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2,4-decadienal downregulates TNF-α gene expression in THP-1 human macrophages
Thursday June 29, 2000: Poster Abstracts P: W29 Oxidation and Atherogenesis
260
the results were showed by absorbance at 405 nm wavelength under substrate integrated. Results: The results showed that the titer of anti-oxidized LDL antibody against oxidized LDL purified from high-LDL serum in patients with AMI was 133% higher than that in normal subjects. But the titer of antibody in AMI was only 68% higher than that in normal subjects when the normal LDL serum was used. Conclusions: We assume that the binding motif of oxidized-LDL from high-LDL serum is different from that of normal range LDL serum. Therefore, it may be important to choose the source of oxidized-LDL in the assay of antoantibody against oxidized LDL. And we found that the titer of autoantibody against oxidized LDL was higher in AMI patients than in normal subjects. This result indicated that the expression of this autoantibody could be related with the process of acute myocardial infarction.
ThP20:W29 I LDL oxidises in presence of plasma antioxidants after aggregation with chandroitin-4-sulfate Peter M. Abuja. Institute of Molecular Biology, Biochemistry and
Microbiology University of Graz, Austria Although the oxidation of low-density lipoprotein (LDL) appears to be an important event in the development of atherosclerosis, it is unclear to date, how extensive lipid peroxidation could occur in the arterial intima. It has been shown that in the presence of high concentrations of water-soluble antioxidants, such as urate and ascorbate in plasma and endothelial lining fluid the oxidation of isolated LDL particles is inhibited very efficiently, both by nletal-catalysed oxidation and by lipid peroxidation initiated by other radicals, e.g. water-soluble peroxyl radicals. Therefore, it was of interest whether aggregation by constituents of the arterial extracellular matrix could facilitate oxidation under these conditions. We incubated human LDL with 300 /xM urate and 20 # M ascorbate, chondroitin-4-sulfate (C4S), chondroitin-6-sulfate (0.25 to 1.0 mg/mL each) and sphingomyelinase (SMase, 50-150 mU/mL), and monitored copper catalysed LDL oxidation by low-level chemiluminescence. Oxidation was inhibited in the control containing only urate and ascorbate, and in the incubations with C6S and SMase. However, in presence of C4S, oxidation proceeded nearly as rapidly as in an antioxidantfree control, and was considerably enhanced by pre-incubation of LDL with 15-lipoxygenase (15-LOX). C4S, C6S and SMase have been reported to induce aggregation of LDL, in addition, C4S is able to bind Cu-ions very efficiently. The prooxidant effect of C4S in Cu-catalysed oxidation can explained be inclusion of copper ions into the aggregates, which can then oxidise LDL very efficiently, in the absence of water-soluble antioxidants. Together with the prooxidant action of 15-LOX, this constitutes a possible way of LDL oxidation in the presence of plasma antioxidants in vivo. 1
ThP21 -W29 [ HDL prevents the formation of aldehyde adducts on
apo-B during the oxidation of LDL M.I. Mackness, P. Sangvanich I , B. Mackness, P.N. Durrington, S. Gaskill I .
University Department of Medicine, Manchester Royal Infirmary, Oxford Road, Manchester, and Department of Chemistry, UMIST, Manchester, UK
Objectives: During the oxidation of LDL, aldehyde break-down products of oxidised lipids for adducts with amino-acids present on apo-B. This adducted apo-B is the ligand form oxidised LDL receptors on macrophages and is therefore an important step in atherugenesis. We have studied the effect of HDL on this process. Methods: 0.5 mg LDL in PBS was oxidised using 5/~M Cu 2+ for 4 hr at 37°Cin the absence or presence of 0.5 mg HDL. Oxidation was stopped by the addition of 0.5 mM BHT and 2 mM EDTA. Samples were delipidated and apo-B subjected to tryptic digestion. The peptides were separated by reverse-phase HPLC and analysed be electrospray ionisation mass spectrometry. HNE-modifled angiotensin II was used as a control. Results: In the absence of HDL several peptides of apo-B were found to be adducted with HNE with a major one of 7 amino-acids at 527.5 m/z and other peaks at 393.6 m/z, 499.9 m/z, 577.3 m/z and 654.8 m/z. In the presence of HDL no aldehyde adducts were found. Condnsion: HDL can protect apo-B from the formation of aldehyde adducts during oxidation. Whether the mechanism involves the prevention of aldehyde formation or the prevention of the adduction process itself is the subject of current research in our laboratory.
