Cytochrome P450 2C9-induced angiogenesis is dependent on EphB4.
OBJECTIVE
Cytochrome P450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids (EETs) are known to stimulate angiogenesis, but the mechanisms involved are incompletely understood. Because EphB4 is involved in vascular development, the aim of this study was to investigate whether, and to what extent, EphB4 is part of the signaling cascade that results in CYP2C9-mediated angiogenesis.


RESULTS
CYP2C9 overexpression as well as stimulation with 11,12-EET (up to 48 hours) time-dependently increased EphB4 expression in endothelial cells. This effect and the activation of the EphB4 promoter were mediated by the phosphatidylinositol-3-kinase (P13-K)/Akt pathway and sensitive to the P13-K inhibitor LY 294002 as well as to simultaneous transfection with dominant-negative Akt. 11,12-EET treatment also increased EphB4 expression in isolated mouse mesenteric arteries as well as in the vessels that developed in 11,12-EET-impregnated Matrigel plugs. Moreover, the CYP2C9-stimulated formation of capillary-like structures in a modified spheroid assay was markedly attenuated by EphB4 downregulation (antisense oligonucleotides). Using a parallel approach in vivo, the inclusion of siRNA directed against EphB4 in EET-impregnated Matrigel plugs prevented endothelial cell invasion and vascularization.


CONCLUSIONS
Our data indicate that EphB4 is a critical component of the CYP2C9- and 11,12-EET-activated signaling cascade that promotes angiogenesis in vitro as well as in vivo.
Webler
Anke C
AC
Vascular Signalling Group, Institut für Kardiovaskuläre Physiologie, Johann Wolfgang Goethe-Universität, Frankfurt, Germany.
Popp
Rüdiger
R
Korff
Thomas
T
Michaelis
U Ruth
UR
Urbich
Carmen
C
Busse
Rudi
R
Fleming
Ingrid
I
D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001011:Aorta / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008815:Mice, Inbred Strains; D009025:Morpholines / Q000494:pharmacology; D018919:Neovascularization, Physiologic / Q000235:genetics / Q000502:physiology*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D036224:Receptor, EphB4 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology*; D013552:Swine; D014471:Umbilical Veins / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D014665:Vasodilator Agents / Q000494:pharmacology
D002867:Chromones; D004791:Enzyme Inhibitors; D009025:Morpholines; D016376:Oligonucleotides, Antisense; D034741:RNA, Small Interfering; D014665:Vasodilator Agents; C400322:cytochrome P-450 CYP2C subfamily; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C046783:11,12-epoxy-5,8,14-eicosatrienoic acid; D003577:Cytochrome P-450 Enzyme System; D019869:Phosphatidylinositol 3-Kinases; D036224:Receptor, EphB4; D051057:Proto-Oncogene Proteins c-akt; D015126:8,11,14-Eicosatrienoic Acid