15(S)-HETE production in human retinal microvascular endothelial cells by hypoxia: Novel role for MEK1 in 15(S)-HETE-Induced angiogenesis

PURPOSE. To examine for the expression of 15-lipoxygenase 1 ( 15-LOX1) and 15-LOX2 in human retinal microvascular endothelial cells ( HRMVECs) and study the role of arachidonic acid metabolites of these enzymes in angiogenesis. METHODS. Quantitative RT-PCR and reverse-phase HPLC analyses were used to determine 15-LOX1/2 expression and their arachidonic acid metabolites in HRMVECs. The role of MEK1 in 15( S)-HETE-induced angiogenesis was studied using HRMVEC migration, tube formation, and basement membrane matrix plug angiogenesis. RESULTS. HRMVECs expressed both 15-LOX1 and 15-LOX2. Hypoxia induced the expression of 15-LOX1 and the production of its arachidonic acid metabolites 15( S)-hydroxyeicosatetraenoic acid ( 15( S)-HETE) and 12( S)-hydroxyeicosatetraenoic acid ( 12( S)-HETE). 15( S)-HETE stimulated HRMVEC migration and tube formation as potently as 20 ng/mL fibroblast growth factor-2 ( FGF-2). In addition, 15( S)-HETE stimulated the phosphorylation of ERK1/2, JNK1, p38 MAPK, and MEK1 in a time-dependent manner in these cells. Inhibition of MEK1 by pharmacologic and dominant-negative mutant approaches attenuated 15( S)-HETE-induced phosphorylation of ERK1/2 and JNK1 but not p38 MAPK. Blockade of ERK1/2 and JNK1 activation suppressed 15( S)-HETE-induced HRMVEC migration and tube formation and basement membrane matrix plug angiogenesis. Inhibition of p38 MAPK attenuated 15( S)-HETE induced HRMVEC migration only. Inhibition of MEK1 also blocked 15( S)-HETE-induced HRMVEC migration and tube formation and basement membrane matrix plug angiogenesis. CONCLUSIONS. These results suggest that hypoxia, through the induction of 15-LOX1 expression, leads to the production of 15( S)-HETE in HRMVECs. In addition, 15( S)-HETE, through MEK1-dependent activation of ERK1/2 and JNK1, stimulates the angiogenic differentiation of HRMVECs and basement membrane matrix plug angiogenesis.