Role of cytochrome P450 2C epoxygenases in hypoxia-induced cell migration and angiogenesis in retinal endothelial cells

U. Ruth Michaelis, Ning Xia, Eduardo Barbosa-Sicard, J R Falck, Ingrid Fleming

Research output: Contribution to journalArticle

32 Citations (Scopus)

Abstract

PURPOSE. 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.

Original languageEnglish (US)
Pages (from-to)1242-1247
Number of pages6
JournalInvestigative Ophthalmology and Visual Science
Volume49
Issue number3
DOIs
StatePublished - Mar 2008

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Cytochrome P-450 Enzyme System
Cell Movement
Endothelial Cells
Antisense Oligonucleotides
Sulfaphenazole
cytochrome P-450 CYP2C subfamily
Hypoxia
Cell Hypoxia
Basement Membrane
Retina
Proteins
Western Blotting
Cell Proliferation
Acids
Serum

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Role of cytochrome P450 2C epoxygenases in hypoxia-induced cell migration and angiogenesis in retinal endothelial cells. / Michaelis, U. Ruth; Xia, Ning; Barbosa-Sicard, Eduardo; Falck, J R; Fleming, Ingrid.

In: Investigative Ophthalmology and Visual Science, Vol. 49, No. 3, 03.2008, p. 1242-1247.

Research output: Contribution to journalArticle

Michaelis, U. Ruth ; Xia, Ning ; Barbosa-Sicard, Eduardo ; Falck, J R ; Fleming, Ingrid. / Role of cytochrome P450 2C epoxygenases in hypoxia-induced cell migration and angiogenesis in retinal endothelial cells. In: Investigative Ophthalmology and Visual Science. 2008 ; Vol. 49, No. 3. pp. 1242-1247.
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N2 - PURPOSE. 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.

AB - PURPOSE. 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.

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