- Email: [email protected]
W13.328 Aggregated LDL uptake induces membrane tissue factor procoagulant activity and microparticle release in human vascular smooth muscle cells
76
Llorente-Cort s
W13
Workshops Cellular lipid and lipoprotein transport
is mainly expressed in liver where it is highly regulated, indicating that mtGPAT may have a unique role in hepatic fatty acid metabolism. Since both mtGPAT and carnitine palmitoyl transferase-1 (CPT-1) are located on the outer mitochondrial membrane, we hypothesized that mtGPAT dh'ects fatty acyl-CoA away fl'om ~-oxidation and towards glycerolipid synthesis. Adenovh'al-mediated overexpression of murine mtGPAT in primary cultures of rat hepatocytes increased mtGPAT activity 2.7-fold with no compensatory effect on microsomal GPAT activity. MtGPAT overexpression resulted in a dramatic 80% reduction in fatty acid oxidation and a significant increase in hepatic diacylglycerol and phospholipid biosynthesis. Following lipid loading of the cells, intracellular triacylglycerol biosynthesis was also induced by mtGPAT overexpression. Changing an invariant aspartic acid residue to a glycine [D235G] in mtGPAT resulted in an inactive enzyme which helps define the active site requh'ed for mammalian mtGPAT function. To determine if obesity increases hepatic mtGPAT activity, two models of rodent obesity were examined and shown to have over 2-fold increased enzyme activity. Overall, these results support the concept that increased hepatic mtGPAT activity associated with obesity positively contributes to lipid disorders by reducing oxidative processes and promoting de novo glycerolipid synthesis.
dothelial cells was observed by confocal microscopy. Both cell lines showed an LPL mediated augmentation of lipoprotein uptake. Quantification of cellular uptake with 125I tagged chylomicron remnants showed a 2.5 fold LPL mediated increase in both wild type and VLDLR-KO cells. To proof a dfl'ectional transport of lipoproteins through endothelial cells, a transcytosis assay was performed. Transport of chylomicron remnants was 2-3 fold increased by LPL in a time dependent manner without differences between wild type and VLDLR-KO cells. These data dh'ectly proof an LPL mediated transendothelial transport of lipoproteins. Ftu'thelxnore it is suggested, that the VLDL receptor does not play an important role in this metabolic pathway.
•
Background: ATP-binding cassette U'ansporter A1 (ABCA1), the defective molecule in Tangier disease, has been recently shown to stimulate cholesterol effiux to apolipoprotein A-I (apoA-I) fl'om cells. However, little is known how ABCA1 mediates the cholesterol effiux fl'om late endosomes/lysosomes as a source of cholesterol. In this study, we examined whether vesicle acidification is requfl'ed for ABCAl-mediated phospholipid and cholesterol effiux. Methods and Results: Fibroblasts were cultured for 48 hours with [3H] labeled-cholesterol and [14C] labeled-choline, cultured for 24 hours without radiolabeled reagents to equilibrate inta'acellulal" phospholipid and cholesterol and then apo A-I mediated phopsholipid and cholesterol effiux fl'om loaded fibroblasts was measured with various neuta'alizing agents. Apo A-I mediated phospholipid and cholesterol effiux was inhibited by the co-incubation with ammonium chloride and chloroquine, which could neuta'alize the pH of acidic compartments of cells, inclulding late endosomes/lysosomes. This inhibition of phospholipid and cholesterol effiux was also confirmed by more specific neuta'alizing agent, bafilomycin A1 which inhibits vacuolar type H+-ATPase in a dose dependent manner. This suppression of apo A-I mediated phospholipid and cholesterol effiux was observed also in ABCAl-ta'ansfected COS-7 cells, suggesting that overexpression of ABCA1 could not recover the effects of neu~alizing agents on phospholipid and cholesterol effiux. Interestingly, this effect was specific for apo A-I mediated cholesterol effiux and these agents did not suppress the HDL-mediated cholesterol effiux. Conclusion: We conclude that ABCAl-mediated phospholipid and cholesterol effiux needs acidification of vesicles, endosomes/lysosomes.
