- Email: [email protected]
Th-P17:436 Proteomic and metabolomic approaches to normal and diseased vessels: Role of oxidative stress
P17
Thurs&ty, June 22, 2006: Poster Session Novel technologies for risk determination
I
I Th-P 17:4331 A M E T A B O N O M I C A P P R O A C H D I S C R I M I N A T E S THE SEVERITY OF THE ATHEROGENIC OUTCOME AMONG HYPERLIPIDEMIC HAMSTERS I
G. Gottaxdi I , C. Canlet I , B. Delplanque 2 , J.C. Martin 3, K. Bensharif 3, • o .o . . o • 1 A. Thalmny-, G. Agnalu-, D. G n p o l s - , A. Paxls . 1 Umr Xenobiotique,
Toulouse, France: 2Len, Orsay, France: 3 Umr Lipides Nutrition Humaine, Marseille, France Objective: metabonomic examines all the detectable metabolites in body fluids to determine changes of homeostasis in relationship with health. We used this approach in a hamster model of nutritionnally-induced atherogenesis. M e t h o d : 5 groups of 9 adult hamsters were fed for 12wks experimental high-fat diets. Four were made by blending lyophilized cheeses with a chowbased diet to provide 20% (by weight) of lipids. The cheeses differed in the fat used in the making process: high saturated cow milk-fat (CS), trioleine milk-fat fraction (CT), canola based oil (CV), raw milk-fat (CR). A fith group was given a fat extract from the CS cheese (CE). The lipid loading in the aorta (determined by GLC) measured the severity of the lesions. Metabonomic analyses of both plasma and urine samples were performed by NMR, followed by discriminant factor analysis. Results: The cholesteryl-ester loading in the aorta was cleaxly influenced by the diets: the CV high-fat diet procuded less lipid deposition than the low-fat C-diet, then both the CR and CT-diets, then the CS diet, and finally the CE diet. This was clearly associated to the nonHDL/HDL-cholesterol ratio (r2=0.42, p < 0.001), and less to the paxaoxonase activity (r2=-0.19, p < 0.05). Metabonomic analysis of the plasma samples allowed to discriminate the hamsters according tho their atherogenic feature even better than the lipoprotein profile. Conclusion: metabonomic analysis of plasma appears highly efficienct to predict the severity of the early steps of atherogenesis produced by a nutritionnal stress, and warrant examination in human. Funding: partly funded by LACTALIS.
I Th P 17:4341 D E V E L O P M E N T O F A HTS A S S A Y F O R A C Y L - C O A C H O L E S T E R O L A C Y L T R A N S F E R A S E 2, A C A T 2 l
I
L. Redaelli, P. Arioli, L. Iuzzolino, D. Caxettoni. Axxam Srl, Milan, Italy Objective: ACAT2-derived cholesterol esters play a key role in the development of atherosclerosis, but the accessibility of ACAT2 to the drug discovery process has been hindered by the complex biochemistry of the target and by the lack of an assay compatible with high-throughput screening. The objective of our study is the design of a fluorescent assay suitable for the identification of novel ACAT2 inhibitors. M e t h o d s : Molecular modelling of ACAT2 predicted a structural motif responsible for multimerization and negative modulation of ACAT2. This motif was removed by mutagenesis and the resulting protein EA-ACAT2 was recombinantly expressed and purified from insect cells. Gel filtration chromatography was used to characterize the structure of the enzyme. ACAT2 catalytic activity was analyzed both by TLC and by an innovative fluorogenic assay in 384 MTP. Results: The bioinformatic prediction was validated by demonstrating that ACAT2 defective of the negative modulation motif was 4 times more active than the native ACAT2. Moreover, the mutant displayed an oligomeric structure in respect with the multimeric assembly of native ACAT2. Finally, a fluorescent assay was developed to follow cholesterol esterification. The kinetic parameters of EA-ACAT2 were calculated and the possibility to detect specific hits by HTS was demonstrated by using FR179254 as reference inhibitor. Conclusions: We have identified a motif responsible for the structural and functional modulation of ACAT2 activity and we have provided evidence for the first time that ACAT2 reaction can be detected with a homogeneous fluorescent assay suitable for HTS.
