Epoxyeicosatrienoic acids are part of the VEGF-activated signaling cascade leading to angiogenesis

Anke C. Webler, U. Ruth Michaelis, Rüdiger Popp, Eduardo Barbosa-Sicard, Andiappan Murugan, John R. Falck, Beate Fisslthaler, Ingrid Fleming

Research output: Contribution to journalArticle

80 Citations (Scopus)

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.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Cell Physiology
Volume295
Issue number5
DOIs
StatePublished - Nov 2008

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Vascular Endothelial Growth Factor A
Acids
AMP-Activated Protein Kinases
Endothelial Cells
Cytochrome P-450 Enzyme System
Fibroblast Growth Factor 2
Phosphorylation
Antisense Oligonucleotides
Second Messenger Systems
Arachidonic Acid
Small Interfering RNA
Cell Movement
Signal Transduction
Proteins
Cell Proliferation
cytochrome P-450 CYP2C subfamily
RNA
Messenger RNA

Keywords

  • Cytochrome P-450
  • Matrigel plug assay

ASJC Scopus subject areas

  • Cell Biology
  • Physiology

Cite this

Epoxyeicosatrienoic acids are part of the VEGF-activated signaling cascade leading to angiogenesis. / Webler, Anke C.; Michaelis, U. Ruth; Popp, Rüdiger; Barbosa-Sicard, Eduardo; Murugan, Andiappan; Falck, John R.; Fisslthaler, Beate; Fleming, Ingrid.

In: American Journal of Physiology - Cell Physiology, Vol. 295, No. 5, 11.2008.

Research output: Contribution to journalArticle

Webler, Anke C. ; Michaelis, U. Ruth ; Popp, Rüdiger ; Barbosa-Sicard, Eduardo ; Murugan, Andiappan ; Falck, John R. ; Fisslthaler, Beate ; Fleming, Ingrid. / Epoxyeicosatrienoic acids are part of the VEGF-activated signaling cascade leading to angiogenesis. In: American Journal of Physiology - Cell Physiology. 2008 ; Vol. 295, No. 5.
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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.",
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T1 - Epoxyeicosatrienoic acids are part of the VEGF-activated signaling cascade leading to angiogenesis

AU - Webler, Anke C.

AU - Michaelis, U. Ruth

AU - Popp, Rüdiger

AU - Barbosa-Sicard, Eduardo

AU - Murugan, Andiappan

AU - Falck, John R.

AU - Fisslthaler, Beate

AU - Fleming, Ingrid

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AB - 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.

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