I ThP22:W29 ] 2.4-decadienal downregulates TNF-= gene expression in THP-1 human macrophages J. Girona, J.C. Vallvt, J. Ribalta, M. Heras, S. Olivt, L. Masana. Unitat de Recerca de Lipids i Arteriosclerosi, Facultat de Medicina, Universitat Rovira i Virgili. Reus, Spain Oxidized lipoproteins inhibit TNF-~x secretion by human THP- 1 macrophages due, at least in part, to aldehydes derived from the oxidation of polyunsaturated fatty acids. This study extends these fndings by investigating the effect of three aldehydes (2.4-decadienal (2.4-DDE), hexanal and 4-hydroxynonenal (4-HNE)) on TNF-~ and IL-1/~ mRNA expression. The 2.4-DDE and 4-HNE showed considerable biological activity which induced cytotoxicity on THP- 1 macrophages at concentrations of 50 #M. Hexanal, on the other hand, had a lower cytotoxic capacity and concentrations of 1000 # M were needed. Exposure of THP-1 macrophages to aldehydes for 24 h inhibited TNF-~ mRNA expression but increased or did not affect IL-I/~ mRNA levels. The inhibitory action of 2.4-DDE was the most dose dependent and began at 5 # M (46%, p < 0.001). The effect of 4-HNE was less inhibitory than 4-DDE but only when cytotoxic concentrations were used (50/~M). Very high concentrations of hexanai (200 /~M) were needed to inhibit TNF-c~ expression (23%, p < 0.001). The inhibitory effect of 2.4-DDE was greatest after 2 hours of incubation and recovered partially after 24 h. This downregulation of TNF-a gene expression by 2.4-DDE was parallel to a lower protein production. These data indicate that low levels of 2.4-DDE may modulate inflammatory action by inhibiting TNF-a mRNA gene expression and that the biological activity of 2.4-DDE may be involved in the development of atherosclerosis.
ThP23:W29 ]I Modulation of basal and IL-l-induced adhesion molecule expression by PHGPX and 15-1ipoxygenase in rabbit aortic smooth muscle cells K. Schnurr 1, I. Petermann 2, R. Brigelius-Floh~ 1'2 . 1German Institute of
Human Nutrition, Potsdam-Rehbriicke; 2 University of Potsdam, Germany
Objective: Expression of cell adhesion molecules is an early event during atherogenesis. They are induced by cytokines and also by conditions of oxidative stress. We recently found that 13-HPODE was able to induce ICAM-1 in HUVEC. Surprisingly 13-HODE, the reduction product of 13-HPODE was even more effective. Both compounds worked additively with IL-1 and TNE We were therefore interested in the contribution of 15-LO which produces 13-HPODE and PHGPx which reduces 13-HPODE to 13-HODE in the induction of VCAM-1 and ICAM-1 by IL-1 in smooth muscle cells overexpressing either PHGPx (SMCPHGPx) or 15-LO (SMCLO). Methods: Cells were cultured for 48 h without FCS and stimulated with IL-1 for 4 h. Thereafter, total cellular RNA was prepared, analysed by RT-PCR and standardized to GAPDH mRNA. Results: RT-PCR showed a decreased basal expression of ICAM-I and VCAM-1 in both SMCPHGPx and SMCLO. Based on the high basal expression an induction of ICAM-1 by IL-1 was not observed in controls (SMC). In contrast, ICAM-1 was induced by IL-1 in SMCLO cells but not in SMCPHGPx cells. Constitutive expression of VCAM-1 in SMC was almost abolished in SMCPHGPx and SMCLO. VCAM-1 was not enhanced in controls whereas in transfected cells VCAM-1 could be induced by IL-1. The effect was much higher in SMCPHGPx than in SMCLO. Condnsions: The modulation of basal and IL-l-induced expression of adhesion molecules by PHGPx and 15-LO will be discussed in the context of the interference of these enzymes with a balanced cellular redox state.
I ThP24:W29
]I Association between LDL constituents and its resistance against copper induced oxidation
P.G. Scheffer 1 , E.E. Musch 1 , S.J.L. Bakker 2, C. Popp-Sni~ders 1'2, R.J. Heine 2, T. Teerlink I . ~Depts. of Clinical Chemistry; ZEndocrinology,
Academic Hospital Vrije Universiteit, Amsterdam, The Netherlands
Objective: Diabetes patients have an increased risk of developing cardiovascular disease. Oxidation of LDL is supposed to play a role in the development of atherosclerosis. The aim of this study was to explore how different LDL constituents contribute to the resistance of LDL to oxidation. Methods: In this study 101 normolipidemic type 2 diabetes patients with acceptable glycemic control were included. The resistance of LDL to in vitro oxidation, expressed as lag time, was measured by monitoring the formation of conjugated dienes. The explained variability in lag time was estimated using stepwise multiple linear regression models with the lag time as the dependent variable.
