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Protective role of phospholipase A2 in processes of LDL modification
Monday June 26, 2000: Poster Abstracts P:W29 Oxidation and Atherogenesis
58
10 weeks the animals with the combined HC + IVH showed 30--40% more of the inner aortic surface covered with lesions than the animals with only HC. The animals with only IVH or the controls with normal cholesterol showed no lesions. Western blots showed that hemin oxidase (HO-1) expression in aorta and other tissues was markedly increased by phenylhydrazine and it was correlated with the extent of IVH. However this enzyme, that is the main agent for heine conversion to bilirubin and biliverdin appeared not to protect the animals from the atherogenicity of hypercholesterolemia. Condnsions: The data suggest that the increased oxidative stress associated with IVH and its associated heme formation potentiate the atherogenicity of the hypercholesterolemia. Induction of HO-1 appear not to be sufficient to counteract this condition
MoP22:W29 I The expression of human ATP-binding cassette transporter 1 mRNA increased by oxidized LDL Y. Wanaka, S. Boh-oka, Y. Sakai, M. Hamada, T. Hano, I. Nishio. Div. of Cardiology, Dept. of Medicine, Wakayama Medical College, Wakayama, Japan
Objectives: The aim of this study is to evaluate the regulation of human ATP-binding cassette transporter I (ABC1) mRNA expression by oxidized LDL. Methods: Human monocytic THP-1 cells were cultured and differentiated into macrophage with PMA. We incubated them with LDL, oxidized LDL, or Troglitazone in various conditions. Total RNA was obtained by AGPC method. Quantitative RT-PCR and Northern blot analysis for ABC 1 mRNA were performed. Results: Stimulation of LDL and oxidized LDL increased the expression of ABC 1 mRNA in dose and time dependent mannars. The stimulation of oxidized LDL is storonger than that of LDL for the expression of ABC 1 mRNA. Troglitazone which is thought to be an agonist for PPARy did not increase the expression of ABC 1 mRNA. Conclusion: These results indicate that oxidized LDL and LDL induced the expression of ABC 1 mRNA of cultured macrophage. This might relate the regulation of intracellular LDL cholesterol and blood HDL cholesterol levels. MoP23:W29 I An oxidized derivative of cholesterol increases the release of soluble vascular cell adhesion molecule-1 from human umbilical vein endotherial cells in culture I
N. Tamasawa 1, H. Murakami I , J. Matsui I , T. Imaizumi2, Kei Satoh 2, T. Suda I . 1Third Department of Internal Medicine; 2Department of Vascular
Biology, Institute of Brain Science, Hirosaki University School of Medicine, Hirosaki, Japan Oxidative modification of low density lipoprotein (LDL) is regarded as one of early features of atherogenesis. We have examined the effect of oxysterols, a group of bioactive oxidized lipids in modified LDL, on the expression of vascular cell adhesion molecule-1 (VCAM-1) in cultured human umbilical vein endothelial cells (HUVECs). Treatment of HUVECs with 7-ketocholesterol, a product of cholesterol autoxidation, resulted in an increased release of soluble VCAM-1 into the medium of the cells. At 0.125 #M, 7-ketocholesterol increased soluble VCAM-1 levels by 100%. 7-Keto-cholesterol did not enhance the expression of mRNA for VCAM-1. Other molecular species of oxysterols such as 7/~-hydroxy- or 25-hydroxycholesterol had no effect on soluble VCAM-1 levels as well as on VCAM-1 mRNA. Western blot analysis revealed that the soluble VCAM-1 in the conditioned medium, as well as in the control medium, had a molecular size of 100 kDa. Stimulation of HUVECs pretreated with TNF-a, which induces the expression of VCAM- 1, further increased the levels of soluble VCAM-1 in the culture medium. Again, 7-ketocholesterol did not affect the VCAM-1 mRNA levels. These results suggest that certain molecular species of oxysterols may release VCAM-1, probably by shedding, from the endothelium and may regulate the interactions between blood cells and the vascular wall.
I MoP24:W29 ] Protective role of phospholipase A2 in processes of LDL modification i
A.A. Korotaeva, I.B. Cbeglakov, N.K. Golovanova, T.N. Vlasik, E.V. Yanushevskaya, V.P. Tsibulsky, V.V. Yakushkin, N.V. Prokazova.
Cardiological Research Centere, Moscow, Russia Objective: Phospholipase A2 modified LDL rich in lysophosphatidylcholine (LPC) have received much attention. The changes caused by LPC accumulation in them, on structure and function of LDL was studied.
