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170 MITOCHONDRIAL INHIBITORY FACTOR 1 (IF1) IS PRESENT IN HUMAN SERUM AND ITS LEVEL IS POSITIVELY ASSOCIATED TO HDL-C
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Atherosclerosis Supplements 12, no. 1 (2011) 13–184
168 ENHANCED BIOSYNTHESIS OF GANGLIOSIDE GM3 IN ATHEROSCLEROSIS N.N. Samovilova1 , E.V. Gracheva1 , N.K. Golovanova1 , O. Kaliya2 , N.V. Prokazova1 . 1 Research Institute of Experimental Cardiology, Russian Cardiology Research Centre, 2 Organic Intermediates and Dyes Institute, Moscow, Russia Our previous immunohistochemical studies in human atherosclerotic aorta revealed high expression levels of ganglioside GM3 and GM3 synthase in cells of monocyte/macrophage origin abundantly infiltrating aortic intima in atherosclerosis. The aim of our study was to assess ganglioside GM3 biosynthesis in monocytes of atherosclerotic patients and differentiated monocyte-derived macrophages. We found that GM3 levels and GM3 synthase activity in human monocytederived macrophages were 5- and 10-fold higher, respectively, compared to freshly isolated human monocytes. GM3 synthase mRNA levels were also significantly higher in differentiated monocyte-derived macrophages compared to monocytes and in atherosclerotic aorta compared to normal aorta. GM3 synthase activity in monocytes from atherosclerotic patients was about 3.5fold higher compared to monocytes from healthy subjects. Monocytes treated with GM3 (40 uM) showed an increase in cellular GM3 levels from 1.5 to 5.2 mg per cell protein and a 1.1−4.4-fold enhancement of their adhesion to HUVEC depending on the subject. Our data suggest a role for biosynthesis of ganglioside GM3 in monocyte activation and monocyte/macrophage differentiation in atherosclerosis. The authors would like to acknowledge financial support from Russian Foundation for Basic Research (grant No. 10−04–00888a). 169 POSSIBLE ROLE OF SPHINGOLIPIDS IN BETA-CAROTENEINDUCED ENDOTHELIA PROANGIOGENIC ACTIVITY A. Polus1 , B. Kiec-Wilk1 , M. Mikołajczyk1 , J. Hartwich1 , R. Goralczyk2 , A. Dembinska-Kiec1 . 1 Clinical Biochemistry, Jagiellonian University, Medical College, Krakow, Poland, 2 DSM Nutritional Products Ltd., R&D, Human Nutrition and Health, Basel, Switzerland Different type of carotenoids function as: free radical scavengers, immunomodulators, regulators of gap junction communication and apoptosis, are claimed in the cardiovascular diseases and cancer prevention. Beta-carotene is the most efficient source of retinol. It has been demonstrated that the activity of fenretinide a synthetic retinoid could be mediated by sphingolipids: ceramide and its derivatives. HUVEC cells were incubated with BC (3mM) for 24 hrs. The effect on cell proliferation was measured by BrdU incorporation. BC uptake was measured by HPLC. The effect on cell proliferation was measured by the BrdU incorporation. Chemotaxis was performed in the Boyden’s chamber. Changes of gene expression were analyzed using microarray hybridization method and confirmed using the real-time PCR method. To measure the activity of following kinases: AKT, p38 Fast Activated Cell-based ELISA Kits were used. BC activated chemotaxis of HUVECs. The microarray data analysis revealed that the genes, involved in proliferation, cell/cell; cell/matrix adhesion; matrix reorganization, and activation of chemotaxis were the most group of affected by BC genes in HUVEC. This effect was also confirmed by the in vitro and in vivo angiogenesis models. Inhibition of AKT kinase and activation of p38 kinase phosphorylation was observed. BC in the physiological concentration range stimulates early steps of angiogeniesis. Observed effects of BC influence could confirmed participation of ceramide or its derivatives. Partially supported by FP7 EU 202272 LIPIDOMICNETproject 170 MITOCHONDRIAL INHIBITORY FACTOR 1 (IF1) IS PRESENT IN HUMAN SERUM AND ITS LEVEL IS POSITIVELY ASSOCIATED TO HDL-C A. Genoux1 , V. Pons1 , M. Laffargue1 , G. Combes1 , J.-B. Ruidavets2 , 2 , B. Perret1 , L.O. Martinez1 . 1 Institute of Metabolic and J. Ferrieres ` Cardiovascular Diseases, INSERM U1048, University of Toulouse III, 2 Epidemiology and Analysis in Public Health, INSERM U1027, University of Toulouse III, Toulouse, France Specific object of study: Mitochondrial ATP synthase is expressed as a plasma membrane receptor for apolipoprotein A-I (apoA-I), the major protein component in high-density lipoproteins (HDL). On hepatocytes, apoA-I binding to cell surface ATP synthase (namely ecto-F1 -ATPase) stimulates the ATP hydrolytic activity of this ecto-enzyme and this production of extracellular ADP subsequently activates a P2Y13 -mediated HDL endocytosis pathway. Conversely, exogenous IF1 (Inhibitory Factor 1), classically known as a natural mitochondrial specific inhibitor of F1 -ATPase activity, inhibits ecto-F1 -ATPase activity and decreases HDL uptake by human hepatocytes and perfused rat liver. Since recent reports also described the presence of IF1 at the plasma membrane of different cell types, we investigated whether IF1 was present in the systemic circulation in human.
