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First published online 15 November 2005
doi: 10.1242/jcs.02674

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

¹ Institut für Kardiovaskuläre Physiologie, Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany
² Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-9038, USA

View larger version (54K): [in a new window]   Fig. 1. Effect of hypoxia on CYP 2C expression and EET formation in human endothelial cells. (A) Human umbilical vein endothelial cells were transfected with the CYP 2C9 promoter construct and then incubated under normoxic or hypoxic (1% O2) conditions for 1 hour. (B) Time-dependent effect of hypoxia on the expression of CYP 2C mRNA in human endothelial cells as assessed by RT-PCR. (C) Western blot and immunocytochemistry showing the effect of hypoxia on CYP 2C protein levels (green). Expression of HIF-1 (red) is shown as a positive control. Similar data were obtained in two additional experiments. (D) Effect of the epoxygenase inhibitor MS-PPOH (MS, 10 µmol/l), on the hypoxia-induced increase in 11,12-EET and 11,12-DHET formation. The bar graphs show the mean±s.e.m. of three to six independent experiments; *P<0.05, **P<0.01 vs levels in cells treated with solvent under normoxia (N). Bar, 100 µm.

View larger version (21K): [in a new window]   Fig. 2. Effect of CYP 2C antisense oligonucleotides on the hypoxia-induced expression of CYP 2C and endothelial cell migration. (A,B) Human endothelial cells were treated with sense or antisense (As) CYP 2C oligonucleotides and either maintained under normoxic conditions or exposed to hypoxia (1% O2) for 24 hours. Thereafter, either CYP 2C protein expression was determined (A) or cells were seeded onto Transwell filters and cells that migrated were counted after 20 hours (B). (C) Effect of the CYP 2C inhibitor MS-PPOH (MS, 10 µmol/l) and the EET antagonist 14,15-epoxyeicosa-5(Z) enoic acid (EEZE, 10 µmol/l) on the migration of human umbilical vein endothelial cells pre-exposed to hypoxia (1% O2, 24 hours) to increase CYP 2C expression. Sol, solvent-only control. The bar graphs summarize the results from three to five independent experiments (mean±s.e.m.); *P<0.05, **P<0.01 vs levels in the normoxia controls in the presence of solvent.

View larger version (36K): [in a new window]   Fig. 3. Effect of CYP 2C9 overexpression on endothelial cell migration. Human endothelial cells were infected with CYP 2C9 sense (2C9) or antisense (CTL) adenoviruses, seeded onto Transwell filters and cell migration was assessed after 20 hours. (A) Representative images of the migrated endothelial cells stained with DAPI and (B) a bar graph summarizing the effect of sulfaphenazole (Sulfa, 30 µmol/l) on CYP-2C9-induced cell migration. The western blot shows CYP 2C9 expression in human umbilical vein endothelial cells after infection with CYP 2C9 antisense (CTL) or sense (2C9) adenoviral vectors. The bar graph shows the mean±s.e.m. of eight to ten independent experiments; **P<0.01 vs the level in the control (CTL).

View larger version (23K): [in a new window]   Fig. 4. Rac-Pak pull-down assay showing the effect of CYP 2C9 and 11,12-EET on Rac activity in human endothelial cells. The amount of PAK-1-bound active Rac was determined by western blotting and compared with the amount of total Rac determined in the whole cell lysates. (A) Effect of CYP 2C9 and sulfaphenazole (Sulfa, 30 µmol/l) on Rac activity 24 hours after infection. (B) Representative western blot and bar graph showing the effect of 11,12-EET (1 µmol/l, 30 minutes) and VEGF (30 ng/ml, 30 minutes) on Rac activity. The bar graphs summarize the results of three to five independent experiments (mean±s.e.m.); *P<0.05 vs the level in the control (CTL).

