JCS ePress online publication date 15 November 2005

Research Article

5489

Cytochrome P450 epoxygenases 2C8 and 2C9 are

implicated in hypoxia-induced endothelial cell
migration and angiogenesis
U. Ruth Michaelis1, Beate Fisslthaler1, Eduardo Barbosa-Sicard1, John R. Falck2, Ingrid Fleming1,* and
Rudi Busse1
1

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

*Author for correspondence (e-mail: fleming@em.uni-frankfurt.de)

Journal of Cell Science

Accepted 5 September 2005

Journal of Cell Science 118, 5489-5498 Published by The Company of Biologists 2005
doi:10.1242/jcs.02674

Summary
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 2C8/9derived 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-

Key words: Angiogenesis, Epoxyeicosatrienoic acid, 14,15Epoxyeicosa-5(Z)-enoic acid, Hypoxia, Matrix degradation

Introduction
Cytochrome P450 (CYP) epoxygenases of the 2B, 2C and 2J
subfamilies are expressed in endothelial cells and metabolize
arachidonic acid to four regio-isomeric epoxides (5,6-, 8,9-,
11,12- and 14,15-epoxyeicosatrienoic acid or EETs). CYP 2J
enzymes seem to be constitutively expressed, whereas the
expression of CYP 2C isoforms can be induced by a number
of different pharmacological and mechanical stimuli (for a
review, see Fleming, 2001). Interest in the vascular actions of
EETs was initially linked to their identification as endotheliumderived hyperpolarizing factors (EDHFs) (Campbell et al.,
1996; Fisslthaler et al., 1999), but it is now generally
appreciated that these compounds mediate a number of
membrane-potential-independent effects. The consequences of
an increased intracellular production of EETs range from
changes in the intracellular concentration of Ca2+ (Graier et al.,
1995), and the activation of protein kinase A (Imig et al., 1999)
and tyrosine kinases and phosphatases (Hoebel and Graier,
1998; Fleming et al., 2001), to the activation of MAP kinase
phosphatases and inhibition of the c-Jun N-terminal kinase
(Potente et al., 2002). Although some of these responses may
result from the direct interaction of an EET with the target

molecule; for example the binding of EETs to the TrpV4

channel may account for effects on Ca2+ signaling (Watanabe
et al., 2003), some of the consequences of increased EET
production can be attributed to the transactivation of the
epidermal growth factor (EGF) receptor (Chen et al., 2002;
Michaelis et al., 2003). Indeed, the release of heparin-binding
EGF-like growth factor from the endothelial cell surface and
the subsequent activation of EGF-receptor-dependent signaling
have been suggested to underlie the proliferative and
angiogenic effects of CYP-derived EETs (Michaelis et al.,
2003).
Of the CYP 2C epoxygenase family, CYP 2C8 and 2C9 are
expressed in native human endothelial cells (Lin et al., 1996;
Hillig et al., 2003). However, the expression of these enzymes
(mRNA and protein) decreases rapidly following cell isolation,
which means that to assess the consequences of CYP 2C
activation in cultured cells, it is necessary to either induce CYP
expression pharmacologically or to transfect the cells with an
expression vector encoding the isoform of interest. To achieve
high transfection efficiencies, adenoviral vectors are generally
used but although this approach facilitates the elucidation of
EET-dependent signaling, it does not accurately reflect a

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.

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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

