- Email: [email protected]
139 LOW DENSITY LIPOPROTEIN ENHANCES C-REACTIVE PROTEIN-MEDIATED FCγ RECEPTOR SIGNAL TRANSDUCTION IN MACROPHAGES BUT NOT IN MONOCYTES
32
Atherosclerosis Supplements 12, no. 1 (2011) 13–184
macrophages to promote mass efflux of cellular cholesterol to lipid-free apoA1 and HDL, respectively. Our data therefore indicate that dynasore represents a new interesting drug for studying cholesterol intracellular trafficking. 138 CHOLESTEROL LOADING OF HUMAN MACROPHAGES REDUCES GROWTH FACTOR-INDUCED HETEROGENEITY OF GENE EXPRESSION INVOLVED IN FOAM CELL FORMATION J. Lappalainen1 , N. Yeung1 , M. Jauhiainen2 , P.T. Kovanen1 , M. Lee-Rueckert1 . 1 Wihuri Research Institute, 2 National Institute of Health and Welfare, Helsinki, Finland Background: Distinct subpopulations of macrophages have been identified in the human arterial intima, with varying effects on the development of atherosclerosis. Granulocyte-macrophage colony-stimulating factor (GMCSF) and macrophage colony-stimulating factor (M-CSF) are hematopoietic growth factors that regulate the generation and function of monocyte-derived macrophages. Methods: A gene expression profile related to cholesterol uptake and efflux, and the cholesterol contents were analyzed in cultured human macrophages differentiated with either GM- or M-CSF, and after pre-exposure to acetylated LDL in the absence or presence of cholesterol acceptors. Results: Macrophages differentiated with M-CSF expressed higher mRNA levels of CD36 and SR-AI, the two scavenger receptors for modified LDL. This correlated with a greater ability of macrophages to accumulate cholesteryl esters in response to incubation with acetylated LDL. Macrophages differentiated with GM-CSF, again, showed higher expression of SR-BI and ABCG1 mRNA, two receptors capable of mediating cholesterol efflux. However, such divergent gene expression profile favoring a pro-atherogenic role for M-CSF, rather than for GM-CSF, was attenuated in the foam cell phenotype. Furthermore, HDL-2 and human blood plasma induced similar degrees of cholesterol efflux in both types of macrophages. Conclusions: A protective compensatory upregulation of some components of the cholesterol efflux machinery was observed in the M-CSF-subtype of macrophages upon foam cell formation. Thus, the gene profile of human macrophages is influenced, in addition to growth factors, also by the relative availability of modified LDL particles and of HDL particles. Ultimately, the composite effect of growth factors and lipoproteins determine cholesterol balance of the macrophages. 139 LOW DENSITY LIPOPROTEIN ENHANCES C-REACTIVE PROTEIN-MEDIATED FCg RECEPTOR SIGNAL TRANSDUCTION IN MACROPHAGES BUT NOT IN MONOCYTES 1 1 K. Li1,2 , M. Radermacher1 , M. Kachele ¨ , C. Rocker ¨ , A. Pott1 , D. Manolov1 , W. Ito3 , U. Nienhaus1,4 , J. Torzewski1,3 , O. Zimmermann1 , J. Wiehe1 , M. Bienek-Ziolkowski1 , H. Ruland1 , K. Vogt1 . 1 University of Ulm, Ulm, Germany, 2 Sino-German Heart Center, Shanghai, China, 3 Cardiovascular ¨ Immenstadt, 4 Institue of Applied Physics (KIT) Karlsruhe, Center Oberallgau, Karlsruhe, Germany Background and Aims: C-reactive protein (CRP) and low density lipoprotein (LDL) are risk markers for cardiovascular disease. Both of them deposit in the arterial wall during atherogenesis. CRP binds to LDL and has been implicated to play a pivotal role in cardiovascular disease. Previous reports proposed that FcgRI and FcgRIIa are the major receptors for CRP. Syk kinase is essential for FcgR signaling. Our objectives is to examine whether CRP can trigger Syk kinase phosphorylation in human monocytes and macrophages and whether this effect can be enhanced by the involvement of LDL. Methods: CRP-LDL complex formation was evaluated by fluorescence correlation spectroscopy. CRP interacts with LDL in solution with a equilibrium dissociation constant of 8.0 mM. Peripheral human blood monocytes (PBMC) and mature macrophages were exposed to CRP and LDL. Equal amounts of protein were assayed by immunoblotting in order to investigate Syk kinase phosphorylation. Results: Induction of Syk kinase phosphorylation was not observed in monocytes. In macrophages, however, Syk kinase phosphorylation in response to CRP (100 mg/mL) is equal to that induced by human IgG. Significant, and CRP dose-dependent, induction of the signal was observed in response to the CRP-LDL complex (50 mg/mL LDL). LDL by itself (50 mg/mL) only induced trivial Syk kinase signaling. Conclusions: Our experiments suggest that human CRP-mediated tyrosine phosphorylation of the immunoreceptor-tyrosine based activation motif (ITAM) of Syk kinase in human macrophages is enhanced by LDL. CRP may recognize human tissue-deposited LDL as a foreign antigen.
