, June 10–13, 2007, Helsinki, Finland
wild type mice but not in FXRKO mice. In dyslipidemic male LDLRKO mice consuming a Western diet, FXR-450 decreased serum triglyceride and VLDL, LDL and HDL cholesterol levels. The Western diet elevations of hepatic total cholesterol, free cholesterol, or triglyceride content along with the repressions of cholesterol-regulated genes (HMG-CoA synthase and PCSK9) were all attenuated by FXR-450. In dyslipidemic rats consuming a high fructose/cholesterol diet, FXR-450 decreased triglyceride and VLDL cholesterol levels but significantly increased HDL cholesterol levels, in contrast to mice. Although FXR has been suggested to modulate apoAI expression, FXR-450 did not repress apoA gene expression in LDLRKO mice, human apoAI transgenic mice, or rats. Gene expression profiling studies indicated that FXR-450 treatment induced endothelial lipase expression in mice but not in rats, suggesting a possible basis for the differing effects on HDL cholesterol in the two species. Together these results suggest that synthetic FXR agonists may have clinical utility in the treatment of dyslipidemia. PO4-98
ABSTRACT WITHDRAWN
PO4-96
UPREGULATION OF CD36 EXPRESSION MEDIATED BY OXLDL IN MURINE DIFFERENTIATING PREADIPOCYTES INVOLVES NUCLEAR FACTOR ERYTHROID 2 (NF-E2)-RELATED FACTOR 2 (NRF2)
M. D’Archivio, C. Santangelo, R. Vari, C. Filesi, B. Scazzocchio, R. Masella. National Centre for Food Quality and Risk Assessment Istituto Superiore Di Sanita, Roma, Italy The scavenger receptor CD36 has been implicated in many biological processes. It acts as receptor for oxidized LDL (oxLDL) and apoptotic cells on macrophages and as fatty acid translocase in adipocytes. Overexpression of CD36, recently demonstrated in adipocytes of patients with type 2 diabetes and hyperlipidemia, has been associated with insulin resistance. OxLDL, found in the plasma of obese patients, are able to modulate intracellular factors and cell activities. We evaluated oxLDL effects, and the related molecular mechanisms, on CD36 expression in differentiating preadipocytes. 3T3-L1 preadipocytes, induced to differentiate, were treated with oxLDL (50 mg protein/l). CD36 and PPARgamma gene expression was detected by quantitative RT-PCR. Protein expression of CD36, PPARgamma, Nrf2, PKC and phospho-PKC was evaluated by Western blotting. Our findings demonstrated that oxLDL induced CD36 upregulation. This increase seemed not to be mediated by PPARgamma, the key regulator of adipocyte differentiation, but rather by an increased Nrf2 nuclear translocation. The involvement of Nrf2 was confirmed by the rapid upregulation of phosphorylated-PKC protein, the major kinase involved in Nrf2 activation. Moreover, by incubating the cells with the PKC inhibitor staurosporine, the oxLDL-induced upregulation of Nrf2 and, worth of note, the subsequent CD36 increase were significantly counteracted. Our results pointed out Nrf2 activation as an alternative CD36-induction pathway. Since PKC is activated by lipid peroxidation products, we hypothesized that oxLDL activated PKC that, in turn, led to Nrf2 nuclear translocation and CD36 overexpression. These findings suggest that oxLDL could contribute to induce insulin resistance by modulating adipose tissue functions. PO4-97
ACTIVATION OF FARNESOID X RECEPTOR (FXR) PROTECTS AGAINST DIET-INDUCED DYSLIPIDEMIA
M.J. Evans 1 , P.E. Mahaney 2 , H.B. Hartman 1 , L. Borges-Marcucci 1 , C. Huard 3 , K. Lai 1 , R. Martinez 3 , S. Wang 1 , D.C. Harnish 1 . 1 Cardiovascular & Metabolic Diseases Research, Wyeth, Collegeville, PA, USA; 2 Chemical & Screening Sciences, Wyeth, Collegeville, PA, USA; 3 Biological Technologies, Wyeth, Cambridge, MA, USA The nuclear hormone receptor farnesoid X Receptor (FXR) plays a critical role in the regulation of bile acid synthesis and cholesterol homeostasis. Here we demonstrate the synthetic ligand FXR-450 is a potent FXR agonist using both in vitro and cell based coactivator recruitment assays. FXR-450 activated known FXR target genes following treatment of human HepG2 or mouse AML12 hepatocyte cell lines, and induced expression of FXR target genes in both liver (SHP, BSEP, apoC2) and ileum (SHP, FGF15) in