AGGREGATED LDL UPTAKE INDUCES MEMBRANE TISSUE FACTOR PROCOAGULANT ACTIVITY AND MICROPARTICLE RELEASE IN HUMAN VASCULAR SMOOTH MUSCLE CELLS
V. Llorente-Cort s, M. Otero-Vi as, S. Camino-L pez, O. Llampayas, L. Badimon. Cardiovascular Research Center, CSIC-ICCC, Hospital Sta.
Creu i S. Pau, Barcelona, Spain Background-Thrombosis on atherosclerotic lesions triggers clinical events. The lipid-rich microenvfl'onment seems to influence the prothrombotic transformation of vascular lesions. Tissue factor (TF) is the main initiator of the blood system and aggregated LDL (agLDL) are found in the arterial intima. Our hypothesis is that agLDL internalization by vascular smooth muscle cells (VSMC) may trigger TF-procoagulant activity (PCA). Methods-Cultured human VSMC were obtained fi'om human coronary arteries of explanted hearts at transplant operations. VSMC were incubated with native LDL (nLDL) o1" agLDL. TF mRNA was analyzed by real time PCR, cellular and released TF protein antigen by western blot. TF microparticle (TF-MP) content was analyzed by flow cytometry and TF procoagulant activity (PCA) by a factor Xa generation test. Results-Both nLDL and agLDL strongly and equally increased TF mRNA and cell membrane protein expression by approximately 5 and 9-fold respectively. A sustained TF-PCA was induced by agLDL but not by nLDL (agLDL: 2.46-4-0.22 mU/mg protein vs nLDL: 0.72-4-0.12 mU/mg protein at 12 hours). AgLDL increased TF antigen release (agLDL: 5.64-4-0.4 AU vs nLDL: 3.28-4-0.22 AU) and TF-MP release (agLDL: 89.85-4-8.51 vs nLDL: 19.69-4-4.59 TF-MP/103 cells). Blockade of Low density lipoprotein receptol:related protein (LRP), receptor for agLDL, prevented agLDLinduced release of TF protein and TF-MP. Conclusions- VSMC-TF expression is upregulated by both nLDL and agLDL. However, only agLDL engagement to its receptor LRP induced cellular TF PCA and TF release by human VSMC.
~
TRANSENDOTHELIAL LIPOPROTEIN LIPASE MEDIATED LIPOPROTEIN TRANSFER IS NOT VLDL RECEPTOR DEPENDENT IN PRIMARY ENDOTHELIAL CELLS
B. Loeffler, J. Heeren, H. Greten, M. Merkel. Department of Internal Medicine I, Department of Molecular Cell Biology, Hamburg, Germany Lipoprotein lipase (LPL) is a key enzyme in lipoprotein metabolism. Synthesized primarily in myocytes and adipocytes, it hydrolyses chylomicrons and VLDL particles while bound to proteoglycans on the luminal surface of vascular endothelial cells. In vita'o experiments revealed dfl'ect interactions between LPL and lipoprotein receptors, especially LDL receptol~related protein and the VLDL receptor. However, LPL mediated uptake of lipoproteins into pfimm'y endothelial cells and transendothelial transport was not dh'ectly shown. To investigate the role of endothelial receptors during LPL mediated lipoprotein transport, primm'y endothelial cells fi'om wild type and VLDL receptor knock out (VLDLR-KO) mice were isolated by antibody coupled magnetic beads. Endothelial cells were identified by immune fluorescence and FACS analysis. The uptake of chylomicron remnants into primary ell-
I
I w l 3.330 iI VESICULAR ACIDIFICATION IS REQUIRED FOR ATP-BINDING CASSETTE TRANSPORTER A1-MEDIATED PHOSPHOLIPID AND CHOLESTEROL EFFLUX A. Matsuyama, N. Sakai, M. Koseki, T. Ohama, K. Hfl'ano, H. Hfl'aoka, S. Yamashita. Department of Internal Medicine and Molecular Science,
Osaka University Graduate School of Medicine, Suita. Japan
I
w13.3311
DIFFERENT TYPES OF ENZYMATIC MODIFICATION CAUSE AGGREGATION OF LOW DENSITY LIPOPROTEIN AND INTRACELLULAR CHOLESTEROL ACCUMULATION
