Cytochrome P450 epoxygenases 2C8 and 2C9 are implicated in hypoxia-induced endothelial cell migration and angiogenesis

Abstract

Recent studies suggest that cytochrome P450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids (EETs) elicit cell proliferation and promote angiogenesis. The aim of this study was to determine the role of CYP 2C8/9-derived EETs in the process of angiogenesis under hypoxic conditions. In human endothelial cells, hypoxia enhanced the activity of the CYP 2C9 promoter, increased the expression of CYP 2C mRNA and protein and augmented 11,12-EET production. In Transwell assays, the migration of endothelial cells pre-exposed to hypoxia to increase CYP expression was abolished by CYP 2C antisense oligonucleotides as well as by the CYP inhibitor MS-PPOH and the EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (EEZE). Similar findings were obtained in porcine coronary artery endothelial cells. CYP 2C9 overexpression in endothelial cells increased the association of PAK-1 with Rac, a response also elicited by the CYP 2C9 product 11,12-EET. Matrix metalloprotease (MMP) activity was increased in CYP-2C9-overexpressing cells and correlated with increased invasion through Matrigel-coated Transwell chambers: an effect sensitive to the CYP 2C9 inhibitor sulfaphenazole as well as to EEZE and the MMP inhibitor GM6001. In in vitro angiogenesis models, the EET antagonist inhibited tube formation induced by CYP 2C9 overexpression as well as that in endothelial cells exposed to hypoxia to increase CYP 2C expression. Furthermore, in the chick chorioallantoic membrane assay, EEZE abolished hypoxia-induced angiogenesis. Taken together, these data indicate that CYP 2C-derived EETs significantly affect the sequence of angiogenic events under hypoxic conditions. Journal of Cell Science 5490 physiological/pathophysiological response. The aim of the present investigation was to determine the effects of a potent angiogenic stimulus (hypoxia) on the expression of CYP 2C protein in endothelial cells and to further elucidate the mechanisms by which CYP 2C-derived EETs promote the degradation of the extracellular matrix and endothelial cell migration: two essential steps in the process of angiogenesis. Materials and Methods MaterialsMatrigel was from BD Biosciences, 11,12-EET was purchased from Cayman Chemicals (Massy, France), KT 5720 and AG 1478 were from Calbiochem (Darmstadt, Germany) and thrombin was from Haemochrom Diagnostica GmbH (Essen, Germany). MS-PPOH and 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) were synthesized as described (Gauthier et al., 2002). Sulfaphenazole, myelin basic protein, the antibody recognizing ␤-actin and all other chemicals were from Sigma. Cell cultureHuman umbilical vein endothelial cells (HUVEC) were either isolated as described (Busse and Lamontagne, 1991) or purchased from Cell Systems/Clonetics (Solingen, Germany). Cells were cultured in MCDB 131 (Gibco Life Technology, Karlsruhe, Germany), supplemented with 8% fetal calf serum (FCS), L-glutamine (10 mmol/l), basic fibroblast growth factor (1 ng/ml), epidermal growth factor (0.1 ng/ml), endothelial cell growth-stimulating factor from bovine brain (ECGS/H...

Hepatic Cytochrome P450 Enzyme Alterations in Humans with Progressive Stages of Nonalcoholic Fatty Liver Disease

“…Several studies have shown that CYP2C9 activity is increased during hypoxia, potentiating metabolism of arachidonic acid into 11,12-epoxyeicosatrienoic acid (Michaelis et al, 2005). 11,12-Epoxyeicosatrienoic acid in turn attenuates vascular smooth muscle cell hyperpolarization and resultant vasoconstriction during acute and chronic hypoxic conditions (Earley et al, 2003).…”

Müller glia cells regulate Notch signaling and retinal angiogenesis via the generation of 19,20-dihydroxydocosapentaenoic acid

“…1:2). The eicosanoid profiles generated were determined with a mass spectrometer (API4000; AB Sciex) operating in multiple reaction monitoring (MRM) mode as previously described (Michaelis et al, 2005;Frömel et al, 2012). Chromatography was performed on a Gemini C18 column (150 mm length, 2 mm inner diameter; particle size, 5 µm; Phenomenex).…”

Inhibition of arachidonic acid metabolism and its implication on cell proliferation and tumour-angiogenesis

“…In addition, it was shown to affect mitogen-activated protein kinase phosphatase-1 (MKP-1) mediated inactivation of c-Jun N-terminal kinase (JNK), which ultimately leads to the expression of cyclin D1 and cell proliferation [89]. In addition, there is evidence that EETs not only elicit cell proliferation but also promote neo-angiogenesis under hypoxia-induced enhanced activity of CYP 450 epoxygenase [90]. In addition, 14,15-EET was found to inhibit apoptosis by a PI3/Akt signalling pathway [91].…”