Xllth International Symposium on Atherosclerosis, Stockholm, Sweden, June 25-29, 2000
260
the results were showed by absorbance at 405 nm wavelength under substrate integrated. Results: The results showed that the titer of anti-oxidized LDL antibody against oxidized LDL purified from high-LDL serum in patients with AMI was 133% higher than that in normal subjects. But the titer of antibody in AMI was only 68% higher than that in normal subjects when the normal LDL serum was used. Conclusions: We assume that the binding motif of oxidized-LDL from high-LDL serum is different from that of normal range LDL serum. Therefore, it may be important to choose the source of oxidized-LDL in the assay of antoantibody against oxidized LDL. And we found that the titer of autoantibody against oxidized LDL was higher in AMI patients than in normal subjects. This result indicated that the expression of this autoantibody could be related with the process of acute myocardial infarction.
ThP20:W29 I LDL oxidises in presence of plasma antioxidants after aggregation with chandroitin-4-sulfate Peter M. Abuja. Institute of Molecular Biology, Biochemistry and
Microbiology University of Graz, Austria Although the oxidation of low-density lipoprotein (LDL) appears to be an important event in the development of atherosclerosis, it is unclear to date, how extensive lipid peroxidation could occur in the arterial intima. It has been shown that in the presence of high concentrations of water-soluble antioxidants, such as urate and ascorbate in plasma and endothelial lining fluid the oxidation of isolated LDL particles is inhibited very efficiently, both by nletal-catalysed oxidation and by lipid peroxidation initiated by other radicals, e.g. water-soluble peroxyl radicals. Therefore, it was of interest whether aggregation by constituents of the arterial extracellular matrix could facilitate oxidation under these conditions. We incubated human LDL with 300 /xM urate and 20 # M ascorbate, chondroitin-4-sulfate (C4S), chondroitin-6-sulfate (0.25 to 1.0 mg/mL each) and sphingomyelinase (SMase, 50-150 mU/mL), and monitored copper catalysed LDL oxidation by low-level chemiluminescence. Oxidation was inhibited in the control containing only urate and ascorbate, and in the incubations with C6S and SMase. However, in presence of C4S, oxidation proceeded nearly as rapidly as in an antioxidantfree control, and was considerably enhanced by pre-incubation of LDL with 15-lipoxygenase (15-LOX). C4S, C6S and SMase have been reported to induce aggregation of LDL, in addition, C4S is able to bind Cu-ions very efficiently. The prooxidant effect of C4S in Cu-catalysed oxidation can explained be inclusion of copper ions into the aggregates, which can then oxidise LDL very efficiently, in the absence of water-soluble antioxidants. Together with the prooxidant action of 15-LOX, this constitutes a possible way of LDL oxidation in the presence of plasma antioxidants in vivo. 1
ThP21 -W29 [ HDL prevents the formation of aldehyde adducts on
apo-B during the oxidation of LDL M.I. Mackness, P. Sangvanich I , B. Mackness, P.N. Durrington, S. Gaskill I .
University Department of Medicine, Manchester Royal Infirmary, Oxford Road, Manchester, and Department of Chemistry, UMIST, Manchester, UK
Objectives: During the oxidation of LDL, aldehyde break-down products of oxidised lipids for adducts with amino-acids present on apo-B. This adducted apo-B is the ligand form oxidised LDL receptors on macrophages and is therefore an important step in atherugenesis. We have studied the effect of HDL on this process. Methods: 0.5 mg LDL in PBS was oxidised using 5/~M Cu 2+ for 4 hr at 37°Cin the absence or presence of 0.5 mg HDL. Oxidation was stopped by the addition of 0.5 mM BHT and 2 mM EDTA. Samples were delipidated and apo-B subjected to tryptic digestion. The peptides were separated by reverse-phase HPLC and analysed be electrospray ionisation mass spectrometry. HNE-modifled angiotensin II was used as a control. Results: In the absence of HDL several peptides of apo-B were found to be adducted with HNE with a major one of 7 amino-acids at 527.5 m/z and other peaks at 393.6 m/z, 499.9 m/z, 577.3 m/z and 654.8 m/z. In the presence of HDL no aldehyde adducts were found. Condnsion: HDL can protect apo-B from the formation of aldehyde adducts during oxidation. Whether the mechanism involves the prevention of aldehyde formation or the prevention of the adduction process itself is the subject of current research in our laboratory.
