Evaluation of antioxidant activity against atherosclerosis using co-culture system

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To analyze in vitro the migration of monocytes to the subendothelial space, their differentiation into macrophages, and the subsequent formation of form cells in vitro, we developed a 2-coculture system with rabbit aortic endothelial cells, aortic smooth muscle cells, and a mixture of matrix proteins on polyethylene folters in chemotaxis chambers. When oxidized low density lipoprotein (LDL) was added to the matrixlayer of coculture, on day 4 transmigrant cells exhibited lipid deposit droplets, and by day 7, they had the appearance of typical foam cells.To follow lipid oxidation of low density lipoprotein andwithin cells we developed a novel method using diphenyl-1-pyrenylphosphine (DPPP) as a fluorescent probe. DPPP reacted with organic hydroperoxides and hydrogen peroxide stoichiometrically to give DPPP oxide (DPPP=O). DPPP incorporated into phosphatidylcholine liposomal membranes and polymorphonuclear leukocytes (PMNs) reacted with methyl linoleate hydroperoxide rapidly but not with … More hydrogen peroxide nor with tert-butyl hydroperoxide. This novel method revealed that lipid peroxidation proceeded within membranes of PMN stimulated by phorbol-12-myristate-13-acetate, which is known to produce several kinds of free radicals. It was found that DPPP is a suitable fluorescent probe to monitor lipid peroxidation within the cell membranes specifically. DPPP incorporated into LDL gave a strong fluo-rescence within macrophage after macrophage took up DPPP-containing LDL.We examined antioxidant activity of 4 isomers of vitamin E, (α- and γ-) tocopherol and tocotrienol, and inhibitory effect on expression of adhesion molecule of human endothelial cell. There were not significant deffereces in antioxidant activity and inhibitory effect on adhesion molecule expression. However, the concentrations of tocotrienols within cell were more than 10 times higher than that of tocopherol due to double bond in side chain of tocotrienol. By taking the defference in the concentrations of these antioxidants into considerations , tocopherols were more efficient in inhibition of the expression of adhesion molecules than tocotrienols. It is interesting to investigate these effects in coculture system we developed. Less