Role of cytochrome P450 2C epoxygenases in hypoxia-induced cell migration and angiogenesis in retinal endothelial cells.
OBJECTIVE
Cytochrome P450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids (EETs) elicit cell proliferation and promote angiogenesis. The aim of this study was to determine the expression of CYP epoxygenases in the bovine retina and the potential role of EETs in hypoxia-induced angiogenesis in bovine retinal endothelial cells.


METHODS
Bovine retinal endothelial cells were cultured under normoxic (21% O(2)) or hypoxic (1% O(2)) conditions, and CYP2C expression was determined by Western blot analysis. The effect of hypoxia on EET levels was determined by LC-MS/MS. Cell migration (Transwell filter assays) and endothelial cell tube formation (on basement membrane matrix) were assessed in vitro in the absence and presence of pharmacologic inhibitors and CYP2C antisense oligonucleotides.


RESULTS
Bovine retinal endothelial cells expressed CYP2C protein in culture and generated detectable levels of EETs under basal conditions. Hypoxia (6-48 hours) enhanced CYP2C protein expression (2-fold) and EET formation (1.5-fold). Moreover, endothelial cells preexposed to hypoxia demonstrated an increase in serum-induced cell migration that was sensitive to the CYP2C inhibitors sulfaphenazole and MS-PPOH and the EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid. Furthermore, preventing the hypoxia-induced expression of CYP2C (antisense oligonucleotides) suppressed hypoxia-induced cell migration. In an in vitro angiogenesis model, the preexposure of endothelial cells to hypoxia increased CYP2C expression and enhanced endothelial tube formation, which was blocked by the EET antagonist and by the CYP2C antisense oligonucleotides.


CONCLUSIONS
Taken together, these data indicate that CYP2C-derived EETs are implicated in angiogenesis by retinal endothelial cells, especially under hypoxic conditions.
Michaelis
U Ruth
UR
Institut für Kardiovaskuläre Physiologie, Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany.
Xia
Ning
N
Barbosa-Sicard
Eduardo
E
Falck
John R
JR
Fleming
Ingrid
I
D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D000577:Amides / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D018929:Cell Culture Techniques; D015687:Cell Hypoxia; D002465:Cell Movement / Q000502:physiology*; D002851:Chromatography, High Pressure Liquid; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000502:physiology*; D015777:Eicosanoids / Q000378:metabolism; D004730:Endothelium, Vascular / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D013058:Mass Spectrometry; D018919:Neovascularization, Physiologic / Q000502:physiology*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D012171:Retinal Vessels / Q000166:cytology*; D013426:Sulfaphenazole / Q000494:pharmacology
D000577:Amides; D065607:Cytochrome P-450 Enzyme Inhibitors; D015777:Eicosanoids; D004791:Enzyme Inhibitors; C121756:N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide; D016376:Oligonucleotides, Antisense; C400322:cytochrome P-450 CYP2C subfamily; D013426:Sulfaphenazole; C046782:14,15-epoxy-5,8,11-eicosatrienoic acid; D003577:Cytochrome P-450 Enzyme System; D015126:8,11,14-Eicosatrienoic Acid