I Th P 17:435 I V I R T U A L G E L O F A T H E R O M A T O U S P L A Q U E 1
C. Felici I , B. Porcelli I , I. Ciari I , C. Setacci 2 , R. Guerranti I , R. Pagani I , L. Terzuoli 1 . 1 Dept btterTtal Medicine, EtMocino-Metabolic Sciences and
Biochemistry, Universi~ of Siena, Siena, Italy: "-Dept Surgery, University of Siena, Siena, Italy Objective: to create a virtual gel that reflects the protein pattern of atheromatous plaque. M e t h o d s : analysis of plaque of ten subjects undergoing carotid endoarterectomy and of the corresponding plasma, as reference material. Plaque
589
and plasma proteins were separated by two-dimensional electrophoresis according to Guerranti et al. (1). The gels were analysed by ImageMaster 2-D Platinum and two synthetic gels were constructed for plaque and plasma as the theoretical mean of the ten gels analysed. To be included in the synthetic gel, spots had to be present in at least three gels (triple pairs). Results: comparison of the spots on the synthetic gels of plaque and plasma showed 194 common spots. When the percentage volume of spots was calculated, 32 were found to have a very high plaque:plasma ratio (>4) and some were identified as ceruloplasmin, antitrypsin, haptoglobin, apolipoprotein J, A1, C2, AIV, seroamyloid protein P, kininogen, Hemoglobin B and A, fibrinogen, IgG and albumin by comparison with the SWISS-2DPAGE proteome map. Conclusions: comparison of only two virtual gels respresenting the ten gels analysed was an advantage. Many of the spots in the plaque gel were presumably of plasma origin by simple accumulation from the blood. Upregulated spots could represent a phenomenon intrinsic to the damaged tunica intima. Future characterization of these 32 spots by mass spectrometry could reveal new markers that could explain the pathogenesis of atherosderosis. 1) Guerranti R et al. Biochem Biophys Res Commun 2004; 323:484-90
Th-P17:436 ] PROTEOMIC AND METABOLOMIC APPROACHES TO NORMAL AND DISEASED VESSELS: ROLE OF OXIDATIVE STRESS M. Mayr, Y.-L. Chung, Q. Xu. St George's Universi~ of London, London,
United Kingdom Objectives: We applied proteomic and metabolomic approaches to vessels of apoE-L and apoE+/+ mice to elucidate the mechanisms of the disease development. M e t h o d s : For 2-DE, protein extracts from aortas of ApoE-L mice were used, and protein profiles were visualised by silver staining. Tandem mass spectrometry was performed for protein sequencing. For metabolomic profile, NMR spectra were obtained using a Bruker spectrometer. Results: We identified about 80 protein species that were dynamically altered during various stages of atherogenesis. Immune activation, redox imbalance and impaired energy metabolism preceded lesion formation in apoE-L mice. Some of the deposited antibodies are directed against phosphocholine. Oxidative stress in the vasculature was reflected by the oxidation status of the redox-sensitive protein 1-Cys peroxiredoxin and correlated to the extent of lesion formation in 12 month-old apoE-L mice. N M R analysis revealed diminished glucose metabolites and a marked depletion of the adenosine nucleotide and creatine pool in vessels of young apoE-L mice. Attenuation of lesion formation was associated with alterations of NADPH generating malic enzyme, highlighting the intimate connection between oxidative stress and vascular energy metabolism. C o n d u s i o n s : Our study is the first proteomic and metabolomic analysis of aortas from apoE-L mice and provides the most comprehensive dataset of protein and metabolite changes during atherogenesis published so fax, by which we can reveal the multiple facets of a single pathogenesis. Funding: BHF and Oak Foundation. 1
Th-P17:4371 S E L D I - T O F $
MS: A H I G H - T H R O U G H P U T T O O L F O R HIGH-DENSITY LIPOPROTEIN PROTEIN FINGERPRINTING ..