View larger version (28K): [in a new window]   Fig. 5. Effect of CYP 2C9-overexpression on matrix metalloprotease (MMP) activity. (A) Human endothelial cells were infected with either CYP 2C9 sense (2C9) or antisense (CTL) adenoviruses in the absence or presence of sulfaphenazole (Sulfa, 30 µmol/l) for 24 hours. MMP activity was determined using a commercially available MMP activity assay. Results are presented as the mean±s.e.m. of data obtained in five to seven independent experiments. Zymography using the supernatant of the cells (B) infected with CYP 2C9 sense (2C9) or antisense (CTL) adenoviruses and cultured for 48 hours or (C) treated with 11,12-EET (1 µmol/l) for the indicated times and myelin basic protein (MBP) as a substrate. The bar graph summarizes the results of five independent experiments; *P<0.05 vs the level in the control (CTL).

View larger version (58K): [in a new window]   Fig. 6. Effect of CYP 2C9 overexpression on endothelial cell invasion and tube formation. Human endothelial cells infected with CYP 2C9 sense (2C9) or antisense (CTL) viruses were seeded on Matrigel-coated Transwell filters (pore size 8 µm). Twenty-four hours after seeding the migrated cells were counted. Effects of (A) sulfaphenazole (Sulfa, 30 µmol/l) and 14,15-epoxyeicosa-5(Z)-enoic acid (EEZE, 10 µmol/l) and (B) the non-selective matrix metalloprotease inhibitor GM 6001 (GM, 30 nmol/l) on CYP-2C9-induced endothelial cell invasion. (C) Representative photographs and bar graph showing the effect of EEZE on tube formation by human endothelial cells infected with CYP 2C9 sense (2C9) or antisense (CTL) adenoviruses and seeded on fibrin gels. Tube formation was assessed after 14 days and total tube length calculated using a computer-assisted microscope. The bar graphs summarize data (mean±s.e.m.) obtained in four to five independent experiments; *P<0.05, **P<0.01, ***P<0.001 vs levels in the respective controls (CTL).

View larger version (61K): [in a new window]   Fig. 7. Effect of 14,15-EEZE on tube formation under hypoxic conditions. Human endothelial cells were cultured in the absence or presence of 14,15-EEZE (EEZE, 10 µmol/l) under either normoxic or hypoxic conditions for 14 hours before branching points were counted. The bar graph summarizes the results of four independent experiments; **P<0.01 vs the level in solvent-treated cells (Sol) maintained in normoxia.

View larger version (27K): [in a new window]   Fig. 8. Effect of hypoxia on CYP 2C expression, migration and tube formation by porcine coronary artery endothelial cells. (A) Representative western blot showing the time-dependent induction of CYP 2C by hypoxia. Porcine liver served as a positive control (pc). (B) Endothelial cells were cultured under normoxic or hypoxic conditions in the absence or presence of 14,15-EEZE (EEZE, 10 µmol/l) or MS-PPOH (MS, 10 µmol/l) for 24 hours and then seeded onto Transwell filters. The number of cells that had migrated through the filters was counted after 20 hours. (C) Endothelial cells, cultured under normoxic or hypoxic conditions, were seeded onto Matrigel and branching points were counted after 14 hours. The bar graphs show the mean±s.e.m. of three to four independent experiments; *P<0.05, **P<0.01 vs the level in solvent-treated cells (Sol) maintained in normoxia.

View larger version (48K): [in a new window]   Fig. 9. Effect of 14,15-EEZE on hypoxia-induced angiogenesis in vivo in the chick chorioallantoic membrane. Methylcellulose discs containing solvent (0.03% DMSO) or 14,15-EEZE (30 µmol/l) were placed on 9-day-old CAMs. After 3 days of incubation under normoxic or hypoxic (14% O2) conditions vessels were stained and photographed and the vessel density calculated. The dashed line on the representative images marks the border of the discs. The bar graph summarizes the results (mean±s.e.m.) obtained from eight eggs per group; **P<0.01 vs the level in solvent-treated eggs (Sol) maintained in hypoxia; *P<0.05 vs the level in solvent-treated eggs maintained in normoxia.