Journal of Cell Science

Materials
Matrigel 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 culture
Human 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,
0.4%), penicillin (50 U/ml) and streptomycin (50 g/ml). First or
second passage endothelial cells were used throughout. Porcine
coronary artery endothelial cells were isolated and cultured as described
previously (Popp et al., 1996). Where appropriate, hypoxic conditions
were achieved by incubating cells in an air-tight Heraeus incubator
(Hanau, Germany) with 5% CO2 and 1% O2 balanced with N2.
Adenoviral infection and transfection
Endothelial cells (80-90% confluent) were serum-starved for 24 hours
prior to infection with adenoviral vectors. Cells were incubated with
recombinant adenoviruses (10 pfU/cell) expressing CYP 2C9 sense or
antisense cDNA in medium without antibiotics for 4 hours at 37C
followed by recovery in the presence of 2% FCS. The infection
efficiency was between 90 and 100% and, as reported previously
(Michaelis et al., 2005), endothelial cells infected with the CYP 2C9
sense adenovirus generated approximately twofold more 11,12- and
14,15-EET under basal conditions than cells infected with the control
(CYP 2C9 antisense) virus and the increase in EET production was
sensitive to the CYP 2C9 inhibitor, sulfaphenazole.
In some experiments, an antisense oligonucleotide approach was
used to prevent the hypoxia-induced expression of CYP 2C8/9;
HUVEC were treated with the oligonucleotides (2 mol/l) using
GeneTrans II according to the manufacturers protocol (MobiTec,
Gttingen, Germany) with CYP 2C sense and antisense
oligonucleotides (antisense, 5-TCC ATT GAA GCC TTC TCT TCT
T-3; sense, 5-AAG AAG AGA AGG CTT CAA TGG A-3, in both
cases the three 5 nucleotides were modified with phosphothioate;
MWG-Biotech, Ebersberg, Germany). The sequence of these
oligonucleotides spans the ATG and is 100% identical with CYP 2C8
and contains one mismatch to the other three human CYP 2C
isoforms. As a consequence of the high homology between the 2C
enzymes the oligonucleotides used were not able to differentiate
between isoforms.
LC-MS/MS measurements
Following exposure to hypoxia for 16 hours, endothelial cells were

cultured for a further 2 hours in normoxic conditions in the absence

or presence of MS-PPOH. Thereafter, the cells were harvested by
scraping and the cell pellets (approximately 8106 cells) were
suspended in 100 l of 0.2 M KCl and 100 l methanol. The cells
were hydrolyzed for 2 hours using NaOH (0.5 N), then neutralized
with HCl (2M) and deuterated internal standards (5-HETE-d8, 12HETE-d8, 15-HETE-d8, 20-HETE-d6, 8,9-EET-d8, 11,12-EET-d8 and
14,15-EET-d8) were added. Liquid-liquid extraction was performed
twice using 1 ml hexane/ethyl acetate (1:1). After evaporation of the
solvent under a gentle stream of nitrogen in a vacuum block, residues
were reconstituted with 50 l methanol/water (1:1, v/v) and
determined with a Sciex API4000 mass spectrometer operating in
multiple reaction monitoring (MRM) mode. Chromatographic
separation was performed on a Gemini C18 column (1502 mm I.D.,
5 m particle size; Phenomenex, Aschaffenburg, Germany).
Reporter gene assay
Endothelial cells were transiently co-transfected with the non-coding
5 region (2088 to +21, kindly provided by P. Maurel, Montpellier,
France) of CYP 2C9 subcloned into pGL3basic (Promega) and
pcDNA3.1myc-His/LacZ (Invitrogen, Karlsruhe, Germany). After 2
hours, the cells were exposed to either normoxia or hypoxia for 60
minutes. Thereafter, the cells were lysed, and luciferase and
galactosidase activities were assayed according to the manufacturers
protocols (Promega, Mannheim, Germany and Applied Biosystems,
Darmstadt, Germany). Promoter activity was determined as luciferase
activity relative to that of galactosidase. Preliminary experiments
using an empty vector demonstrated that luciferase production from
the vector construct was not influenced by hypoxia per se.
Isolation of RNA and RT-PCR
Total RNA was isolated from cultured endothelial cells using phenol
and guanidine isothiocyanate (TriReagenz, Sigma, Germany).
Random hexanucleotide primers were used for reverse transcription
of equal amounts of RNA. The cDNA was used for real-time PCR
using Taqman probes for the detection of the specific amplification
products. The oligonucleotides used were derived from the human
CYP 2C8 sequence (2C8 forward, 5-GGACTTTATCGATTGCTTCCTG-3; reverse, 5-CCATATCTCAGAGTGGTGCTTG-3; FAM
and dabcyl-labeled Taqman probe, 5-TTGGCACTGTAGCTGATCTATTTGTTGCTGGA-3). To ensure equal amounts of cDNA were
used, the 18S RNA was amplified (Assay on Demand, Applied
Biosystems) and the amount of cDNA in the samples was calculated
on the basis of the amplification of a serial dilution of a plasmid (CYP
2C8) or the serial dilution of the cDNA (18S RNA). The CYP 2C8
levels were normalized to that of 18S. At least two RT reactions were
performed using each RNA preparation and at least two PCR reactions
were performed with each cDNA sample.
Immunofluorescence
Cells were treated as described in the Results section, fixed in
phosphate-buffered formaldehyde solution (4%), permeabilized using
Triton X-100 (0.2%) and blocked with bovine serum albumin solution
(3%) and horse serum (5%) in phosphate-buffered saline. CYP 2C
was detected using a specific polyclonal CYP 2C antibody (kindly
provided by E. Morgan, Atlanta, GA) and HIF 1 using a monoclonal
antibody (BD Transduction Laboratories). The cells were then washed
and incubated with fluorescent secondary antibodies (Alexa),
mounted and images were acquired by laser-scanning microscopy
(LSM 510 meta, Carl Zeiss, Jena, Germany).
Cell migration and invasion assay
Endothelial cell migration was investigated using a modified
Transwell chamber system. After pre-treatment of the cells as