Poster presentations
140 IMPACT OF OMEGA 3 AND OMEGA 6 FATTY ACIDS MEMBRANE INCORPORATION ON CHOLESTEROL EFFLUX FROM CULTURED RAT CARDIOMYOCYTES N. Fournier1,2 , A. Reboulleau1 , V. Robert1 , B. Vedie2 , J.-L. Paul1,2 , A. Grynberg1 . 1 Laboratoire de Biochimie, UMR INRA 1154, Faculte des Sciences Pharmaceutiques, Universite Paris-Sud 11, Chatenay-Malabry, 2 Laboratoire de Biochimie, Hopital Europeen Georges Pompidou, Paris, France Although cholesterol-rich microdomains are highly involved in the functions of cardiomyocytes, the cholesterol (C) homeostasis is largely unknown in these cells. We previously reported that C efflux to apolipoprotein AI (apo AI) or to HDL is primarily mediated by ATP Binding Cassette ABCA1 and ABCG1 transporters, respectively, both being upregulated by LXR/RXR activation (unpublished data). We investigated the impact of omega 3 (docosahexaenoate) and omega 6 (arachidonate) polyunsaturated fatty acids (PUFAs) membrane enrichment (80 mM, 72 h) on isotopic C efflux from cultured rat cardiomyocytes. The C efflux was appreciated by measuring the cellular [3 H] free C release from primary cultures of cardiac myocytes obtained from 2−4 days-old Wistar rats to the extracellular acceptors after 4 h of incubation. Under basal conditions, docosahexaenoate membrane incorporation induced a significant increase of C efflux to apo AI (25%) whereas arachidonate induced a significant decrease of C efflux to both apo AI (25%) and HDL (14%). Under activated conditions, only the ABCA1 activity remained slightly although significantly increased by docosahexaenoate (8%) and decreased by arachidonate (10%). Moreover, the ABCA1 and ABCG1 mRNA expression was not affected by PUFA treatments. These results show that omega 3 supply to cardiomyocytes may be beneficial on C homeostasis by enhancing C efflux active processes whereas omega 6 supply may be deleterious, likely by modulating the chemo-physical properties of the cellular membrane. We are currently quantitating ABCA1 and ABCG1 protein levels as well as their cellular localization to investigate the potential mechanism(s) by which PUFA affect the ABC functions. 141 NEW LMF1 VARIANTS IDENTIFIED IN HYPERTRIGLYCERIDEMIC PATIENTS I. De Castro-Oros ´ 1,2 , E. Ros3 , F. Civeira2,4 , M. Pocov´ı1,5 . 1 Dpto. Bioqu´ımica, Biolog´ıa Molecular y Celular, University of Zaragoza, 2 RECAVA, Instituto ´ de Ciencias de la Salud, Zaragoza, 3 Unitat de Lipids, Servei Aragones ´ Institut d’Investigacions Biomediques ` d’Endocrinologia i Nutricio. August Pi Sunyer (IDIBAPS), Hospital Cl´ınic de Barcelona and Ciber Fisiopatolog´ıa de la 4 ´ (CIBERobn), Barcelona, Unidad de Lipidos. Laboratorio Obesidad y Nutricion ´ Molecular, Hospital Universitario Miguel Servet, 5 CIBERER, de Investigacion Zaragoza, Spain Lipase maturing factor 1 (LMF1) plays an essential role in the formation of catalytically active Lipoprotein Lipase (LpL). Mutations in LMF1 gene have been described as cause of combined deficiency lipase affecting the enzyme activity for both LpL and Hepatic Lipase and leading to severe hypertriglyceridemia (HTG). The aim of our study was to identify the contribution of LMF1 gene in severe HTG. For this purpose, we have selected 37 unrelated subjects with severe forms of HTG, previously analysed for LPL, APOA5, APOC2 and GPIHBP1 genes. The LMF1 11 exons and the boundaries exon-intron were sequenced. We have identified 9 heterozygote variants not previously described. The R450W (exon 9) variant was found in a 48 y.o. male with TG: 3,757 mg/dl affected of a pancreatitis episode. The L44L (exon 2) was found in a 31 y.o. male with TG: 3,584 mg/dl with pancreatitis and diabetes. The R350Q was found in a 7 y.o. girl with TG: 346 mg/dl. Two subjects with TG levels >3,000 