title: Epoxyeicosatrienoic acids are part of the VEGF-activated signaling cascade leading to angiogenesis.
abstract: 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.
authors.affiliation: Vascular Signalling Group, Institut für Kardiovaskuläre Physiologie, Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany.
authors.forename: Anke C
authors.initials: AC
authors.lastname: Webler
authors.forename: U Ruth
authors.initials: UR
authors.lastname: Michaelis
authors.forename: Rüdiger
authors.initials: R
authors.lastname: Popp
authors.forename: Eduardo
authors.initials: E
authors.lastname: Barbosa-Sicard
authors.forename: Andiappan
authors.initials: A
authors.lastname: Murugan
authors.forename: John R
authors.initials: JR
authors.lastname: Falck
authors.forename: Beate
authors.initials: B
authors.lastname: Fisslthaler
authors.forename: Ingrid
authors.initials: I
authors.lastname: Fleming
mesh_terms: 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*
chemical_list: 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
references.citation: Nature. 1999 Sep 30;401(6752):493-7
references.pmid: 10519554
references.citation: Hypertension. 2001 Dec 1;38(6):1427-32
references.pmid: 11751730
references.citation: J Biol Chem. 2001 Feb 2;276(5):3222-30
references.pmid: 11058584
references.citation: Invest Ophthalmol Vis Sci. 2008 Mar;49(3):1242-7
references.pmid: 18326754
references.citation: Hypertension. 2000 Aug;36(2):270-5
references.pmid: 10948089
references.citation: J Cell Sci. 2005 Sep 15;118(Pt 18):4103-11
references.pmid: 16118242
references.citation: J Biol Chem. 2005 Feb 11;280(6):4367-73
references.pmid: 15572372
references.citation: J Biol Chem. 1995 Jun 2;270(22):13333-40
references.pmid: 7768934
references.citation: Arterioscler Thromb Vasc Biol. 2007 Dec;27(12):2612-8
references.pmid: 17872452
references.citation: Circ Res. 1996 Mar;78(3):415-23
references.pmid: 8593700
references.citation: Eur J Pharmacol. 1998 Apr 3;346(1):115-7
references.pmid: 9617761
references.citation: Stroke. 2002 Dec;33(12):2957-64
references.pmid: 12468797
references.citation: Nature. 1995 Jul 6;376(6535):62-6
references.pmid: 7596435
references.citation: J Biol Chem. 2004 Jan 9;279(2):1304-9
references.pmid: 14557259
references.citation: Nature. 1995 Jul 6;376(6535):66-70
references.pmid: 7596436
references.citation: Am J Physiol Lung Cell Mol Physiol. 2006 Sep;291(3):L369-77
references.pmid: 16679379
references.citation: J Biol Chem. 1999 Feb 19;274(8):4764-9
references.pmid: 9988714
references.citation: Am J Physiol Cell Physiol. 2007 Mar;292(3):C996-1012
references.pmid: 16987999
references.citation: Am J Physiol Heart Circ Physiol. 2003 Jan;284(1):H215-24
references.pmid: 12388259
references.citation: J Biol Chem. 2002 May 3;277(18):15671-6
references.pmid: 11867622
references.citation: Proc Natl Acad Sci U S A. 2007 Jan 16;104(3):1045-50
references.pmid: 17213310
references.citation: FASEB J. 2003 Apr;17(6):770-2
references.pmid: 12586744
references.citation: Virchows Arch. 1999 May;434(5):401-11
references.pmid: 10389623
references.citation: Biochem Biophys Res Commun. 2007 Mar 23;354(4):1084-8
references.pmid: 17276402
references.citation: Arterioscler Thromb Vasc Biol. 2008 Jun;28(6):1123-9
references.pmid: 18340006
references.citation: J Biol Chem. 1998 Oct 30;273(44):29254-61
references.pmid: 9786938
references.citation: Proc Natl Acad Sci U S A. 2002 Apr 30;99(9):6029-34
references.pmid: 11983897
references.citation: Circ Res. 2002 Dec 13;91(12):1160-7
references.pmid: 12480817
references.citation: Cardiovasc Res. 2008 Jan;77(1):160-8
references.pmid: 18006475
references.citation: Endocr Rev. 1997 Feb;18(1):4-25
references.pmid: 9034784
references.citation: Circ Res. 2002 May 17;90(9):1028-36
references.pmid: 12016270
references.citation: J Biol Chem. 2003 Aug 15;278(33):31000-6
references.pmid: 12788940
references.citation: Med Res Rev. 2003 Nov;23(6):657-68
references.pmid: 12939788
references.citation: J Cell Sci. 2005 Dec 1;118(Pt 23):5489-98
references.pmid: 16291720
references.citation: Pflugers Arch. 2001 Jul;442(4):511-8
references.pmid: 11510882
references.citation: Cancer Res. 2007 Jul 15;67(14):6665-74
references.pmid: 17638876
references.citation: J Biol Chem. 2002 Feb 1;277(5):3101-8
references.pmid: 11719508
references.citation: Arterioscler Thromb Vasc Biol. 2006 Mar;26(3):468-74
references.pmid: 16357318
references.citation: Cell Mol Life Sci. 2006 Mar;63(5):601-15
references.pmid: 16465447
references.citation: Cancer Res. 2005 Jun 1;65(11):4707-15
references.pmid: 15930289
references.citation: Circ Res. 2001 Jan 19;88(1):44-51
references.pmid: 11139472
references.citation: ACS Chem Biol. 2007 Apr 24;2(4):263-70
references.pmid: 17432820
references.citation: Pharmacol Ther. 2006 Sep;111(3):584-95
references.pmid: 16380164