CYP 2C9 in angiogenesis

described in the Results section, endothelial cells were counted and
seeded on membrane inserts (FluoroBlok, 3 m pore size, BD
Bioscience, Heidelberg, Germany) in the presence of MCDB 131
supplemented with 0.1% BSA. The lower chamber contained MCDB
131 supplemented with 4% FCS, L-glutamine (10 mmol/l), basic
fibroblast growth factor (0.5 ng/ml), epidermal growth factor (0.05
ng/ml) and endothelial cell growth-stimulating factor from bovine
brain (ECGS/H, 0.2%). After 20 hours, the cells on the upper surface
of the filter were removed mechanically and cells that had migrated
into the lower compartment were fixed (4% paraformaldehyde in
PBS), stained with DAPI and counted (10 images per well, 30
magnification). Invasion assays were performed in the same manner
but using Matrigel-coated membrane inserts with a pore size of 8 m
(BD Bioscience, Heidelberg, Germany).

Journal of Cell Science

Rac activity assay

Endothelial cells were treated as described in the Results section and
Rac activity was determined using a Rac-Pak pull-down assay
according to the manufacturers instructions (Upstate Biotechnology,
Lake Placid, NY).
MT-MMP activity assay
Endothelial cells were infected with CYP 2C9 sense or antisense
adenoviruses and cultured with or without sulfaphenazole (30 mol/l)
for 24 hours. Crude membrane fractions were prepared by repeated
cycles of freezing and thawing and MT-MMP activity assessed using

5491

a commercially available kit (Chemicon International, Temecula,

CA).
Zymography
Zymography was performed as described (Bouloumi et al., 2001)
using myelin basic protein (MBP) polymerized in an SDS gel as the
substrate. Cell supernatants were separated by SDS-PAGE, and
following renaturation of the proteins with 2.5% Triton-X100, the gels
were incubated for 22 hours in a buffer containing Tris-HCl (50
mmol/l), CaCl2 (5 mmol/l) and NaN3 (0.02%). Gels were then stained
with Coomassie Blue and MBP degradation assessed
densitometrically.
In vitro angiogenesis assays
Fibrin gels were prepared using thrombin (0.5 U/l)-polymerized
fibrinogen (1.5 mg/ml in MCDB 131). Endothelial cells were infected
with CYP 2C9 sense or antisense adenoviruses 24 hours after seeding
onto the fibrin gels and cultured as described above, in the absence or
presence of 14,15-EEZE (10 mol/l). Total tube length was
determined after 14 days.
To investigate the effect of hypoxia-induced CYP 2C expression on
tube formation, cells were cultured in normoxic or hypoxic conditions
for 24 hours in the absence and presence of 14,15-EEZE (10 mol/l).
Thereafter, the cells were seeded onto Matrigel (3104 cells/cm2) and
cultured under normoxic or hypoxic conditions, in the absence and
presence of 14,15-EEZE. Tube formation was assessed after 14 hours
and quantified by counting the number of
branch points.
Chick chorioallantoic membrane
assay
All experiments with chick embryos were
performed in ovo. A window (7-10 mm in
diameter) was cut into the eggshell of 3day-old
embryos,
resealed
with
transparent film, and incubated for a
further 6 days. For the chorioallantoic
membrane (CAM) assay, solvent or
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 mol/l), on the hypoxia-induced
increase in 11,12-EET and 11,12-DHET
formation. The bar graphs show the
means.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 m.