A. Mel'nichenko, D. Aksenov, I. Sobenin, O. Panasenko, A. Orekhov.
Research Institute of Physico-Chemical Medicine, Institute of Experimental Cardiology, Institute of General Pathology and Pathophysiology, Moscow, Russia We have early found a subfi'action of low density lipoprotein (LDL) with low level of sialic acid. Desialylated LDL particles aggregated spontaneously and was capable to accumulate cholesteryl esters in cultured human aortic intimal smooth muscle cells and macrophages. It has been suggested that enzymatic desialylation of LDL and treatment with other enzymes could lead to enhanced LDL-induced intracellular cholesterol accumulation mediated through increased aggregation of lipoprotein particles. In the present study we have demonstrated that after sialic acid removal by neuraminidase (2,3sialidase and 2,6-sialidase) LDL increases the intracellular cholesteryl ester content. In vitro tmatrnent of LDL by proteases (trypsin and chymotrypsin) and lipases (phospholipase AII and phospholipase C) also resulted in intracellular cholesterol accumulation. In all cases, modification of LDL with any enzyme was accompanied with increased spontaneous aggregation of lipoprotein pm'ticles. There was a strong positive COla'elation between the degree of LDL aggregation and its ability to accumulate intracellulm" cholesterol. Using anti-apoB monoclonal antibodies we have also found that any kind of enzymatic modification of LDL involving either protein,
74th EAS Congress, 17-20 April 2004, Seville, Spain
Llorente-Cort s
W13
Workshops Cellular lipid and lipoprotein transport
is mainly expressed in liver where it is highly regulated, indicating that mtGPAT may have a unique role in hepatic fatty acid metabolism. Since both mtGPAT and carnitine palmitoyl transferase-1 (CPT-1) are located on the outer mitochondrial membrane, we hypothesized that mtGPAT dh'ects fatty acyl-CoA away fl'om ~-oxidation and towards glycerolipid synthesis. Adenovh'al-mediated overexpression of murine mtGPAT in primary cultures of rat hepatocytes increased mtGPAT activity 2.7-fold with no compensatory effect on microsomal GPAT activity. MtGPAT overexpression resulted in a dramatic 80% reduction in fatty acid oxidation and a significant increase in hepatic diacylglycerol and phospholipid biosynthesis. Following lipid loading of the cells, intracellular triacylglycerol biosynthesis was also induced by mtGPAT overexpression. Changing an invariant aspartic acid residue to a glycine [D235G] in mtGPAT resulted in an inactive enzyme which helps define the active site requh'ed for mammalian mtGPAT function. To determine if obesity increases hepatic mtGPAT activity, two models of rodent obesity were examined and shown to have over 2-fold increased enzyme activity. Overall, these results support the concept that increased hepatic mtGPAT activity associated with obesity positively contributes to lipid disorders by reducing oxidative processes and promoting de novo glycerolipid synthesis.
dothelial cells was observed by confocal microscopy. Both cell lines showed an LPL mediated augmentation of lipoprotein uptake. Quantification of cellular uptake with 125I tagged chylomicron remnants showed a 2.5 fold LPL mediated increase in both wild type and VLDLR-KO cells. To proof a dfl'ectional transport of lipoproteins through endothelial cells, a transcytosis assay was performed. Transport of chylomicron remnants was 2-3 fold increased by LPL in a time dependent manner without differences between wild type and VLDLR-KO cells. These data dh'ectly proof an LPL mediated transendothelial transport of lipoproteins. Ftu'thelxnore it is suggested, that the VLDL receptor does not play an important role in this metabolic pathway.
