Epoxyeicosatrienoic acids are part of the VEGF-activated signaling cascade leading to angiogenesis.
Cytochrome P-450 (CYP) epoxygenases metabolize arachidonic acid to epoxyeicosatrienoic acid (EET) regioisomers, which activate several signaling pathways to promote endothelial cell proliferation, migration, and angiogenesis. Since vascular endothelial growth factor (VEGF) plays a key role in angiogenesis, we assessed a possible role of EETs in the VEGF-activated signal transduction cascade. Stimulation with VEGF increased CYP2C promoter activity in endothelial cells and enhanced CYP2C8 mRNA and protein expression resulting in increased intracellular EET levels. VEGF-induced endothelial cell tube formation was inhibited by the EET antagonist 14,15-epoxyeicosa-5(Z)-enoicacid (14,15-EEZE), which did not affect the VEGF-induced phosphorylation of its receptor or basic fibroblast growth factor (bFGF)-stimulated tube formation. Moreover, VEGF-stimulated endothelial cell sprouting in a modified spheroid assay was reduced by CYP2C antisense oligonucleotides. Mechanistically, VEGF stimulated the phosphorylation of the AMP-activated protein kinase (AMPK), which has also been linked to CYP induction, and the overexpression of a constitutively active AMPK mutant increased CYP2C expression. On the other hand, a dominant-negative AMPK mutant prevented the VEGF-induced increase in CYP2C RNA and protein expression in human endothelial cells. In vivo (Matrigel plug assay) in mice, endothelial cells were recruited into VEGF-impregnated plugs; an effect that was sensitive to 14,15-EEZE and the inclusion of small interfering RNA directed against the AMPK. The EET antagonist did not affect responses observed in plugs containing bFGF. Taken together, our data indicate that CYP2C-derived EETs participate as second messengers in the angiogenic response initiated by VEGF and that preventing the increase in CYP expression curtails the angiogenic response to VEGF.
Webler
Anke C
AC
Vascular Signalling Group, Institut für Kardiovaskuläre Physiologie, Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany.
Michaelis
U Ruth
UR
Popp
Rüdiger
R
Barbosa-Sicard
Eduardo
E
Murugan
Andiappan
A
Falck
John R
JR
Fisslthaler
Beate
B
Fleming
Ingrid
I
D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology; D055372:AMP-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D002478:Cells, Cultured; D065727:Cytochrome P-450 CYP2C8; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D004790:Enzyme Induction; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D051379:Mice; D018919:Neovascularization, Physiologic / Q000187:drug effects; D016376:Oligonucleotides, Antisense / Q000378:metabolism; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D040262:Receptors, Vascular Endothelial Growth Factor / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013552:Swine; D013997:Time Factors; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*
C494022:14,15-eicosa-5-enoic acid; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D016376:Oligonucleotides, Antisense; D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A; C400322:cytochrome P-450 CYP2C subfamily; C046783:11,12-epoxy-5,8,14-eicosatrienoic acid; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; C450259:CYP2C8 protein, human; D065727:Cytochrome P-450 CYP2C8; D040262:Receptors, Vascular Endothelial Growth Factor; D055372:AMP-Activated Protein Kinases; D015126:8,11,14-Eicosatrienoic Acid
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