o
1
o
J.H.M. Levels 1, B. Bleulevens-, J.A. Kuivenhoven , J.M.F.G. Aerts-, J.C. Meijers 1.1Academic Medical Center; Dept. of Vascular Medicine,
Amsterdam, The Netherlands: "-Academic Medical Center; Dept. of Clinical Proteomics And Biochemistry, Amsterdam, The Netherlamls Objectives: HDL affects inflammation, coagulation, oxidation, and lipid metabolism. HDL cholesterol levels are inversely related to the risk of CAD, but it is not known how HDL exerts its atheroprotection. This underlines the need for tools to asses HDL function, especially in the context of novel HDL cholesterol increasing drugs. We set out to evaluate the applicability of SELDI-TOF mass-spectrometry to obtain fingerprints of HDL-associated proteins. M e t h o d s : Antibodies against apoAI and apoAII were covalently bound to a PS20 protein chip and HDL of normolipemic subjects was immuno captured and subjected to mass spectrometry. Results: On-chip capture of HDL either from plasma or from gel-filtration pre-purified HDL resulted in similar fingerprints. Albumin contamination was significantly reduced by 1) stringent washing (without loss of HDL specific components), 2) the use of EDTA and citrate plasma (compared to serum
XIV bzterTtational Symposium on Atherosclerosis, Rome, Italy, June 18-22, 2006
Thurs&ty, June 22, 2006: Poster Session Novel technologies for risk determination
I
I Th-P 17:4331 A M E T A B O N O M I C A P P R O A C H D I S C R I M I N A T E S THE SEVERITY OF THE ATHEROGENIC OUTCOME AMONG HYPERLIPIDEMIC HAMSTERS I
G. Gottaxdi I , C. Canlet I , B. Delplanque 2 , J.C. Martin 3, K. Bensharif 3, • o .o . . o • 1 A. Thalmny-, G. Agnalu-, D. G n p o l s - , A. Paxls . 1 Umr Xenobiotique,
Toulouse, France: 2Len, Orsay, France: 3 Umr Lipides Nutrition Humaine, Marseille, France Objective: metabonomic examines all the detectable metabolites in body fluids to determine changes of homeostasis in relationship with health. We used this approach in a hamster model of nutritionnally-induced atherogenesis. M e t h o d : 5 groups of 9 adult hamsters were fed for 12wks experimental high-fat diets. Four were made by blending lyophilized cheeses with a chowbased diet to provide 20% (by weight) of lipids. The cheeses differed in the fat used in the making process: high saturated cow milk-fat (CS), trioleine milk-fat fraction (CT), canola based oil (CV), raw milk-fat (CR). A fith group was given a fat extract from the CS cheese (CE). The lipid loading in the aorta (determined by GLC) measured the severity of the lesions. Metabonomic analyses of both plasma and urine samples were performed by NMR, followed by discriminant factor analysis. Results: The cholesteryl-ester loading in the aorta was cleaxly influenced by the diets: the CV high-fat diet procuded less lipid deposition than the low-fat C-diet, then both the CR and CT-diets, then the CS diet, and finally the CE diet. This was clearly associated to the nonHDL/HDL-cholesterol ratio (r2=0.42, p < 0.001), and less to the paxaoxonase activity (r2=-0.19, p < 0.05). Metabonomic analysis of the plasma samples allowed to discriminate the hamsters according tho their atherogenic feature even better than the lipoprotein profile. Conclusion: metabonomic analysis of plasma appears highly efficienct to predict the severity of the early steps of atherogenesis produced by a nutritionnal stress, and warrant examination in human. Funding: partly funded by LACTALIS.
I Th P 17:4341 D E V E L O P M E N T O F A HTS A S S A Y F O R A C Y L - C O A C H O L E S T E R O L A C Y L T R A N S F E R A S E 2, A C A T 2 l
I
L. Redaelli, P. Arioli, L. Iuzzolino, D. Caxettoni. Axxam Srl, Milan, Italy Objective: ACAT2-derived cholesterol esters play a key role in the development of atherosclerosis, but the accessibility of ACAT2 to the drug discovery process has been hindered by the complex biochemistry of the target and by the lack of an assay compatible with high-throughput screening. The objective of our study is the design of a fluorescent assay suitable for the identification of novel ACAT2 inhibitors. M e t h o d s : Molecular modelling of ACAT2 predicted a structural motif responsible for multimerization and negative modulation of ACAT2. This motif was removed by mutagenesis and the resulting protein EA-ACAT2 was recombinantly expressed and purified from insect cells. Gel filtration chromatography was used to characterize the structure of the enzyme. ACAT2 catalytic activity was analyzed both by TLC and by an innovative fluorogenic assay in 384 MTP. Results: The bioinformatic prediction was validated by demonstrating that ACAT2 defective of the negative modulation motif was 4 times more active than the native ACAT2. Moreover, the mutant displayed an oligomeric structure in respect with the multimeric assembly of native ACAT2. Finally, a fluorescent assay was developed to follow cholesterol esterification. The kinetic parameters of EA-ACAT2 were calculated and the possibility to detect specific hits by HTS was demonstrated by using FR179254 as reference inhibitor. Conclusions: We have identified a motif responsible for the structural and functional modulation of ACAT2 activity and we have provided evidence for the first time that ACAT2 reaction can be detected with a homogeneous fluorescent assay suitable for HTS.