•
Background: ATP-binding cassette U'ansporter A1 (ABCA1), the defective molecule in Tangier disease, has been recently shown to stimulate cholesterol effiux to apolipoprotein A-I (apoA-I) fl'om cells. However, little is known how ABCA1 mediates the cholesterol effiux fl'om late endosomes/lysosomes as a source of cholesterol. In this study, we examined whether vesicle acidification is requfl'ed for ABCAl-mediated phospholipid and cholesterol effiux. Methods and Results: Fibroblasts were cultured for 48 hours with [3H] labeled-cholesterol and [14C] labeled-choline, cultured for 24 hours without radiolabeled reagents to equilibrate inta'acellulal" phospholipid and cholesterol and then apo A-I mediated phopsholipid and cholesterol effiux fl'om loaded fibroblasts was measured with various neuta'alizing agents. Apo A-I mediated phospholipid and cholesterol effiux was inhibited by the co-incubation with ammonium chloride and chloroquine, which could neuta'alize the pH of acidic compartments of cells, inclulding late endosomes/lysosomes. This inhibition of phospholipid and cholesterol effiux was also confirmed by more specific neuta'alizing agent, bafilomycin A1 which inhibits vacuolar type H+-ATPase in a dose dependent manner. This suppression of apo A-I mediated phospholipid and cholesterol effiux was observed also in ABCAl-ta'ansfected COS-7 cells, suggesting that overexpression of ABCA1 could not recover the effects of neu~alizing agents on phospholipid and cholesterol effiux. Interestingly, this effect was specific for apo A-I mediated cholesterol effiux and these agents did not suppress the HDL-mediated cholesterol effiux. Conclusion: We conclude that ABCAl-mediated phospholipid and cholesterol effiux needs acidification of vesicles, endosomes/lysosomes.
AGGREGATED LDL UPTAKE INDUCES MEMBRANE TISSUE FACTOR PROCOAGULANT ACTIVITY AND MICROPARTICLE RELEASE IN HUMAN VASCULAR SMOOTH MUSCLE CELLS
V. Llorente-Cort s, M. Otero-Vi as, S. Camino-L pez, O. Llampayas, L. Badimon. Cardiovascular Research Center, CSIC-ICCC, Hospital Sta.
Creu i S. Pau, Barcelona, Spain Background-Thrombosis on atherosclerotic lesions triggers clinical events. The lipid-rich microenvfl'onment seems to influence the prothrombotic transformation of vascular lesions. Tissue factor (TF) is the main initiator of the blood system and aggregated LDL (agLDL) are found in the arterial intima. Our hypothesis is that agLDL internalization by vascular smooth muscle cells (VSMC) may trigger TF-procoagulant activity (PCA). Methods-Cultured human VSMC were obtained fi'om human coronary arteries of explanted hearts at transplant operations. VSMC were incubated with native LDL (nLDL) o1" agLDL. TF mRNA was analyzed by real time PCR, cellular and released TF protein antigen by western blot. TF microparticle (TF-MP) content was analyzed by flow cytometry and TF procoagulant activity (PCA) by a factor Xa generation test. Results-Both nLDL and agLDL strongly and equally increased TF mRNA and cell membrane protein expression by approximately 5 and 9-fold respectively. A sustained TF-PCA was induced by agLDL but not by nLDL (agLDL: 2.46-4-0.22 mU/mg protein vs nLDL: 0.72-4-0.12 mU/mg protein at 12 hours). AgLDL increased TF antigen release (agLDL: 5.64-4-0.4 AU vs nLDL: 3.28-4-0.22 AU) and TF-MP release (agLDL: 89.85-4-8.51 vs nLDL: 19.69-4-4.59 TF-MP/103 cells). Blockade of Low density lipoprotein receptol:related protein (LRP), receptor for agLDL, prevented agLDLinduced release of TF protein and TF-MP. Conclusions- VSMC-TF expression is upregulated by both nLDL and agLDL. However, only agLDL engagement to its receptor LRP induced cellular TF PCA and TF release by human VSMC.