Poster presentations
Methodology and Results: We first unambiguously detected IF1 in human serum by mass spectrometry and then set up a competitive Elisa assay in order to quantify its level in human serum. Analyse of IF1 levels in 50 normolipemic male subjects evidenced a normal distribution, with a median value at 0.49 mg/mL and a 95% confidence interval, 0.44–0.54 mg/L. Correlations between IF1 levels and plasma lipid level parameters were investigated and we observed that plasma IF1 levels were positively correlated with HDL-cholesterol and negatively with triglycerides. Conclusions: Altogether, these data support the view that, in human, circulating IF1 might impact HDL levels by inhibiting hepatic HDL uptake through its inhibitory activity on ecto-F1 -ATPase. 171 HDL FUNCTION IS IMPAIRED IN PATIENTS WITH ACUTE MYOCARDIAL INFARCTION INDEPENDENT OF PLASMA HDL CHOLESTEROL LEVELS W. Annema1 , H.M. Willemsen2 , R.A. Tio2 , U.J.F. Tietge1 . 1 Department of Pediatrics, 2 Department of Cardiology, University Medical Center Groningen, Groningen, The Netherlands Objective: To determine the impact of acute myocardial infarction on three key anti-atherogenic functions of HDL. Patients and Methods: Patients with either acute non-ST segment elevation myocardial infarction (non-STEMI, n = 41) or STEMI (n = 37) were compared with controls experiencing non-cardiac acute chest pain (n = 33). Groups were matched for age, sex, CRP, multiple cardiovascular risk factors, and importantly plasma HDL cholesterol. The in vitro potential of HDL to (i) mediate cholesterol efflux, (ii) prevent LDL oxidation, and (iii) inhibit cytokine-induced adhesion molecule expression in endothelial cells was measured. Results: HDL from STEMI patients displayed reduced cholesterol efflux versus controls (P < 0.001) and non-STEMI patients (P < 0.001). Anti-oxidative properties of HDL to inhibit LDL oxidation did not differ among groups. Relative to controls, adhesion molecule expression remained 1.5-fold higher with HDL from non-STEMI and 3.2-fold higher with HDL from STEMI patients (P < 0.001), indicating impaired anti-inflammatory HDL functionality in the STEMI group. Cholesterol efflux correlated with anti-inflammatory activity in MI patients (P < 0.001) but not in controls. CRP levels, a systemic inflammation marker, were not linked to HDL function. Nevertheless, higher plasma myeloperoxidase levels were associated with impaired HDL function in MI patients (efflux: P < 0.05; anti-inflammation: P < 0.05) but not in controls. Conclusions: Independent of plasma HDL cholesterol, two out of three anti-atherogenic HDL functions tested were significantly impaired in STEMI patients, namely cholesterol efflux and anti-inflammatory properties. Specifically myeloperoxidase might contribute causally to decreased HDL functionality. Assessment of HDL function might reveal important clinical information over mere HDL cholesterol mass measurements. 172 NIACIN, ATORVASTATIN AND FENOFIBRATE DECREASE PLASMA CETP BY REDUCTION OF THE HEPATIC MACROPHAGE CONTENT IN APOE*3-LEIDEN.CETP MICE Y. Wang1 , Z. Li2 , M. Hoekstra2 , V. Bieghs3 , R. Shiri-Sverdlov3 , T.J.C. Van Berkel2 , J.W.A. Smit1 , H.M.G. Princen4 , J.A. Romijn1 , L.M. Havekes1,4 , P.C.N. Rensen1 . 