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Tissue factor induced in rat smooth muscle cells by oxidized low density lipoprotein and lysophosphatidylcholine
Oral session abstracts / Atherosclerosis 115 ( SuppL ) (1995) $3-$42 026 Oxidative Modification of LDL and its Implication in Atherogenesis
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SIMULATION OF LIPID PEROXIDATION IN LOW DENSITY LIPOPROTEIN BY A BASIC "SKELETON" OF REACTIONS P.M. Abuja, H. Esterbauer Institute of Biochemistry, University of Graz, Graz, Austria
OXIDIZED LDL AND ATHEROSCLEROSIS S. Yl~.-Herttula, A.I. Virtanen Institute of Molecular-Sciences, University of Kuopio, P . O B o x 1627, FIN-70211 Kuopio, Finland
A minimal kinetic model describing lipid peroxidation on low-density lipoprotein (LDL) has been set up. Models have been calculated by numeric integration of the differential equations describing this system consisting of seven reactions and eleven reactants in a single compartment. The model describes the usually observed behaviour of the reaction system showing that the crucial intermediate is the lipid peroxyl radical (LOO). During different stages of the reaction, depending on the presence of antioxidants (~-tocopherol), different pathways in the reaction scheme become active. Simulation also demonstrates that tocopherolmediated propagation can occur under certain conditions, i.e. low rate of initiation. This, however, does not mean that tocopherol enhances lipid peroxidation in LDL, as without tocopherol the process would be much faster. Further extension of the basic model by inclusion of a hypothetical antioxidant leads to a model which is capable of describing Cu2+-induced LPO over the whole lag-phase up to full propagation.
Oxidized LDL plays an important role in atherogenesis. LDL oxidation takes place in the arterial wall, where several enzymatic and nonenzymatic systems may contribute to the formation of oxidized LDL. In vitro, 15-1ipoxygenase and NADPH oxidase can catalyze LDL oxidation. Both of these enzymes/enzyme complexes are expressed in atherosclerotic lesions, but are absent or inactive in normal artery wall. Other enzymes which could be involved in LDL oxidation are nitric oxide synthases and myeloperoxidase, both of which have been shown to be expressed in atherosclerotic lesions. Constitutively expressed nitric oxide synthase is also active in normal arteries. Various other enzymes, such as cytochrome P-450 and xanthine oxidase, could contribute to oxidative stress in the arteries, but their role regarding LDL oxidation is uncertain. Enzymes with antioxidative activity include various superoxide dismutases, glutathione peroxidases and catalase, all of which are expressed in normal arteries and in atherosclerotic lesions. Under certain conditions NO radical produced by nitric oxide synthases may also scavenge other radicals. LDL oxidation in the arterial wall is a microenvironment phenomenon. Thus, a local balance between oxidative and antioxidative mechanisms determines a net outcome of the process. However, further studies are needed to characterize anatomical, functional and temporal relationships between various oxidative and antioxidative systems in the developing athernsclerotic lesions.
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TISSUE FACTOR INDUCED IN RAT SMOOTH MUSCLE CELLS BY OXIDIZED LOW DENSITY LIPOPROTEIN AND LYSOPl-IOSPHATIDYLCHOLINE G.M. Cttisolm, M.S. Penn, J. Bethea, P . E DiCorleto, T.A. Hamilton Cleveland Clinic Foundation, Cleveland, Ohio, USA
TARGETING GENE EXPRESSION TO THE VASCULAR WALL IN TRANSGENIC MICE USING THE MURINE PREPROENDOTHELIN-I PROMOTER E. Sigal, H. Kurihara, P. Belloni, G. Cain, R. Lawn, D. Harats Syntex Discovery Research, Palo Alto, CA 94303 USA; Div. of Cardiovascular Med, Stanford Univ Sch of Med, Stanford, CA 94305; Third Dept of Med, Univ of Tokyo, Hongo, Tokyo, 113 Japan
Tissue factor (TF) activates Factor VI1 and ultimately leads to thrombin production, which can promote not only coagulation, but also smooth muscle cell (SMC) proliferation. Influenced by reports of oxidized LDL (oLDL) stimulation of TE in endothelial cells (EC), we tested SMC fbr TF induction, oLDL was a particularly potent inducer of cell surface TF activity in rat aortic SMC, rivaling the potency of stimulation reported for serum and thrombin. As with other stimulants of TF in SMC, TF activity was maximal near 4 hr after oLDL. Steady state mRNA levels were increased to maximum levels by 60-90 min. We examined whether the action of oLDL was due to known oLDL constituents, 7aOH-. 7BOH- and 7 keto cholesterol and lysophosphatidylcholine (lysoPC). Of these, only lysoPC induced TF mRNA and cell surface activity. The induction of TF did not result from mortal cell injury, since cell number measured at 20 hr after a 4 hr exposure to oEDL did not decrease. Interestingly, utocopherol pretreatmem of the cells inhibited the TF induction by both oLDL and lysoPC, but not that induced by serum or thrombin. TF induction in SMC by oLDE constituents may increase risk of vascular lesion complications caused by enhanced thrombosis after plaque fissure or enhanced thrombin mediated SMC proliferation. Funded by NIH (HL 47852).