PERFLUOROHEXANESULFONATE AND PERFLUOROOCTANESULFONATE DECREASE PLASMA CHOLESTEROL AND TRIGLYCERIDES IN APOE*3LEIDEN TRANSGENIC MICE. INDICATION FOR A PPARα AGONIST MECHANISM
E. Pieterman 1 , A.M. van den Hoek 1 , L.M. Havekes 1 , D.J. Ehresman 2 , S. Chang 2 , J.L. Butenhoff 2 , H.M. Princen 1 , L.H. Cohen 1 . 1 TNO BioSciences, Leiden, The Netherlands; 2 3M Company, Medical Department, St. Paul, MN, USA Objective: Perfluorinated alkyl sulphonates are fully fluorinated amphiphilic organic molecules with strong surface-tension reducing properties. They are stable to environmental and metabolic degradation. Perfluorooctanesulfonate (PFOS) is widely dispersed in humans, fish-eating wildlife, and surface waters. Toxicological studies in rats and monkeys have shown a reduction in serum cholesterol after treatment with PFOS; however, such reductions have not been observed among exposed workers. In the present study we investigated the mechanistical background of the effect of perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS) and PFOS on cholesterol and triglyceride metabolism in APOE*3Leiden transgenic mice (a mouse model with a human-like lipoprotein profile). Methods and Results: Whereas PFBS treatment (30 mg/kg/day) had no effect, both PFHS (6 mg/kg/day) and PFOS (3 mg/kg/day) reduced plasma cholesterol (-36% and -31%) and triglycerides (-50% and -22%) in APOE*3Leiden mice, by a decrease in VLDL/IDL, and caused a concomitant shift of HDL towards larger particles. Mice treated for 10 weeks with PFHS and PFOS had increased ALAT levels (2-5 fold), enlarged livers (2-3 fold) and reduced cholesterol 7-α-hydroxylase activities (-60% and -77%) with a decreased fecal excretion of bile acids (-25% and -48%). Furthermore, PFHS and PFOS showed elevated plasma ketone bodies (2-3 fold), decreased respiratory exchange ratios, increased amounts of liver mitochondria and loss of perigonadal fat, all indicative of an increased fatty acid oxidation. Conclusions: Treatment of APOE*3Leiden mice with PFHS and PFOS results in reduced plasma cholesterol and triglycerides, increased liver size, decreased cholesterol 7-α-hydroxylase and increased fatty acid oxidation, suggesting a PPARα agonist activity. PO4-99
HDL AND ATORVASTATIN COUNTERBALANCE PROINFLAMMATORY EFFECT OF OXIDIZED LDL BY INHIBITING NF-KAPPAB ACTIVATION WITH/WITHOUT ENHANCING PPAR-GAMA EXPRESSION IN 3T3-L1 ADIPOCYTES
X. XZ, Z. SP, Y. BL. Department of Cardiology,The Second Xiangya Hospital of Central South University, Changsha, China Background and Aims: Adipose tissue is an important source of endogenous tumor necrosis factor α (TNFα) production, which is an inflammatory cytokine involved in atherogensis. This work aimed to investigate how high-density lipoprotein (HDL) and atorvastatin may prevent TNFα expression in oxidized low-density lipoprotein (oxLDL)-stimulated 3T3-L1 adipocytes. Methods: Fully differentiated 3T3-L1 adipocytes were incubated in the medium containing various concentrations of HDL (10 - 100 μg/ml) or atorvastatin (0.1 -10 μM) with oxLDL (50 μg/ml) stimulated, with/without protein kinase A (PKA) inhibitor H-89 (10 μM) preincubated. TNFα and
76th Congress of the European Atherosclerosis Society, June 10–13, 2007, Helsinki, Finland
POSTER SESSIONS
PO4-95
41
42
Poster Sessions PO4 Receptors and signaling pathways in lipid metabolism
peroxisome proliferator- activated receptor γ (PPARγ) mRNA expression, nuclear factor-κB (NF-κB) activity and IκB protein level were evaluated. Results: OxLDL stimulation induced a significant increase of TNFα expression and NF-κB activity. Both atorvastatin and HDL showed antiinflammatory effects in a dose-dependent manner. Compared with that of oxLDL stimulation, treatment with atorvastatin at 10 μM significantly suppressed TNFα expression by 56.5%, inhibited NF-κB activation by 41.2%, and increased PPARγ expression to 2.83-fold; and treatment with HDL at 100 μg/ml suppressed TNFα expression by 64.5%, inhibited NF-κB activation by 49%, and stabilized IκB significantly, but had no effect on PPARγ expression. The anti-inflammatory effect of HDL could be abolished by H-89 partially. Conclusions: HDL could suppress TNFα expression in oxLDLstimulated 3T3-L1 adipocytes by PKA- IκBα- NF-κB signaling pathway, without any contact between HDL and oxLDL. Atorvastatin inhibited TNFα expression in oxLDL-stimulated 3T3-L1 adipocytes by NF-κB and PPARγ pathways. The anti-inflammatory intensity of HDL was similar with or even more powerful than that of atorvastatin. PO4-100