mg/dl showed combined variants in heterozygosis: K520X and L552R; R100T and A286A, respectively. Other two variants, A251A (exon 6) and R353W (exon 7), were found in HTG subjects and also in normolipaemic individuals, indicating these variants such plausible polymorphisms. In conclusion, we have identified 9 new LMF1 variants in HTG patients which must be further studied in order to determine its contribution to severe HTG. 142 REGULATION OF LIVER AND ADIPOSE TISSUE LIPOGENESIS IN HUMAN OBESITY J. Heeren1 , M. Vossen1 , T. Scherer2 , L. Eissing1 , U. Knippschield3 , K. Toedter1 , C. Buettner2 , A. Wolf3 , L. Scheja1 . 1 IBMII: Molecular Cell Biology, Hamburg, Germany, 2 The Mount Sinai Medical Center, New York, NY, USA, 3 University Hospital of Ulm, Ulm, Germany Objective: De novo lipogenesis (DNL) and DNL-associated fatty acids derived from adipose tissue have been shown to improve systemic insulin sensitivity in rodents while increased hepatic DNL is generally associated with insulin resistant states. DNL is down-regulated in adipose tissue of obese rodents, thereby contributing to systemic insulin resistance. Here we investigated whether human obesity is associated with impaired DNL in adipose tissue and how adipose tissue and hepatic DNL correlates with liver steatosis, insulin resistance and inflammation. Methods: Liver and visceral adipose tissue (VAT) samples from more than 60 surgery patients, comprising lean, overweight and obese subjects, were
Atherosclerosis Supplements 12, no. 1 (2011) 13–184
macrophages to promote mass efflux of cellular cholesterol to lipid-free apoA1 and HDL, respectively. Our data therefore indicate that dynasore represents a new interesting drug for studying cholesterol intracellular trafficking. 138 CHOLESTEROL LOADING OF HUMAN MACROPHAGES REDUCES GROWTH FACTOR-INDUCED HETEROGENEITY OF GENE EXPRESSION INVOLVED IN FOAM CELL FORMATION J. Lappalainen1 , N. Yeung1 , M. Jauhiainen2 , P.T. Kovanen1 , M. Lee-Rueckert1 . 1 Wihuri Research Institute, 2 National Institute of Health and Welfare, Helsinki, Finland Background: Distinct subpopulations of macrophages have been identified in the human arterial intima, with varying effects on the development of atherosclerosis. Granulocyte-macrophage colony-stimulating factor (GMCSF) and macrophage colony-stimulating factor (M-CSF) are hematopoietic growth factors that regulate the generation and function of monocyte-derived macrophages. Methods: A gene expression profile related to cholesterol uptake and efflux, and the cholesterol contents were analyzed in cultured human macrophages differentiated with either GM- or M-CSF, and after pre-exposure to acetylated LDL in the absence or presence of cholesterol acceptors. Results: Macrophages differentiated with M-CSF expressed higher mRNA levels of CD36 and SR-AI, the two scavenger receptors for modified LDL. This correlated with a greater ability of macrophages to accumulate cholesteryl esters in response to incubation with acetylated LDL. Macrophages differentiated with GM-CSF, again, showed higher expression of SR-BI and ABCG1 mRNA, two receptors capable of mediating cholesterol efflux. However, such divergent gene expression profile favoring a pro-atherogenic role for M-CSF, rather than for GM-CSF, was attenuated in the foam cell phenotype. Furthermore, HDL-2 and human blood plasma induced similar degrees of cholesterol efflux in both types of macrophages. Conclusions: A protective compensatory upregulation of some components of the cholesterol efflux machinery was observed in the M-CSF-subtype of macrophages upon foam cell formation. Thus, the gene profile of human macrophages is influenced, in addition to growth factors, also by the relative availability of modified LDL particles and of HDL particles. Ultimately, the composite effect of growth factors and lipoproteins determine cholesterol balance of the macrophages. 