Methods: Lipid-protein particles (pI-LDL) were obtained by treatment of LDL with phospbolipase A2 from bee venom. Results: Half of PC in pl-LDL was changed to LPC, the composition of other lipids and protein structure were unaffected. Three MAbs against different apt B epitopes were used to test immunoreactivity of pl-LDL. MAbs 4C 11 interacting with ape B epitope (residue 2377-2658) placed near receptor site, showed significant decrease immunoreactivity. In contrast, increase in 4C11 binding was demonstrated to be function of oxidation extent of LDL. Thus changing of half PC to LPC modified a p t B translocation in lipoprotein globule in opposite manner than oxidative modification did it. Two fold increase of pl-LDL affinity to immobilized LDL-receptor was shown in contrast to LDL. Pl-LDL as well as LPC abolished hormon induced Ca 2+ elevation in platelets and platelet aggregation induced by PAF, ADP and thrombine. The effect persisted in Ca free medium, indicating that pl-LDL and LPC did not abolish mobilisation of intracellular stores with above mentioned inductors. Neither LPC no pl-LDL suppressed platelet aggregation. Inhibited effect depended of LPC concentration and platelet incubation time with pI-LDL or LPC. Half-maximum effective LPC concentrations were identical for itself LPC and phl-LDL and were 2-4 uM. Conclusion: It was concluded that LPC incorporation in LDL can act as a metabolite reconstructing LDL properties deranged by their oxidation. So, phospholipase A2 can be supposed to play a protective role in the processes of LDL modification.
MoP25:W29 ]
Oral vitamin BI2 lowers homocysteine in patients with ESRD more effectively in the homozygous T/l" MTHFR 677 patients
M.E. Hyndman, B. Manns, P. Bridge, N. Scott-Douglas, E. Burgess, J. Schaefer, H. Parsons. Department of Medicine, University of Calgary,
Canada Objective: The efficacy of vitamin therapy with B12 and folic acid in addition to a daily multivitamin dose containing 1 mg/day of folic acid was studied in 82 end-stage renal disease (ESRD) patients with homocysteine (hcys) levels > 16/zmol. Before enrolment all patients were receiving a multivitamin daily, containing 1 mg of folate and 6 ug of B12. All patients then received 1 mg of oral vitamin Bl2 daily for four weeks, followed by random assignment to 0, 5, or 15 mg of folic acid daily. MTHFR 677 C - + T genotype was measured and the effect of vitamin therapy on hey levels was examined within the genotypes. Results: The distribution of the MTHFR 677 genotype was 52%, 37% and 12% and the mean homocysteine at base line was 21.19 (4.88) 22.38 (4.69) and 29.842 (12.30) #mol/L for the C/C, C/T and T/T genotypes respectively. Patients with the T/T genotype had significantly elevated baseline bcys levels compared to either the CIC and C/T patients (P = 0.008). BI2 therapy reduced the hcys levels by 2.77 (3.20), 3.46 (3.2) and 9.12 (8.35) in the MTHFR C/C, C/T and T/T patients respectively (P = 0.001 T/T vs C/T, C/C). Folate supplementation with 5 mg/15 mg did not further reduce homocysteine level in any of the genotypes (p = 0.35). Serum B12 arid RBC folate levels were not different between groups at baseline or after folate supplementation except that RBC folate levels were significantly elevated in the T f f patients after four weeks of B12 supplementation. Atherosclerosis was present in 29% of the patients and did not differ between the MTHFR 677 genotypes. Conclusion: Patients with the homozygous 677 C--*T MTHFR mutation responded to Bl2 therapy significantly better than those with the C/C or C/T genotypes. Extra supplementation with 5 mg or 15 mg of folic acid did not lower homocysteine levels significantly in any of the MTHFR 677 genotypes compared to the placebo group.
