Role of Cytochrome P450 2C Epoxygenases in Hypoxia-Induced Cell Migration and Angiogenesis in Retinal Endothelial Cells

• Published in: Investigative ophthalmology & visual science Vol. 49; no. 3; pp. 1242 - 1247
• Main Authors: Michaelis, U. Ruth, Xia, Ning, Barbosa-Sicard, Eduardo, Falck, John R, Fleming, Ingrid
• Format: Journal Article
• Language: English
• Published: Rockville, MD ARVO 01.03.2008; Association for Research in Vision and Ophtalmology
• Subjects: 8,11,14-Eicosatrienoic Acid - analogs & derivatives; 8,11,14-Eicosatrienoic Acid - metabolism; 8,11,14-Eicosatrienoic Acid - pharmacology; Amides - pharmacology; Animals; Biological and medical sciences; Blotting, Western; Cattle; Cell Culture Techniques; Cell Hypoxia; Cell Movement - physiology; Chromatography, High Pressure Liquid; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System - physiology; Eicosanoids - metabolism; Endothelium, Vascular - enzymology; Enzyme Inhibitors - pharmacology; Eye and associated structures. Visual pathways and centers. Vision; Fluorescent Antibody Technique, Indirect; Fundamental and applied biological sciences. Psychology; Mass Spectrometry; Medical sciences; Neovascularization, Physiologic - physiology; Oligonucleotides, Antisense - pharmacology; Ophthalmology; Retinal Vessels - cytology; Sulfaphenazole - pharmacology; Vertebrates: nervous system and sense organs; Angiogenesis; Endothelial cell; Oxygen; Enzyme; Cytochrome P450; Migration; Hypoxia; Retina; Ophthalmology
• ISSN: 0146-0404; 1552-5783; 1552-5783
• DOI: 10.1167/iovs.07-1087

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. 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. 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. Taken together, these data indicate that CYP2C-derived EETs are implicated in angiogenesis by retinal endothelial cells, especially under hypoxic conditions.