139 LOW DENSITY LIPOPROTEIN ENHANCES C-REACTIVE PROTEIN-MEDIATED FCg RECEPTOR SIGNAL TRANSDUCTION IN MACROPHAGES BUT NOT IN MONOCYTES 1 1 K. Li1,2 , M. Radermacher1 , M. Kachele ¨ , C. Rocker ¨ , A. Pott1 , D. Manolov1 , W. Ito3 , U. Nienhaus1,4 , J. Torzewski1,3 , O. Zimmermann1 , J. Wiehe1 , M. Bienek-Ziolkowski1 , H. Ruland1 , K. Vogt1 . 1 University of Ulm, Ulm, Germany, 2 Sino-German Heart Center, Shanghai, China, 3 Cardiovascular ¨ Immenstadt, 4 Institue of Applied Physics (KIT) Karlsruhe, Center Oberallgau, Karlsruhe, Germany Background and Aims: C-reactive protein (CRP) and low density lipoprotein (LDL) are risk markers for cardiovascular disease. Both of them deposit in the arterial wall during atherogenesis. CRP binds to LDL and has been implicated to play a pivotal role in cardiovascular disease. Previous reports proposed that FcgRI and FcgRIIa are the major receptors for CRP. Syk kinase is essential for FcgR signaling. Our objectives is to examine whether CRP can trigger Syk kinase phosphorylation in human monocytes and macrophages and whether this effect can be enhanced by the involvement of LDL. Methods: CRP-LDL complex formation was evaluated by fluorescence correlation spectroscopy. CRP interacts with LDL in solution with a equilibrium dissociation constant of 8.0 mM. Peripheral human blood monocytes (PBMC) and mature macrophages were exposed to CRP and LDL. Equal amounts of protein were assayed by immunoblotting in order to investigate Syk kinase phosphorylation. Results: Induction of Syk kinase phosphorylation was not observed in monocytes. In macrophages, however, Syk kinase phosphorylation in response to CRP (100 mg/mL) is equal to that induced by human IgG. Significant, and CRP dose-dependent, induction of the signal was observed in response to the CRP-LDL complex (50 mg/mL LDL). LDL by itself (50 mg/mL) only induced trivial Syk kinase signaling. Conclusions: Our experiments suggest that human CRP-mediated tyrosine phosphorylation of the immunoreceptor-tyrosine based activation motif (ITAM) of Syk kinase in human macrophages is enhanced by LDL. CRP may recognize human tissue-deposited LDL as a foreign antigen.
Poster presentations
140 IMPACT OF OMEGA 3 AND OMEGA 6 FATTY ACIDS MEMBRANE INCORPORATION ON CHOLESTEROL EFFLUX FROM CULTURED RAT CARDIOMYOCYTES N. Fournier1,2 , A. Reboulleau1 , V. Robert1 , B. Vedie2 , J.-L. Paul1,2 , A. Grynberg1 . 1 Laboratoire de Biochimie, UMR INRA 1154, Faculte des Sciences Pharmaceutiques, Universite Paris-Sud 11, Chatenay-Malabry, 2 Laboratoire de Biochimie, Hopital Europeen Georges Pompidou, Paris, France Although cholesterol-rich microdomains are highly involved in the functions of cardiomyocytes, the cholesterol (C) homeostasis is largely unknown in these cells. We previously reported that C efflux to apolipoprotein AI (apo AI) or to HDL is primarily mediated by ATP Binding Cassette ABCA1 and ABCG1 transporters, respectively, both being upregulated by LXR/RXR activation (unpublished data). We investigated the impact of omega 3 (docosahexaenoate) and omega 6 (arachidonate) polyunsaturated fatty acids (PUFAs) membrane enrichment (80 mM, 72 h) on isotopic C efflux from cultured rat cardiomyocytes. The C efflux was appreciated by measuring the cellular [3 H] free C release from primary cultures of cardiac myocytes obtained from 2−4 days-old Wistar rats to the extracellular acceptors after 4 h of incubation. Under basal conditions, docosahexaenoate membrane incorporation induced a significant increase of C efflux to apo AI (25%) whereas arachidonate induced a significant decrease of C efflux to both apo AI (25%) and HDL (14%). Under