1 Leiden University Medical Center, 2 Leiden Amsterdam Center for Drug Research, Leiden, 3 Maastricht University, Maastricht, 4 Netherlands Organization for Applied Scientific Research, Leiden, The Netherlands Introduction: Hypolipidemic drugs decrease (V)LDL-C and increase HDL-C. We previously demonstrated that niacin, atorvastatin and fenofibrate increase HDL-C by reducing hepatic CETP expression and consequently plasma CETP levels. Since hepatic CETP is expressed by both hepatocytes and liver macrophages, the aim of the present study was to evaluate the contribution of hepatic macrophages to the CETP-lowering effect of these hypolipidemic drugs by using APOE*3-Leiden (E3L).CETP mice, a unique mouse model for human-like lipoprotein metabolism. Methods and Results: Female mice (n = 8 per group) were fed a Western-type diet containing 0.15% cholesterol without or with niacin (1% w/w), atorvastatin (0.01% w/w) or fenofibrate (0.04% w/w) for 3 weeks. As compared to vehicle, niacin, atorvastatin and fenofibrate reduced plasma (V)LDL-C (−72%, −58% and −80%; all P < 0.01) and elevated HDL-C (+56%, +48% and +131%) as related to reduced plasma CETP mass (−51%, −45% and −49%; all P < 0.001). Concomitantly, they all reduced hepatic CD68 gene expression, and reduced the number of F4/80-positive macrophages (−28%, −29% and −54%; all P < 0.05) as well as Mac1-positive macrophages (−36%, P < 0.001; −7%, n.s; and −22%; P < 0.05). The hepatic mRNA expression of CETP strongly correlated with that of CD68 (R2 = 0.444; P < 0.001), which strongly suggests that niacin, atorvastatin and fenofibrate decrease hepatic CETP expression by reducing the hepatic macrophage content. Conclusion: These data suggest that atorvastatin and fenofibrate decrease the macrophage content of the liver, leading to a decreased hepatic CETP expression, thereby reducing plasma CETP and increasing HDL-C.
Atherosclerosis Supplements 12, no. 1 (2011) 13–184
168 ENHANCED BIOSYNTHESIS OF GANGLIOSIDE GM3 IN ATHEROSCLEROSIS N.N. Samovilova1 , E.V. Gracheva1 , N.K. Golovanova1 , O. Kaliya2 , N.V. Prokazova1 . 1 Research Institute of Experimental Cardiology, Russian Cardiology Research Centre, 2 Organic Intermediates and Dyes Institute, Moscow, Russia Our previous immunohistochemical studies in human atherosclerotic aorta revealed high expression levels of ganglioside GM3 and GM3 synthase in cells of monocyte/macrophage origin abundantly infiltrating aortic intima in atherosclerosis. The aim of our study was to assess ganglioside GM3 biosynthesis in monocytes of atherosclerotic patients and differentiated monocyte-derived macrophages. We found that GM3 levels and GM3 synthase activity in human monocytederived macrophages were 5- and 10-fold higher, respectively, compared to freshly isolated human monocytes. GM3 synthase mRNA levels were also significantly higher in differentiated monocyte-derived macrophages compared to monocytes and in atherosclerotic aorta compared to normal aorta. GM3 synthase activity in monocytes from atherosclerotic patients was about 3.5fold higher compared to monocytes from healthy subjects. Monocytes treated with GM3 (40 uM) showed an increase in cellular GM3 levels from 1.5 to 5.2 mg per cell protein and a 1.1−4.4-fold enhancement of their adhesion to HUVEC depending on the subject. Our data suggest a role for biosynthesis of ganglioside GM3 in monocyte activation and monocyte/macrophage differentiation in atherosclerosis. The authors would like to acknowledge financial support from Russian Foundation for Basic Research (grant No. 10−04–00888a). 