To develop a system for overexpressing genes in the vascular wall, we created transgenic mice using the murine preproendothelin-1 promoter and the reporter gene luciferase. In vitro analysis suggested that the murine 5'-flanking region contained endothelial-specific elements in a 5.9 kb fragment. Five transgenic mice colonies established from independent founders all exhibited the highest level of luciferase activity in the aorta with up to 8,540 light units per gg protein. Immunohistochemistry with anti-luciferase antisera revealed high levels of expression in the endothelial cells of both large and small arteries and lower levels of expression in veins and capillaries. Significant expression was also seen in arterial smooth muscle cells and in select epithelial surfaces which is consistent with the known distribution of endothelin-1 in mammals. To further demonstrate the targeting capability of this system, we overexpressed the lipidperoxidating enzyme, human 15-1ipoxygenase, in the vessel wall of transgenic mice. As with luciferase, expression of active enzyme and immunohistochemical localisation in vascular cells were documented in transgenic animals. Hence, this new system can be used to direct expression of molecules to the vascular wall for the purpose of examining the biological significance of either overexpression or inhibition of select proteins, and to study their role in atherosclerosis and other vascular diseases.
$41
153
155
SIMULATION OF LIPID PEROXIDATION IN LOW DENSITY LIPOPROTEIN BY A BASIC "SKELETON" OF REACTIONS P.M. Abuja, H. Esterbauer Institute of Biochemistry, University of Graz, Graz, Austria
OXIDIZED LDL AND ATHEROSCLEROSIS S. Yl~.-Herttula, A.I. Virtanen Institute of Molecular-Sciences, University of Kuopio, P . O B o x 1627, FIN-70211 Kuopio, Finland
A minimal kinetic model describing lipid peroxidation on low-density lipoprotein (LDL) has been set up. Models have been calculated by numeric integration of the differential equations describing this system consisting of seven reactions and eleven reactants in a single compartment. The model describes the usually observed behaviour of the reaction system showing that the crucial intermediate is the lipid peroxyl radical (LOO). During different stages of the reaction, depending on the presence of antioxidants (~-tocopherol), different pathways in the reaction scheme become active. Simulation also demonstrates that tocopherolmediated propagation can occur under certain conditions, i.e. low rate of initiation. This, however, does not mean that tocopherol enhances lipid peroxidation in LDL, as without tocopherol the process would be much faster. Further extension of the basic model by inclusion of a hypothetical antioxidant leads to a model which is capable of describing Cu2+-induced LPO over the whole lag-phase up to full propagation.