5492

Journal of Cell Science 118 (23)

substances described in the Results section were mixed with a 1%

methylcellulose solution. Aliquots (10 l) of the resulting 0.5%
methylcellulose solution were applied to bacteriological-grade Petri
dishes, air dried for 1 hour and then placed onto the 9-day-old CAM.
Two discs were placed on each CAM ~10-20 mm apart. Eggs were
then incubated under normoxic or hypoxic (14% O2) conditions for 3
days. To better visualize the vascular system of the CAM, Intralipid
was injected underneath the membrane and 20% Luconyl Black
(BASF, Ludwigshafen, Germany) in PBS was injected into a vitelline
vein. Photographs were taken using a Nikon SMZ1000
stereomicroscope. The angiogenic response was quantified as
described (Brunner et al., 2000).

Journal of Cell Science

Statistics
Data are expressed as the means.e.m. Statistical analyses were
performed by one-way ANOVA followed by Bonferronis multiple
comparison test. Values of P<0.05 were considered statistically
significant.

Results
Effect of hypoxia on CYP 2C expression and EET
formation
To assess the effects of hypoxia on CYP 2C8/9 expression we
measured the activity of a reporter construct as well as levels
of endogenous CYP 2C RNA and protein. Exposure of
HUVEC, transfected with a CYP 2C9 promoter-luciferase
construct, to hypoxia (1% O2) for 60 minutes elicited a
significant increase (approximately fivefold) in the activity of
the reporter gene construct (Fig. 1A).
In non-transfected cells, the endogenous expression of CYP
2C8 RNA (Fig. 1B) and CYP 2C protein (Fig. 1C) was timedependently increased following exposure to hypoxia. The
increase in CYP 2C RNA was rapid and reached an elevated
steady-state level after approximately 4 hours. Increased
protein levels were evident after approximately 8 hours and
remained elevated over the entire observation period (up to 24
hours). Hypoxia was also associated with a marked increase in
the expression of HIF 1. The increase in CYP 2C expression
was associated with an elevation in EET production. The
hypoxia (16 hours)-induced increase in intracellular levels of
11,12-EET and 11,12-dihydroxyeicosatrienoic acid (11,12DHET) was inhibited in the presence of the CYP epoxygenase
inhibitor MS-PPOH (Fig. 1D). No effect of hypoxia on the
formation of the other EET regioisomers (5,6-, 8,9- or 14,15EET) was detected (data not shown).
Effect of CYP 2C8/9 on endothelial cell migration
To determine the consequences of an increase in endogenous
CYP 2C8/9 expression and activity on endothelial cell
migration, experiments were performed using endothelial cells
pre-exposed to hypoxia (24 hours) to increase CYP 2C
expression as well as with CYP 2C antisense oligonucleotides.
The hypoxia-induced expression of CYP 2C was unaffected by
sense oligonucleotides but was significantly attenuated in cells
treated with antisense oligonucleotides (Fig. 2A). As reported
previously
(Fisslthaler
et
al.,
1999),
scrambled
oligonucleotides had no effect on CYP expression (data not
shown). Endothelial cell migration under normoxic conditions
tended to be slightly enhanced following incubation with the
antisense oligonucleotides although this effect did not achieve

Fig. 2. Effect of CYP 2C antisense oligonucleotides on the hypoxiainduced 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 mol/l) and the EET antagonist 14,15-epoxyeicosa-5(Z)
enoic acid (EEZE, 10 mol/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 (means.e.m.); *P<0.05, **P<0.01 vs levels in the
normoxia controls in the presence of solvent.

statistical significance. However, treatment with the antisense

oligonucleotides significantly attenuated endothelial cell
migration in cells pre-exposed to hypoxia (Fig. 2B).
Corresponding to a role for a CYP-dependent potentiation of
endothelial cell migration under hypoxia, the migration of cells

CYP 2C9 in angiogenesis

Journal of Cell Science

maintained under normoxic conditions was not significantly

affected by either the epoxygenase inhibitor MS-PPOH or the
EET antagonist 14,15-EEZE whereas the migration of
endothelial cells pretreated with hypoxia was significantly
attenuated by both substances (Fig. 2C).