169 POSSIBLE ROLE OF SPHINGOLIPIDS IN BETA-CAROTENEINDUCED ENDOTHELIA PROANGIOGENIC ACTIVITY A. Polus1 , B. Kiec-Wilk1 , M. Mikołajczyk1 , J. Hartwich1 , R. Goralczyk2 , A. Dembinska-Kiec1 . 1 Clinical Biochemistry, Jagiellonian University, Medical College, Krakow, Poland, 2 DSM Nutritional Products Ltd., R&D, Human Nutrition and Health, Basel, Switzerland Different type of carotenoids function as: free radical scavengers, immunomodulators, regulators of gap junction communication and apoptosis, are claimed in the cardiovascular diseases and cancer prevention. Beta-carotene is the most efficient source of retinol. It has been demonstrated that the activity of fenretinide a synthetic retinoid could be mediated by sphingolipids: ceramide and its derivatives. HUVEC cells were incubated with BC (3mM) for 24 hrs. The effect on cell proliferation was measured by BrdU incorporation. BC uptake was measured by HPLC. The effect on cell proliferation was measured by the BrdU incorporation. Chemotaxis was performed in the Boyden’s chamber. Changes of gene expression were analyzed using microarray hybridization method and confirmed using the real-time PCR method. To measure the activity of following kinases: AKT, p38 Fast Activated Cell-based ELISA Kits were used. BC activated chemotaxis of HUVECs. The microarray data analysis revealed that the genes, involved in proliferation, cell/cell; cell/matrix adhesion; matrix reorganization, and activation of chemotaxis were the most group of affected by BC genes in HUVEC. This effect was also confirmed by the in vitro and in vivo angiogenesis models. Inhibition of AKT kinase and activation of p38 kinase phosphorylation was observed. BC in the physiological concentration range stimulates early steps of angiogeniesis. Observed effects of BC influence could confirmed participation of ceramide or its derivatives. Partially supported by FP7 EU 202272 LIPIDOMICNETproject 170 MITOCHONDRIAL INHIBITORY FACTOR 1 (IF1) IS PRESENT IN HUMAN SERUM AND ITS LEVEL IS POSITIVELY ASSOCIATED TO HDL-C A. Genoux1 , V. Pons1 , M. Laffargue1 , G. Combes1 , J.-B. Ruidavets2 , 2 , B. Perret1 , L.O. Martinez1 . 1 Institute of Metabolic and J. Ferrieres ` Cardiovascular Diseases, INSERM U1048, University of Toulouse III, 2 Epidemiology and Analysis in Public Health, INSERM U1027, University of Toulouse III, Toulouse, France Specific object of study: Mitochondrial ATP synthase is expressed as a plasma membrane receptor for apolipoprotein A-I (apoA-I), the major protein component in high-density lipoproteins (HDL). On hepatocytes, apoA-I binding to cell surface ATP synthase (namely ecto-F1 -ATPase) stimulates the ATP hydrolytic activity of this ecto-enzyme and this production of extracellular ADP subsequently activates a P2Y13 -mediated HDL endocytosis pathway. Conversely, exogenous IF1 (Inhibitory Factor 1), classically known as a natural mitochondrial specific inhibitor of F1 -ATPase activity, inhibits ecto-F1 -ATPase activity and decreases HDL uptake by human hepatocytes and perfused rat liver. Since recent reports also described the presence of IF1 at the plasma membrane of different cell types, we investigated whether IF1 was present in the systemic circulation in human.
