Ϫ ) react with nitric oxide (NO) to form peroxynitrite (ONOO Ϫ ), a process that limits NO availability, results in NO synthase (NOS) uncoupling, and, through the action of ONOO Ϫ , leads to protein and thiol oxidation as well as tyrosine nitration. 1 Hydrogen peroxide (H 2 O 2 ), the dismutation product of O 2 Ϫ , also elicits multiple effects, among them smooth muscle cell hypertrophy, activation of metalloproteinases, and, in higher concentrations, NOS inhibition by phosphorylation of tyrosine 657 through the redox-activated tyrosine kinase Pyk2. 2 Interestingly, H 2 O 2 also induces positive endothelial effects because it can activate protein kinase-G I␣ by thiol oxidation and subsequent dimerization. 3 Moreover, H 2 O 2 induces as well as activates endothelial NOS (eNOS). 4

Abstract-In the porcine coronary artery, a cytochrome P450 (CYP) isozyme homologous to CYP 2C8/9 has been identified as an endothelium-derived hyperpolarizing factor (EDHF) synthase. As some CYP enzymes are reported to generate reactive oxygen species (ROS), we hypothesized that the coronary EDHF synthase may modulate vascular homeostasis by the simultaneous production of ROS and epoxyeicosatrienoic acids. In bradykinin-stimulated coronary arteries, antisense oligonucleotides against CYP 2C almost abolished EDHF-mediated responses but potentiated nitric oxide (NO)-mediated relaxation. The selective CYP 2C9 inhibitor sulfaphenazole and the superoxide anion (O 2 Ϫ ) scavengers Tiron and nordihydroguaretic acid also induced a leftward shift in the NO-mediated concentration-relaxation curve to bradykinin. CYP activity and O 2 Ϫ production, determined in microsomes prepared from cells overexpressing CYP 2C9, were almost completely inhibited by sulfaphenazole. Sulfaphenazole did not alter the activity of either CYP 2C8, the leukocyte NADPH oxidase, or xanthine oxidase. ROS generation in coronary artery rings, visualized using either ethidium or dichlorofluorescein fluorescence, was detected under basal conditions. The endothelial signal was attenuated by CYP 2C antisense treatment as well as by sulfaphenazole. In isolated coronary endothelial cells, bradykinin elicited a sulfaphenazole-sensitive increase in ROS production. Although 11,12 epoxyeicosatrienoic acid attenuated the activity of nuclear factor-B in cultured human endothelial cells, nuclear factor-B activity was enhanced after the induction or overexpression of CYP 2C9, as was the expression of vascular cell adhesion molecule-1. These results suggest that a CYP isozyme homologous to CYP 2C9 is a physiologically relevant generator of ROS in coronary endothelial cells and modulates both vascular tone and homeostasis. Key Words: coronary artery Ⅲ cytochrome P450 Ⅲ endothelium-derived hyperpolarizing factor Ⅲ NADPH oxidase Ⅲ reactive oxygen species T he bioavailability of nitric oxide (NO) within the vascular wall and, as a consequence, NO-mediated relaxation is attenuated by an elevation in superoxide anion (O 2 Ϫ ) production. 1 Enzymes capable of generating reactive oxygen species (ROS) within the vasculature are, for example, NO synthases (NOS), cyclooxygenases, lipoxygenases, xanthine oxidase, and NADPH oxidase, all of which are reported to be functional in endothelial cells. 2 Diphenyleneiodonium has been used extensively to characterize ROS-generating enzymatic systems; however, this compound inactivates flavoproteins during electron-transfer reactions 3 and completely inhibits the activity of all isoforms of NOS, 3-6 xanthine oxidase, 3,7 and NADPH oxidase. 8 Thus, although pharmacological studies using isolated arteries have demonstrated that the production of ROS is markedly increased in animal models of hypertension, atherosclerosis, and heart failure, 9 the relative contribution of each of the potential O 2 Ϫ -producing enzymes to the overall ROS prod...

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Cytochrome P450 (CYP) epoxygenase products, such as 11,12-epoxyeicosatrienoic acid (EET), stimulate endothelial cell proliferation. We set out to identify the signal transduction cascade linking EET generation to enhanced proliferation and angiogenesis. In human endothelial cells overexpressing CYP 2C9, cell number was increased compared with control cells and was inhibited by the CYP 2C9 inhibitor, sulfaphenazole. CYP 2C9 overexpression was associated with the activation of Akt and an increase in cyclin D1 expression, effects that were abolished by the epidermal growth factor (EGF) receptor inhibitor, AG1478, which also prevented the CYP 2C9-induced increase in cell proliferation. Stimulation of EGF receptor overexpressing cells with 11,12-EET or transfection of these cells with CYP 2C9 enhanced the tyrosine phosphorylation of the EGF receptor. Endothelial tube formation in a fibrin gel was significantly enhanced (6-fold) in CYP 2C9 overexpressing cells and was comparable with the tube formation induced by EGF. In the chick chorioallantoic membrane, 11,12-EET stimulated vessel formation (3.5-fold) and induced vessel convergence, an effect that was abolished by cotreatment with either an EGF receptor-neutralizing antibody or AG1478. These results indicate that CYP 2C9-derived EETs stimulate angiogenesis by a mechanism involving the activation of the EGF receptor.

Valproic acid (VPA) is a widely used antiepileptic agent that is undergoing clinical evaluation for anticancer therapy. We assessed the effects of VPA on angiogenesis in vitro and in vivo. In human umbilical vein endothelial cells, therapeutically relevant concentrations of VPA (0.25 to 1 mM) inhibited proliferation, migration, and tube formation. VPA 1 mM inhibited endothelial cell proliferation by 51 Ϯ 5%, migration by 86 Ϯ 11%, and tube formation by 82 Ϯ 3%. These changes were preceded by the hyperacetylation of histone H4, indicating the inhibition of histone deacetylase (HDAC), and a decreased expression of the endothelial nitric-oxide synthase (eNOS). The inhibition of endothelial cell tube formation by VPA was prevented by addition of the nitric oxide donor (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA NONOate). The anticonvulsive active VPA derivative 2-ethyl-4-methylpentanoic acid, which does not inhibit HDAC, did not affect endothelial cell proliferation, tube formation, or eNOS expression. VPA was also found to inhibit angiogenesis in vivo in the chicken chorioallantoic membrane assay and in a Matrigel plug assay in mice. Embryos from VPA-treated mice showed disturbed vessel formation. These results indicate that therapeutic plasma levels of VPA inhibit angiogenesis by a mechanism involving a decrease in eNOS expression preceded by HDAC inhibition.

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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...

We studied 105 patients who received a total hip arthroplasty between June 1985 and August 2001 using freehand positioning of the acetabular cup. Using pelvic CT scan and the hip-plan module of SurgiGATESystem (Medivision, Oberdorf, Switzerland), we measured the angles of inclination and anteversion of the cup. Mean inclination angle was 45.8°±10.1°(range: 23.0-71.5°) and mean anteversion angle was 27.3°±15.0°(range: −23.5°to 59.0°). We compared the results to the "safe" position as defined by Lewinnek et al. and found that only 27/105 cups were implanted within the limits of the safe position.We conclude that a safe position as defined by Lewinnek et al. [13] was only achieved in a minority of the cups that were implanted freehand.