15(S)-HETE production in human retinal microvascular endothelial cells by hypoxia: Novel role for MEK1 in 15(S)-HETE induced angiogenesis.
OBJECTIVE
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.
Bajpai
Arun K
AK
Department of Physiology, University of Tennessee Health Science Center, Memphis, TN 38163, USA.
Blaskova
Eva
E
Pakala
Suresh B
SB
Zhao
Tieqiang
T
Glasgow
Wayne C
WC
Penn
John S
JS
Johnson
Dianna A
DA
Rao
Gadiparthi N
GN
D000256:Adenoviridae / Q000235:genetics; D001093:Arachidonate 15-Lipoxygenase / Q000235:genetics / Q000378:metabolism; D001485:Basement Membrane; D002465:Cell Movement; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D004730:Endothelium, Vascular / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005822:Genetic Vectors; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000096:biosynthesis*; D000860:Hypoxia / Q000378:metabolism*; D048369:MAP Kinase Kinase 1 / Q000502:physiology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism; D048055:Mitogen-Activated Protein Kinase 8 / Q000037:antagonists & inhibitors / Q000378:metabolism; D010766:Phosphorylation; D015861:Retinal Neovascularization / Q000378:metabolism*; D012171:Retinal Vessels / Q000166:cytology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
D004791:Enzyme Inhibitors; D006893:Hydroxyeicosatetraenoic Acids; C025984:15-hydroxy-5,8,11,13-eicosatetraenoic acid; C458528:ALOX15B protein, human; D001093:Arachidonate 15-Lipoxygenase; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048055:Mitogen-Activated Protein Kinase 8; D048051:p38 Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human