~
TRANSENDOTHELIAL LIPOPROTEIN LIPASE MEDIATED LIPOPROTEIN TRANSFER IS NOT VLDL RECEPTOR DEPENDENT IN PRIMARY ENDOTHELIAL CELLS
B. Loeffler, J. Heeren, H. Greten, M. Merkel. Department of Internal Medicine I, Department of Molecular Cell Biology, Hamburg, Germany Lipoprotein lipase (LPL) is a key enzyme in lipoprotein metabolism. Synthesized primarily in myocytes and adipocytes, it hydrolyses chylomicrons and VLDL particles while bound to proteoglycans on the luminal surface of vascular endothelial cells. In vita'o experiments revealed dfl'ect interactions between LPL and lipoprotein receptors, especially LDL receptol~related protein and the VLDL receptor. However, LPL mediated uptake of lipoproteins into pfimm'y endothelial cells and transendothelial transport was not dh'ectly shown. To investigate the role of endothelial receptors during LPL mediated lipoprotein transport, primm'y endothelial cells fi'om wild type and VLDL receptor knock out (VLDLR-KO) mice were isolated by antibody coupled magnetic beads. Endothelial cells were identified by immune fluorescence and FACS analysis. The uptake of chylomicron remnants into primary ell-
I
I w l 3.330 iI VESICULAR ACIDIFICATION IS REQUIRED FOR ATP-BINDING CASSETTE TRANSPORTER A1-MEDIATED PHOSPHOLIPID AND CHOLESTEROL EFFLUX A. Matsuyama, N. Sakai, M. Koseki, T. Ohama, K. Hfl'ano, H. Hfl'aoka, S. Yamashita. Department of Internal Medicine and Molecular Science,
Osaka University Graduate School of Medicine, Suita. Japan
I
w13.3311
DIFFERENT TYPES OF ENZYMATIC MODIFICATION CAUSE AGGREGATION OF LOW DENSITY LIPOPROTEIN AND INTRACELLULAR CHOLESTEROL ACCUMULATION
A. Mel'nichenko, D. Aksenov, I. Sobenin, O. Panasenko, A. Orekhov.
Research Institute of Physico-Chemical Medicine, Institute of Experimental Cardiology, Institute of General Pathology and Pathophysiology, Moscow, Russia We have early found a subfi'action of low density lipoprotein (LDL) with low level of sialic acid. Desialylated LDL particles aggregated spontaneously and was capable to accumulate cholesteryl esters in cultured human aortic intimal smooth muscle cells and macrophages. It has been suggested that enzymatic desialylation of LDL and treatment with other enzymes could lead to enhanced LDL-induced intracellular cholesterol accumulation mediated through increased aggregation of lipoprotein particles. In the present study we have demonstrated that after sialic acid removal by neuraminidase (2,3sialidase and 2,6-sialidase) LDL increases the intracellular cholesteryl ester content. In vitro tmatrnent of LDL by proteases (trypsin and chymotrypsin) and lipases (phospholipase AII and phospholipase C) also resulted in intracellular cholesterol accumulation. In all cases, modification of LDL with any enzyme was accompanied with increased spontaneous aggregation of lipoprotein pm'ticles. There was a strong positive COla'elation between the degree of LDL aggregation and its ability to accumulate intracellulm" cholesterol. Using anti-apoB monoclonal antibodies we have also found that any kind of enzymatic modification of LDL involving either protein,
74th EAS Congress, 17-20 April 2004, Seville, Spain