CONJUGATED LINOLEIC ACID ALTERS THE PRO-ATHEROSCLEROTIC PHENOTYPE OF THE MONOCYTE/MACROPHAGE – EVIDENCE FOR THE INVOLVEMENT OF A NOVEL SIGNALLING PATHWAY
S.E. McClelland, R. O’Connor, P.E. Buckmaster, D.J. Fitzgerald, O.A. Belton. Department of Medicine and Medical Therapeutics, Conway Institute, University College Dublin, Dublin, Ireland Conjugated linoleic acid (CLA), a peroxisome-proliferator-activator receptor (PPAR) agonist, denotes a group of naturally occurring isomers of linoleic acid. We have shown that CLA induces regression of pre-established atherosclerosis, however the mechanism by which CLA mediates this effect is unknown. Monocyte-derived macrophages, critical in atherosclerosis development, are believed to be an important target. Here we describe a novel pathway, induced by CLA, which is associated with an altered macrophage phenotype. CLA treatment of THP-1 macrophages induced expression of PPARtarget genes, as expected. Interestingly, CLA also induced expression of Retinoic-acid-related-Orphan Receptor-α (RORα), a nuclear transcription factor (endogenous ligand cholesterol), involved in inflammation and lipid metabolism. Importantly, RORα-signalling has not previously been associated with CLA. In addition, CLA induced expression of the RORα-target gene, PPARγ-coactivator-1(PGC-1), providing an important link between RORα and PPAR-signalling pathways. Many PPAR target genes are also targets for RORα. CLA induced expression of several PPAR/RORα targets such as CD-36 (the oxLDL scavenger receptor) and ABCA-1 (a cholesterol-efflux mediator) demonstrating a potential vascular-protective effect by coupling LDL-uptake with its efflux to HDL. Monocyte transendothelial migration, an atherosclerosis-initiating event, is preceded by platelet accumulation. We showed that activated-plateletreleasate induces THP-1 migration, linking platelet accumulation with monocyte-migration. CLA inhibited both platelet-releasate and monocyte chemoattractant protein-1(MCP-1)-induced migration. Furthermore, CLA decreased cyclooxygenase-2 expression, prostaglandinE2 and MMP-9 production, and altered PPARγ-dependent monocyte/macrophage differentiation; demonstrating that CLA is associated with a “less-atherogenic” macrophage phenotype. CLA induces RORα and PGC-1 expression and alters PPAR/RORα target genes that regulate cholesterol, fatty acid metabolism, inflammation and monocyte-migration. PO4-101
EFFECT OF ENDOTHELIN RECEPTOR ANTAGONISTS ON SYMPATHETIC INNERVATION IN HYPERLIPIDEMIC RABBITS
N.C. Chang 1 , M.S. Lin 2 , T.M. Lee 3 . 1 Cardiology Section, Department of Internal Medicine, Taipei Medical University and Hospital, Taipei, Taiwan; 2 Department of Pharmacy, National Taiwan University Hospital, Taipei, Taiwan; 3 Cardiology Section, Department of Internal Medicine, Taipei Medical University and Chi-Mei Medical Center, Tainan, Taiwan Background: Activation of the endothelin (ET) receptor exerts an arrhythmogenic effect of ET-1; however, the mechanisms remain unclear. We investigated whether selective ETA or nonselective ETA/ETB recep-