MoP26:W29
] Oxidized low density fipoprotein induces reactive oxigen species in endothelial cells: Effect on the intracelinlar nitric oxide availability [
L. Conainacini, A. Rigoni, U. Garbin, A. Fratta Pasini, A. Davoli, M. Campagnola, M.L. Tosetti. Department of lnternal and Surgical Sciences,
University of Verona, Verona, Italy Objective: The impaired endothelium-dependent relaxation, the earliest alteration in hypertension, is largely due to a reduction in nitric oxide (NO) activity. Oxidized low density/ipoprotein (oxLDL) has been demonstrated to increase reactive oxigen species (ROS) in endothelial cells. Since ROS may reduce NO aim of this study is to evaluate the effect of oxLDL on intracellular ROS formation and NO availability in endothelial cells. Methods: Bovine aortic endothelial cells (BAECs) were incubated with native LDL and 5 /zM Cu++-oxLDL for 5 rain. 2', 7'-dichlorofluorescein
Xllth International Symposium on Atherosclerosis, Stockholm, Sweden, June 25-29, 2000
58
10 weeks the animals with the combined HC + IVH showed 30--40% more of the inner aortic surface covered with lesions than the animals with only HC. The animals with only IVH or the controls with normal cholesterol showed no lesions. Western blots showed that hemin oxidase (HO-1) expression in aorta and other tissues was markedly increased by phenylhydrazine and it was correlated with the extent of IVH. However this enzyme, that is the main agent for heine conversion to bilirubin and biliverdin appeared not to protect the animals from the atherogenicity of hypercholesterolemia. Condnsions: The data suggest that the increased oxidative stress associated with IVH and its associated heme formation potentiate the atherogenicity of the hypercholesterolemia. Induction of HO-1 appear not to be sufficient to counteract this condition
MoP22:W29 I The expression of human ATP-binding cassette transporter 1 mRNA increased by oxidized LDL Y. Wanaka, S. Boh-oka, Y. Sakai, M. Hamada, T. Hano, I. Nishio. Div. of Cardiology, Dept. of Medicine, Wakayama Medical College, Wakayama, Japan
Objectives: The aim of this study is to evaluate the regulation of human ATP-binding cassette transporter I (ABC1) mRNA expression by oxidized LDL. Methods: Human monocytic THP-1 cells were cultured and differentiated into macrophage with PMA. We incubated them with LDL, oxidized LDL, or Troglitazone in various conditions. Total RNA was obtained by AGPC method. Quantitative RT-PCR and Northern blot analysis for ABC 1 mRNA were performed. Results: Stimulation of LDL and oxidized LDL increased the expression of ABC 1 mRNA in dose and time dependent mannars. The stimulation of oxidized LDL is storonger than that of LDL for the expression of ABC 1 mRNA. Troglitazone which is thought to be an agonist for PPARy did not increase the expression of ABC 1 mRNA. Conclusion: These results indicate that oxidized LDL and LDL induced the expression of ABC 1 mRNA of cultured macrophage. This might relate the regulation of intracellular LDL cholesterol and blood HDL cholesterol levels. MoP23:W29 I An oxidized derivative of cholesterol increases the release of soluble vascular cell adhesion molecule-1 from human umbilical vein endotherial cells in culture I
N. Tamasawa 1, H. Murakami I , J. Matsui I , T. Imaizumi2, Kei Satoh 2, T. Suda I . 1Third Department of Internal Medicine; 2Department of Vascular
Biology, Institute of Brain Science, Hirosaki University School of Medicine, Hirosaki, Japan Oxidative modification of low density lipoprotein (LDL) is regarded as one of early features of atherogenesis. We have examined the effect of oxysterols, a group of bioactive oxidized lipids in modified LDL, on the expression of vascular cell adhesion molecule-1 (VCAM-1) in cultured human umbilical vein endothelial cells (HUVECs). Treatment of HUVECs with 7-ketocholesterol, a product of cholesterol autoxidation, resulted in an increased release of soluble VCAM-1 into the medium of the cells. At 0.125 #M, 7-ketocholesterol increased soluble VCAM-1 levels by 100%. 7-Keto-cholesterol did not enhance the expression of mRNA for VCAM-1. Other molecular species of oxysterols such as 7/~-hydroxy- or 25-hydroxycholesterol had no effect on soluble VCAM-1 levels as well as on VCAM-1 mRNA. Western blot analysis revealed that the soluble VCAM-1 in the conditioned medium, as well as in the control medium, had a molecular size of 100 kDa. Stimulation of HUVECs pretreated with TNF-a, which induces the expression of VCAM- 1, further increased the levels of soluble VCAM-1 in the culture medium. Again, 7-ketocholesterol did not affect the VCAM-1 mRNA levels. These results suggest that certain molecular species of oxysterols may release VCAM-1, probably by shedding, from the endothelium and may regulate the interactions between blood cells and the vascular wall.
I MoP24:W29 ] Protective role of phospholipase A2 in processes of LDL modification i
A.A. Korotaeva, I.B. Cbeglakov, N.K. Golovanova, T.N. Vlasik, E.V. Yanushevskaya, V.P. Tsibulsky, V.V. Yakushkin, N.V. Prokazova.
Cardiological Research Centere, Moscow, Russia Objective: Phospholipase A2 modified LDL rich in lysophosphatidylcholine (LPC) have received much attention. The changes caused by LPC accumulation in them, on structure and function of LDL was studied.