Poster presentations
Methodology and Results: We first unambiguously detected IF1 in human serum by mass spectrometry and then set up a competitive Elisa assay in order to quantify its level in human serum. Analyse of IF1 levels in 50 normolipemic male subjects evidenced a normal distribution, with a median value at 0.49 mg/mL and a 95% confidence interval, 0.44–0.54 mg/L. Correlations between IF1 levels and plasma lipid level parameters were investigated and we observed that plasma IF1 levels were positively correlated with HDL-cholesterol and negatively with triglycerides. Conclusions: Altogether, these data support the view that, in human, circulating IF1 might impact HDL levels by inhibiting hepatic HDL uptake through its inhibitory activity on ecto-F1 -ATPase. 171 HDL FUNCTION IS IMPAIRED IN PATIENTS WITH ACUTE MYOCARDIAL INFARCTION INDEPENDENT OF PLASMA HDL CHOLESTEROL LEVELS W. Annema1 , H.M. Willemsen2 , R.A. Tio2 , U.J.F. Tietge1 . 1 Department of Pediatrics, 2 Department of Cardiology, University Medical Center Groningen, Groningen, The Netherlands Objective: To determine the impact of acute myocardial infarction on three key anti-atherogenic functions of HDL. Patients and Methods: Patients with either acute non-ST segment elevation myocardial infarction (non-STEMI, n = 41) or STEMI (n = 37) were compared with controls experiencing non-cardiac acute chest pain (n = 33). Groups were matched for age, sex, CRP, multiple cardiovascular risk factors, and importantly plasma HDL cholesterol. The in vitro potential of HDL to (i) mediate cholesterol efflux, (ii) prevent LDL oxidation, and (iii) inhibit cytokine-induced adhesion molecule expression in endothelial cells was measured. Results: HDL from STEMI patients displayed reduced cholesterol efflux versus controls (P < 0.001) and non-STEMI patients (P < 0.001). Anti-oxidative properties of HDL to inhibit LDL oxidation did not differ among groups. Relative to controls, adhesion molecule expression remained 1.5-fold higher with HDL from non-STEMI and 3.2-fold higher with HDL from STEMI patients (P < 0.001), indicating impaired anti-inflammatory HDL functionality in the STEMI group. Cholesterol efflux correlated with anti-inflammatory activity in MI patients (P < 0.001) but not in controls. CRP levels, a systemic inflammation marker, were not linked to HDL function. Nevertheless, higher plasma myeloperoxidase levels were associated with impaired HDL function in MI patients (efflux: P < 0.05; anti-inflammation: P < 0.05) but not in controls. Conclusions: Independent of plasma HDL cholesterol, two out of three anti-atherogenic HDL functions tested were significantly impaired in STEMI patients, namely cholesterol efflux and anti-inflammatory properties. Specifically myeloperoxidase might contribute causally to decreased HDL functionality. Assessment of HDL function might reveal important clinical information over mere HDL cholesterol mass measurements. 172 NIACIN, ATORVASTATIN AND FENOFIBRATE DECREASE PLASMA CETP BY REDUCTION OF THE HEPATIC MACROPHAGE CONTENT IN APOE*3-LEIDEN.CETP MICE Y. Wang1 , Z. Li2 , M. Hoekstra2 , V. Bieghs3 , R. Shiri-Sverdlov3 , T.J.C. Van Berkel2 , J.W.A. Smit1 , H.M.G. Princen4 , J.A. Romijn1 , L.M. Havekes1,4 , P.C.N. Rensen1 . 1 Leiden University Medical Center, 2 Leiden Amsterdam Center for Drug Research, Leiden, 3 Maastricht University, Maastricht, 4 Netherlands Organization for Applied Scientific Research, Leiden, The Netherlands Introduction: Hypolipidemic drugs decrease (V)LDL-C and increase HDL-C. We previously demonstrated that niacin, atorvastatin and fenofibrate increase HDL-C by reducing hepatic CETP expression and consequently plasma CETP levels. Since hepatic CETP is expressed by both hepatocytes and liver macrophages, the aim of the present study was to evaluate the contribution of hepatic macrophages to the CETP-lowering effect of these hypolipidemic drugs by using APOE*3-Leiden (E3L).CETP mice, a unique mouse model for human-like lipoprotein metabolism. Methods and Results: Female mice (n = 8 per group) were fed a Western-type diet containing 0.15% cholesterol without or with niacin (1% w/w), atorvastatin (0.01% w/w) or fenofibrate (0.04% w/w) for 3 weeks. As compared to vehicle, niacin, atorvastatin and fenofibrate reduced plasma (V)LDL-C (−72%, −58% and −80%; all P < 0.01) and elevated HDL-C (+56%, +48% and +131%) as related to reduced plasma CETP mass (−51%, −45% and −49%; all P < 0.001). Concomitantly, they all reduced hepatic CD68 gene expression, and reduced the number of F4/80-positive macrophages (−28%, −29% and −54%; all P < 0.05) as well as Mac1-positive macrophages (−36%, P < 0.001; −7%, n.s; and −22%; P < 0.05). The hepatic mRNA expression of CETP strongly correlated with that of CD68 (R2 = 0.444; P < 0.001), which strongly suggests that niacin, atorvastatin and fenofibrate decrease hepatic CETP expression by reducing the hepatic macrophage content. Conclusion: These data suggest that atorvastatin and fenofibrate decrease the macrophage content of the liver, leading to a decreased hepatic CETP expression, thereby reducing plasma CETP and increasing HDL-C.