Induction of monocyte binding to endothelial cells by MM-LDL - Role of lipoxygenase metabolites

Treatment of human aortic endothelial cells (EC) with minimally oxidized LDL (or minimally modified LDL, MM-LDL) produces a specific pattern of endothelial cell activation distinct from that produced by LPS, tumor necrosis factor-alpha, and interleukin-1, but similar to other agents that elevate cAMP. The current studies focus on the signal transduction pathways by which MM-LDL activates EC to bind monocytes. We now demonstrate that, in addition to an elevation of cAMP, lipoxygenase products are necessary for the MR I-LDL response. Treatment of EC with inhibitors of the lipoxygenase pathway, 5,8,11,14-eicosatetraynoic acid (ETYA) or cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate (CDC), blocked monocyte binding in MM-LDL-treated EC (MM-LDL = 118 +/- 13%; MM-LDL + ETYA = 33 +/- 4%; MM-LDL + CDC = 23 +/- 4% increase in monocyte binding) without reducing cAMP levels. To further investigate the role of the lipoxygenase pathway, cellular phospholipids were labeled with arachidonic acid. Treatment of cells for 4 hours with 50 to 100 mu g/mL MM-LDL, but not native LDL, caused a 60% increase in arachidonate release into the medium and increased the intracellular formation of 12(S)-HETE (approximate to 100% increase). There was little 15(S)-HETE present, and no increase in its levels was observed. We demonstrated that 12(S)-HETE reversed the inhibitory effect of CDC. We also observed a 70% increase in the formation of 11,12-epoxyeicosatrienoic acid (11,12-EET) in cells treated with MM-LDL. To determine the mechanism of arachidonate release induced by MM-LDL, we examined the effects of MM-LDL on intracellular calcium levels. Treatment of EC with both native LDL and MM-LDL caused a rapid release of intracellular calcium from internal stores. However, several pieces of evidence suggest that calcium release alone does not explain the increased arachidonate release in MM-LDL-treated cells. The present studies suggest that products of 12-lipoxygenase play an important role in MM-LDL action on the induction of monocyte binding to EC.