Methods: Lipid-protein particles (pI-LDL) were obtained by treatment of LDL with phospbolipase A2 from bee venom. Results: Half of PC in pl-LDL was changed to LPC, the composition of other lipids and protein structure were unaffected. Three MAbs against different apt B epitopes were used to test immunoreactivity of pl-LDL. MAbs 4C 11 interacting with ape B epitope (residue 2377-2658) placed near receptor site, showed significant decrease immunoreactivity. In contrast, increase in 4C11 binding was demonstrated to be function of oxidation extent of LDL. Thus changing of half PC to LPC modified a p t B translocation in lipoprotein globule in opposite manner than oxidative modification did it. Two fold increase of pl-LDL affinity to immobilized LDL-receptor was shown in contrast to LDL. Pl-LDL as well as LPC abolished hormon induced Ca 2+ elevation in platelets and platelet aggregation induced by PAF, ADP and thrombine. The effect persisted in Ca free medium, indicating that pl-LDL and LPC did not abolish mobilisation of intracellular stores with above mentioned inductors. Neither LPC no pl-LDL suppressed platelet aggregation. Inhibited effect depended of LPC concentration and platelet incubation time with pI-LDL or LPC. Half-maximum effective LPC concentrations were identical for itself LPC and phl-LDL and were 2-4 uM. Conclusion: It was concluded that LPC incorporation in LDL can act as a metabolite reconstructing LDL properties deranged by their oxidation. So, phospholipase A2 can be supposed to play a protective role in the processes of LDL modification.
MoP25:W29 ]
Oral vitamin BI2 lowers homocysteine in patients with ESRD more effectively in the homozygous T/l" MTHFR 677 patients
M.E. Hyndman, B. Manns, P. Bridge, N. Scott-Douglas, E. Burgess, J. Schaefer, H. Parsons. Department of Medicine, University of Calgary,
Canada Objective: The efficacy of vitamin therapy with B12 and folic acid in addition to a daily multivitamin dose containing 1 mg/day of folic acid was studied in 82 end-stage renal disease (ESRD) patients with homocysteine (hcys) levels > 16/zmol. Before enrolment all patients were receiving a multivitamin daily, containing 1 mg of folate and 6 ug of B12. All patients then received 1 mg of oral vitamin Bl2 daily for four weeks, followed by random assignment to 0, 5, or 15 mg of folic acid daily. MTHFR 677 C - + T genotype was measured and the effect of vitamin therapy on hey levels was examined within the genotypes. Results: The distribution of the MTHFR 677 genotype was 52%, 37% and 12% and the mean homocysteine at base line was 21.19 (4.88) 22.38 (4.69) and 29.842 (12.30) #mol/L for the C/C, C/T and T/T genotypes respectively. Patients with the T/T genotype had significantly elevated baseline bcys levels compared to either the CIC and C/T patients (P = 0.008). BI2 therapy reduced the hcys levels by 2.77 (3.20), 3.46 (3.2) and 9.12 (8.35) in the MTHFR C/C, C/T and T/T patients respectively (P = 0.001 T/T vs C/T, C/C). Folate supplementation with 5 mg/15 mg did not further reduce homocysteine level in any of the genotypes (p = 0.35). Serum B12 arid RBC folate levels were not different between groups at baseline or after folate supplementation except that RBC folate levels were significantly elevated in the T f f patients after four weeks of B12 supplementation. Atherosclerosis was present in 29% of the patients and did not differ between the MTHFR 677 genotypes. Conclusion: Patients with the homozygous 677 C--*T MTHFR mutation responded to Bl2 therapy significantly better than those with the C/C or C/T genotypes. Extra supplementation with 5 mg or 15 mg of folic acid did not lower homocysteine levels significantly in any of the MTHFR 677 genotypes compared to the placebo group.
MoP26:W29
] Oxidized low density fipoprotein induces reactive oxigen species in endothelial cells: Effect on the intracelinlar nitric oxide availability [
L. Conainacini, A. Rigoni, U. Garbin, A. Fratta Pasini, A. Davoli, M. Campagnola, M.L. Tosetti. Department of lnternal and Surgical Sciences,
University of Verona, Verona, Italy Objective: The impaired endothelium-dependent relaxation, the earliest alteration in hypertension, is largely due to a reduction in nitric oxide (NO) activity. Oxidized low density/ipoprotein (oxLDL) has been demonstrated to increase reactive oxigen species (ROS) in endothelial cells. Since ROS may reduce NO aim of this study is to evaluate the effect of oxLDL on intracellular ROS formation and NO availability in endothelial cells. Methods: Bovine aortic endothelial cells (BAECs) were incubated with native LDL and 5 /zM Cu++-oxLDL for 5 rain. 2', 7'-dichlorofluorescein
Xllth International Symposium on Atherosclerosis, Stockholm, Sweden, June 25-29, 2000