tor blockade exerts antiarrhythmic effects through attenuated sympathetic reinnervation in hyperlipidemic rabbits. Methods: We evaluated the hemodynamic, biochemical, morphological, and electrophysiological responses to endothelin receptor blockers in cholesterol-fed (1%) rabbits. We randomized male New Zealand White rabbits to either vehicle, hydralazine, ABT-627 (selective ETA receptor antagonist), or bosentan (nonselective ETA/ETB receptor antagonist) for 8 weeks. Results: Myocardial ET-1 levels revealed a significant elevation after cholesterol-feeding in comparison to that in normocholesterolemic rabbits, consistent with increased activities of ET-1 after hyperlipidemia. Sympathetic reinnervation and nerve function were parallel to ET-1 levels. Sympathetic hyperinnervation was blunted after administering either ETA or ETA/ETB blockade to a similar extent, assessed by immunohistochemical analysis of tyrosine hydroxylase, growth associated protein 43 and neurofilament, and Western blot and real-time quantitative RT-PCR of nerve growth factor. Dissociation between the effects of blood pressure and sympathetic innervation was noted, because ET receptor antagonists and hydralazine reduced arterial pressure similarly. Arrhythmic scores during programmed stimulation in ET receptor antagonists-treated rats were significantly lower than those treated with vehicle. Conclusions: Our data indicate that the ET system, especially via ETA receptors, plays an important role in the sympathetic innervation in hyperlipidemic rabbits. Independent of their hemodynamic effects, chronic use of either ETA or ETA/ETB antagonists may modify the arrhythmogenic response to programmed electrical stimulation (corresponding author: Tsung-Ming Lee). PO4-102
ISOFORM-SPECIFIC INTERFERENCE OF APOE WITH THE REELIN SIGNALING PATHWAY
N. Andrade 1 , M. Butterweck 1 , J. Owen 2 , W.J. Schneider 1 , J. Nimpf 1 . of Medical Biochemistry, Max F. Perutz Laboratories, Medical University of Vienna, Vienna, Austria; 2 Department of Medicine, University College London, London, UK 1 Department
Background and Aims: The Reelin signaling pathway plays a key role in neuronal migration and proper layer formation during embryonic brain development. ApoER2 and VLDL receptor are integral parts of this pathway, inducing phosphorylation of Dab1 upon reelin-induced receptor clustering. Subsequently a complex downstream signaling cascade which involves activation of PI3-kinase and Akt is triggered. Since ApoER2 and VLDL receptor bind apoE we investigated whether apoE containing lipoproteins interfere with the Reelin signaling pathway. Results: ApoE2-, apoE3-, and apoE4-containing lipoprotein particles were prepared using stable transfected CHO cells. Stimulation of primary rat neurons with the apoE-containing particles induced an isoform-specific activation of the Reelin-signaling pathway with E4 having the strongest effect. The signaling effect of apoE was confirmed using subventricular zone explants cultivated in Matrigel. These explants develop chains of migrating neurons which disintegrate upon Reelin stimulation. Addition of apoE4 dissociated the chains equally well as Reelin. The effect of apoE3 was significantly weaker and that of apoE2 was only slightly above background. Conclusion: These results demonstrated that there is an isoform-specific interference of apoE with the Reelin signaling pathway which might have an implication for the development of Alzheimer’s disease which is prevalent in carriers of the ε4 allele. PO4-103
INHIBITION OF CHOLESTEROL BIOSYNTHESIS AFFECTS LIPID RAFT/CAVEOLAE AND SIGNAL TRANSDUCTION IN ADIPOGENESIS
R. Busto 1 , J. Sanchez-Wandelmer 1 , A. Davalos 1 , O. Pastor 1 , M.A. Lasuncion 1,2 . 1 Servicio Bioquimica-Investigacion, Hospital Ramon y Cajal, Madrid, Spain; 2 Departamento de Bioquimica y Biologia Molecular, Universidad de Alcala, Madrid, Spain Background and Aims: Acute cell cholesterol extraction by cyclodextrins results in the reversible disruption and flattering of lipid raft domains, which subsequently alters the interaction between lipid-raft associated proteins, the mediated signalling and cellular function. Adipogenesis has been shown to be inhibited by lipid raft disruption. Adipocyte differentiation is regulated by several signalling pathways such as AKT and mitogen-activated protein kinase (MAPK). The aim of the study was to elucidate whether cholesterol biosynthesis inhibitors disrupt lipid raft domains and signal transduction in 3T3-L1 cells.
76th Congress of the European Atherosclerosis Society, June 10–13, 2007, Helsinki, Finland