activated conditions, only the ABCA1 activity remained slightly although significantly increased by docosahexaenoate (8%) and decreased by arachidonate (10%). Moreover, the ABCA1 and ABCG1 mRNA expression was not affected by PUFA treatments. These results show that omega 3 supply to cardiomyocytes may be beneficial on C homeostasis by enhancing C efflux active processes whereas omega 6 supply may be deleterious, likely by modulating the chemo-physical properties of the cellular membrane. We are currently quantitating ABCA1 and ABCG1 protein levels as well as their cellular localization to investigate the potential mechanism(s) by which PUFA affect the ABC functions. 141 NEW LMF1 VARIANTS IDENTIFIED IN HYPERTRIGLYCERIDEMIC PATIENTS I. De Castro-Oros ´ 1,2 , E. Ros3 , F. Civeira2,4 , M. Pocov´ı1,5 . 1 Dpto. Bioqu´ımica, Biolog´ıa Molecular y Celular, University of Zaragoza, 2 RECAVA, Instituto ´ de Ciencias de la Salud, Zaragoza, 3 Unitat de Lipids, Servei Aragones ´ Institut d’Investigacions Biomediques ` d’Endocrinologia i Nutricio. August Pi Sunyer (IDIBAPS), Hospital Cl´ınic de Barcelona and Ciber Fisiopatolog´ıa de la 4 ´ (CIBERobn), Barcelona, Unidad de Lipidos. Laboratorio Obesidad y Nutricion ´ Molecular, Hospital Universitario Miguel Servet, 5 CIBERER, de Investigacion Zaragoza, Spain Lipase maturing factor 1 (LMF1) plays an essential role in the formation of catalytically active Lipoprotein Lipase (LpL). Mutations in LMF1 gene have been described as cause of combined deficiency lipase affecting the enzyme activity for both LpL and Hepatic Lipase and leading to severe hypertriglyceridemia (HTG). The aim of our study was to identify the contribution of LMF1 gene in severe HTG. For this purpose, we have selected 37 unrelated subjects with severe forms of HTG, previously analysed for LPL, APOA5, APOC2 and GPIHBP1 genes. The LMF1 11 exons and the boundaries exon-intron were sequenced. We have identified 9 heterozygote variants not previously described. The R450W (exon 9) variant was found in a 48 y.o. male with TG: 3,757 mg/dl affected of a pancreatitis episode. The L44L (exon 2) was found in a 31 y.o. male with TG: 3,584 mg/dl with pancreatitis and diabetes. The R350Q was found in a 7 y.o. girl with TG: 346 mg/dl. Two subjects with TG levels >3,000 mg/dl showed combined variants in heterozygosis: K520X and L552R; R100T and A286A, respectively. Other two variants, A251A (exon 6) and R353W (exon 7), were found in HTG subjects and also in normolipaemic individuals, indicating these variants such plausible polymorphisms. In conclusion, we have identified 9 new LMF1 variants in HTG patients which must be further studied in order to determine its contribution to severe HTG. 142 REGULATION OF LIVER AND ADIPOSE TISSUE LIPOGENESIS IN HUMAN OBESITY J. Heeren1 , M. Vossen1 , T. Scherer2 , L. Eissing1 , U. Knippschield3 , K. Toedter1 , C. Buettner2 , A. Wolf3 , L. Scheja1 . 1 IBMII: Molecular Cell Biology, Hamburg, Germany, 2 The Mount Sinai Medical Center, New York, NY, USA, 3 University Hospital of Ulm, Ulm, Germany Objective: De novo lipogenesis (DNL) and DNL-associated fatty acids derived from adipose tissue have been shown to improve systemic insulin sensitivity in rodents while increased hepatic DNL is generally associated with insulin resistant states. DNL is down-regulated in adipose tissue of obese rodents, thereby contributing to systemic insulin resistance. Here we investigated whether human obesity is associated with impaired DNL in adipose tissue and how adipose tissue and hepatic DNL correlates with liver steatosis, insulin resistance and inflammation. Methods: Liver and visceral adipose tissue (VAT) samples from more than 60 surgery patients, comprising lean, overweight and obese subjects, were