Oxidized LDL plays an important role in atherogenesis. LDL oxidation takes place in the arterial wall, where several enzymatic and nonenzymatic systems may contribute to the formation of oxidized LDL. In vitro, 15-1ipoxygenase and NADPH oxidase can catalyze LDL oxidation. Both of these enzymes/enzyme complexes are expressed in atherosclerotic lesions, but are absent or inactive in normal artery wall. Other enzymes which could be involved in LDL oxidation are nitric oxide synthases and myeloperoxidase, both of which have been shown to be expressed in atherosclerotic lesions. Constitutively expressed nitric oxide synthase is also active in normal arteries. Various other enzymes, such as cytochrome P-450 and xanthine oxidase, could contribute to oxidative stress in the arteries, but their role regarding LDL oxidation is uncertain. Enzymes with antioxidative activity include various superoxide dismutases, glutathione peroxidases and catalase, all of which are expressed in normal arteries and in atherosclerotic lesions. Under certain conditions NO radical produced by nitric oxide synthases may also scavenge other radicals. LDL oxidation in the arterial wall is a microenvironment phenomenon. Thus, a local balance between oxidative and antioxidative mechanisms determines a net outcome of the process. However, further studies are needed to characterize anatomical, functional and temporal relationships between various oxidative and antioxidative systems in the developing athernsclerotic lesions.
154
156
TISSUE FACTOR INDUCED IN RAT SMOOTH MUSCLE CELLS BY OXIDIZED LOW DENSITY LIPOPROTEIN AND LYSOPl-IOSPHATIDYLCHOLINE G.M. Cttisolm, M.S. Penn, J. Bethea, P . E DiCorleto, T.A. Hamilton Cleveland Clinic Foundation, Cleveland, Ohio, USA
TARGETING GENE EXPRESSION TO THE VASCULAR WALL IN TRANSGENIC MICE USING THE MURINE PREPROENDOTHELIN-I PROMOTER E. Sigal, H. Kurihara, P. Belloni, G. Cain, R. Lawn, D. Harats Syntex Discovery Research, Palo Alto, CA 94303 USA; Div. of Cardiovascular Med, Stanford Univ Sch of Med, Stanford, CA 94305; Third Dept of Med, Univ of Tokyo, Hongo, Tokyo, 113 Japan
Tissue factor (TF) activates Factor VI1 and ultimately leads to thrombin production, which can promote not only coagulation, but also smooth muscle cell (SMC) proliferation. Influenced by reports of oxidized LDL (oLDL) stimulation of TE in endothelial cells (EC), we tested SMC fbr TF induction, oLDL was a particularly potent inducer of cell surface TF activity in rat aortic SMC, rivaling the potency of stimulation reported for serum and thrombin. As with other stimulants of TF in SMC, TF activity was maximal near 4 hr after oLDL. Steady state mRNA levels were increased to maximum levels by 60-90 min. We examined whether the action of oLDL was due to known oLDL constituents, 7aOH-. 7BOH- and 7 keto cholesterol and lysophosphatidylcholine (lysoPC). Of these, only lysoPC induced TF mRNA and cell surface activity. The induction of TF did not result from mortal cell injury, since cell number measured at 20 hr after a 4 hr exposure to oEDL did not decrease. Interestingly, utocopherol pretreatmem of the cells inhibited the TF induction by both oLDL and lysoPC, but not that induced by serum or thrombin. TF induction in SMC by oLDE constituents may increase risk of vascular lesion complications caused by enhanced thrombosis after plaque fissure or enhanced thrombin mediated SMC proliferation. Funded by NIH (HL 47852).
To develop a system for overexpressing genes in the vascular wall, we created transgenic mice using the murine preproendothelin-1 promoter and the reporter gene luciferase. In vitro analysis suggested that the murine 5'-flanking region contained endothelial-specific elements in a 5.9 kb fragment. Five transgenic mice colonies established from independent founders all exhibited the highest level of luciferase activity in the aorta with up to 8,540 light units per gg protein. Immunohistochemistry with anti-luciferase antisera revealed high levels of expression in the endothelial cells of both large and small arteries and lower levels of expression in veins and capillaries. Significant expression was also seen in arterial smooth muscle cells and in select epithelial surfaces which is consistent with the known distribution of endothelin-1 in mammals. To further demonstrate the targeting capability of this system, we overexpressed the lipidperoxidating enzyme, human 15-1ipoxygenase, in the vessel wall of transgenic mice. As with luciferase, expression of active enzyme and immunohistochemical localisation in vascular cells were documented in transgenic animals. Hence, this new system can be used to direct expression of molecules to the vascular wall for the purpose of examining the biological significance of either overexpression or inhibition of select proteins, and to study their role in atherosclerosis and other vascular diseases.