I ThP22:W29 ] 2.4-decadienal downregulates TNF-= gene expression in THP-1 human macrophages J. Girona, J.C. Vallvt, J. Ribalta, M. Heras, S. Olivt, L. Masana. Unitat de Recerca de Lipids i Arteriosclerosi, Facultat de Medicina, Universitat Rovira i Virgili. Reus, Spain Oxidized lipoproteins inhibit TNF-~x secretion by human THP- 1 macrophages due, at least in part, to aldehydes derived from the oxidation of polyunsaturated fatty acids. This study extends these fndings by investigating the effect of three aldehydes (2.4-decadienal (2.4-DDE), hexanal and 4-hydroxynonenal (4-HNE)) on TNF-~ and IL-1/~ mRNA expression. The 2.4-DDE and 4-HNE showed considerable biological activity which induced cytotoxicity on THP- 1 macrophages at concentrations of 50 #M. Hexanal, on the other hand, had a lower cytotoxic capacity and concentrations of 1000 # M were needed. Exposure of THP-1 macrophages to aldehydes for 24 h inhibited TNF-~ mRNA expression but increased or did not affect IL-I/~ mRNA levels. The inhibitory action of 2.4-DDE was the most dose dependent and began at 5 # M (46%, p < 0.001). The effect of 4-HNE was less inhibitory than 4-DDE but only when cytotoxic concentrations were used (50/~M). Very high concentrations of hexanai (200 /~M) were needed to inhibit TNF-c~ expression (23%, p < 0.001). The inhibitory effect of 2.4-DDE was greatest after 2 hours of incubation and recovered partially after 24 h. This downregulation of TNF-a gene expression by 2.4-DDE was parallel to a lower protein production. These data indicate that low levels of 2.4-DDE may modulate inflammatory action by inhibiting TNF-a mRNA gene expression and that the biological activity of 2.4-DDE may be involved in the development of atherosclerosis.
ThP23:W29 ]I Modulation of basal and IL-l-induced adhesion molecule expression by PHGPX and 15-1ipoxygenase in rabbit aortic smooth muscle cells K. Schnurr 1, I. Petermann 2, R. Brigelius-Floh~ 1'2 . 1German Institute of
Human Nutrition, Potsdam-Rehbriicke; 2 University of Potsdam, Germany
Objective: Expression of cell adhesion molecules is an early event during atherogenesis. They are induced by cytokines and also by conditions of oxidative stress. We recently found that 13-HPODE was able to induce ICAM-1 in HUVEC. Surprisingly 13-HODE, the reduction product of 13-HPODE was even more effective. Both compounds worked additively with IL-1 and TNE We were therefore interested in the contribution of 15-LO which produces 13-HPODE and PHGPx which reduces 13-HPODE to 13-HODE in the induction of VCAM-1 and ICAM-1 by IL-1 in smooth muscle cells overexpressing either PHGPx (SMCPHGPx) or 15-LO (SMCLO). Methods: Cells were cultured for 48 h without FCS and stimulated with IL-1 for 4 h. Thereafter, total cellular RNA was prepared, analysed by RT-PCR and standardized to GAPDH mRNA. Results: RT-PCR showed a decreased basal expression of ICAM-I and VCAM-1 in both SMCPHGPx and SMCLO. Based on the high basal expression an induction of ICAM-1 by IL-1 was not observed in controls (SMC). In contrast, ICAM-1 was induced by IL-1 in SMCLO cells but not in SMCPHGPx cells. Constitutive expression of VCAM-1 in SMC was almost abolished in SMCPHGPx and SMCLO. VCAM-1 was not enhanced in controls whereas in transfected cells VCAM-1 could be induced by IL-1. The effect was much higher in SMCPHGPx than in SMCLO. Condnsions: The modulation of basal and IL-l-induced expression of adhesion molecules by PHGPx and 15-LO will be discussed in the context of the interference of these enzymes with a balanced cellular redox state.
I ThP24:W29
]I Association between LDL constituents and its resistance against copper induced oxidation
P.G. Scheffer 1 , E.E. Musch 1 , S.J.L. Bakker 2, C. Popp-Sni~ders 1'2, R.J. Heine 2, T. Teerlink I . ~Depts. of Clinical Chemistry; ZEndocrinology,
Academic Hospital Vrije Universiteit, Amsterdam, The Netherlands
Objective: Diabetes patients have an increased risk of developing cardiovascular disease. Oxidation of LDL is supposed to play a role in the development of atherosclerosis. The aim of this study was to explore how different LDL constituents contribute to the resistance of LDL to oxidation. Methods: In this study 101 normolipidemic type 2 diabetes patients with acceptable glycemic control were included. The resistance of LDL to in vitro oxidation, expressed as lag time, was measured by monitoring the formation of conjugated dienes. The explained variability in lag time was estimated using stepwise multiple linear regression models with the lag time as the dependent variable.
Xllth International Symposium on Atherosclerosis, Stockholm, Sweden, June 25-29, 2000