15-Lipoxygenase expression in smooth muscle cells from atherosclerotic rabbit aortas

Abstract

To determine the extent and origin of the stimulation of 15-lipoxygenase activity in atherosclerotic aortas, formation of hydroxy-derivatives from arachidonic acid was measured by HPLC-analysis and 15-lipoxygenase mRNA expression was investigated by RNA blot and in situ hybridization in atherosclerotic and normal rabbit aortic tissues. The synthesis of hydroxy-eicosatetraenoic acids (HETE) from exogenously added [ 14C]arachidonic acid was unchanged in atherosclerotic aortas in comparison with healthy aortas, but pretreatment with indomethacin demonstrated that 15-HETE production resulted essentially (75%) from cyclooxygenase activity in healthy aorta and from lipoxygenase activity in atherosclerotic aorta. The RNA blot and in situ hybridization with radiolabelled oligonucleotide probe demonstrated that 15-lipoxygenase mRNA was strictly localized in intimal thickening of atherosclerotic aortas. The immunostaining using anti-α smooth muscle actin, revealed that smooth muscle cell rich areas of the intimal thickening expressed 15-lipoxygenase mRNA. In addition, RNA blot hybridization indicated that cultured smooth muscle cells from atherosclerotic aortas expressed strongly 15-lipoxygenase mRNA. These results demonstrate that augmentation of 15-lipoxygenase activity in atherosclerotic aortas is correlated with 15-lipoxygenase mRNA expression in atherosclerotic plaque, and that intimal smooth muscle cells were involved, in addition to macrophages, in the expression of 15-lipoxygenase.