I Th P 17:435 I V I R T U A L G E L O F A T H E R O M A T O U S P L A Q U E 1
C. Felici I , B. Porcelli I , I. Ciari I , C. Setacci 2 , R. Guerranti I , R. Pagani I , L. Terzuoli 1 . 1 Dept btterTtal Medicine, EtMocino-Metabolic Sciences and
Biochemistry, Universi~ of Siena, Siena, Italy: "-Dept Surgery, University of Siena, Siena, Italy Objective: to create a virtual gel that reflects the protein pattern of atheromatous plaque. M e t h o d s : analysis of plaque of ten subjects undergoing carotid endoarterectomy and of the corresponding plasma, as reference material. Plaque
589
and plasma proteins were separated by two-dimensional electrophoresis according to Guerranti et al. (1). The gels were analysed by ImageMaster 2-D Platinum and two synthetic gels were constructed for plaque and plasma as the theoretical mean of the ten gels analysed. To be included in the synthetic gel, spots had to be present in at least three gels (triple pairs). Results: comparison of the spots on the synthetic gels of plaque and plasma showed 194 common spots. When the percentage volume of spots was calculated, 32 were found to have a very high plaque:plasma ratio (>4) and some were identified as ceruloplasmin, antitrypsin, haptoglobin, apolipoprotein J, A1, C2, AIV, seroamyloid protein P, kininogen, Hemoglobin B and A, fibrinogen, IgG and albumin by comparison with the SWISS-2DPAGE proteome map. Conclusions: comparison of only two virtual gels respresenting the ten gels analysed was an advantage. Many of the spots in the plaque gel were presumably of plasma origin by simple accumulation from the blood. Upregulated spots could represent a phenomenon intrinsic to the damaged tunica intima. Future characterization of these 32 spots by mass spectrometry could reveal new markers that could explain the pathogenesis of atherosderosis. 1) Guerranti R et al. Biochem Biophys Res Commun 2004; 323:484-90
Th-P17:436 ] PROTEOMIC AND METABOLOMIC APPROACHES TO NORMAL AND DISEASED VESSELS: ROLE OF OXIDATIVE STRESS M. Mayr, Y.-L. Chung, Q. Xu. St George's Universi~ of London, London,
United Kingdom Objectives: We applied proteomic and metabolomic approaches to vessels of apoE-L and apoE+/+ mice to elucidate the mechanisms of the disease development. M e t h o d s : For 2-DE, protein extracts from aortas of ApoE-L mice were used, and protein profiles were visualised by silver staining. Tandem mass spectrometry was performed for protein sequencing. For metabolomic profile, NMR spectra were obtained using a Bruker spectrometer. Results: We identified about 80 protein species that were dynamically altered during various stages of atherogenesis. Immune activation, redox imbalance and impaired energy metabolism preceded lesion formation in apoE-L mice. Some of the deposited antibodies are directed against phosphocholine. Oxidative stress in the vasculature was reflected by the oxidation status of the redox-sensitive protein 1-Cys peroxiredoxin and correlated to the extent of lesion formation in 12 month-old apoE-L mice. N M R analysis revealed diminished glucose metabolites and a marked depletion of the adenosine nucleotide and creatine pool in vessels of young apoE-L mice. Attenuation of lesion formation was associated with alterations of NADPH generating malic enzyme, highlighting the intimate connection between oxidative stress and vascular energy metabolism. C o n d u s i o n s : Our study is the first proteomic and metabolomic analysis of aortas from apoE-L mice and provides the most comprehensive dataset of protein and metabolite changes during atherogenesis published so fax, by which we can reveal the multiple facets of a single pathogenesis. Funding: BHF and Oak Foundation. 1
Th-P17:4371 S E L D I - T O F $
MS: A H I G H - T H R O U G H P U T T O O L F O R HIGH-DENSITY LIPOPROTEIN PROTEIN FINGERPRINTING ..
o
1
o
J.H.M. Levels 1, B. Bleulevens-, J.A. Kuivenhoven , J.M.F.G. Aerts-, J.C. Meijers 1.1Academic Medical Center; Dept. of Vascular Medicine,
Amsterdam, The Netherlands: "-Academic Medical Center; Dept. of Clinical Proteomics And Biochemistry, Amsterdam, The Netherlamls Objectives: HDL affects inflammation, coagulation, oxidation, and lipid metabolism. HDL cholesterol levels are inversely related to the risk of CAD, but it is not known how HDL exerts its atheroprotection. This underlines the need for tools to asses HDL function, especially in the context of novel HDL cholesterol increasing drugs. We set out to evaluate the applicability of SELDI-TOF mass-spectrometry to obtain fingerprints of HDL-associated proteins. M e t h o d s : Antibodies against apoAI and apoAII were covalently bound to a PS20 protein chip and HDL of normolipemic subjects was immuno captured and subjected to mass spectrometry. Results: On-chip capture of HDL either from plasma or from gel-filtration pre-purified HDL resulted in similar fingerprints. Albumin contamination was significantly reduced by 1) stringent washing (without loss of HDL specific components), 2) the use of EDTA and citrate plasma (compared to serum
XIV bzterTtational Symposium on Atherosclerosis, Rome, Italy, June 18-22, 2006