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 mol/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 means.e.m. of eight to ten
independent experiments; **P<0.01 vs the level in the control (CTL).

5493

To exclude additional effects of hypoxia on endothelial cells

and to elucidate the specific role of CYP 2C8/9 on endothelial
cell migration, extracellular matrix degradation and
angiogenesis, we used endothelial cells infected with an
adenovirus coding for CYP 2C9. Although CYP 2C8 was
endogenously expressed in human endothelial cells we chose
to overexpress CYP 2C9 (the EET profile generated by CYP
2C8 and 2C9 are similar), as its activity can be inhibited by
sulfaphenazole (Mancy et al., 1996). Human endothelial cells
were infected with adenoviruses expressing CYP 2C9 mRNA
in the sense or antisense orientation and seeded on Transwell
filters. More of the CYP-2C9-overexpressing cells had
migrated to the lower portion of the filter after 20 hours than
cells treated with the control (CYP 2C9 antisense) adenovirus.
The CYP-2C9-induced increase in endothelial cell migration
was inhibited by sulfaphenazole (Fig. 3A,B).
Effect of CYP 2C9 overexpression on Rac and MMP
activity and on cell invasion
As cell migration is generally accompanied by an increase in
Rac activity, we investigated the effect of CYP 2C9 on Rac
using a Rac-PAK pull-down assay. Overexpression of CYP
2C9 markedly increased the association of Rac with PAK-1, an
effect that was sensitive to treatment with sulfaphenazole (Fig.
4A). The exogenous application of the CYP 2C8/9 product
11,12-EET, increased Rac activity to a similar extent as
vascular endothelial growth factor (VEGF) (Fig. 4B).
We next determined the effect of CYP 2C9 on MMP activity
using a commercially available MT-MMP assay. MMP activity
was markedly increased in membrane fractions isolated from
CYP-2C9-overexpressing endothelial cells and was inhibited
by sulfaphenazole (Fig. 5A). MMP activity was also assessed
by zymography using MBP as a substrate. Enhanced
degradation of MBP by a protein with the apparent molecular
mass of ~150 kDa was readily apparent in the lanes containing
the supernatant from CYP-2C9-overexpressing cells (Fig. 5B).
The time-dependent increase in MMP activity was also evident
in cells treated with 11,12-EET (1 mol/l) for 4-8 hours (Fig.
5C).
To determine the consequence of CYP 2C9 overexpression
on endothelial cell invasion, HUVEC were seeded on Matrigelcoated Transwell filters and migration was monitored after 24
hours. Significantly more CYP-2C9-overexpressing cells were
detected in the lower chamber than cells treated with the
control virus. This CYP-2C9-dependent induction of cell

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-1bound 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 mol/l) on
Rac activity 24 hours after infection.
(B) Representative western blot and bar graph
showing the effect of 11,12-EET (1 mol/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 (means.e.m.);
*P<0.05 vs the level in the control (CTL).

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Journal of Cell Science 118 (23)

Journal of Cell Science

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 mol/l) for 24 hours. MMP activity was
determined using a commercially available MMP
activity assay. Results are presented as the
means.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 mol/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).

invasion was sensitive to both sulfaphenazole, and the EETantagonist 14,15-EEZE (Fig. 6A). The non-selective MMP
inhibitor GM 6001 completely abolished CYP-2C9-induced
cell invasion (Fig. 6B).
Effect of 14,15-EEZE on CYP-2C9-induced in vitro
angiogenesis
To determine the effect of 14,15-EEZE on CYP-2C9-induced
angiogenesis, we monitored tube formation in a fibrin gel.
After 14 days, the formation of capillary-like structures was
apparent in cultures containing CYP-2C9-overexpressing cells,
but not in those cells infected with the control (antisense) virus

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 m).
Twenty-four hours after seeding the
migrated cells were counted. Effects of
(A) sulfaphenazole (Sulfa, 30 mol/l)
and 14,15-epoxyeicosa-5(Z)-enoic acid
(EEZE, 10 mol/l) and (B) the nonselective matrix metalloprotease inhibitor
GM 6001 (GM, 30 nmol/l) on CYP-2C9induced 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 computerassisted microscope. The bar graphs
summarize data (means.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).

or CYP-2C9-overexpressing endothelial cells cultured in the

presence of 14,15-EEZE (Fig. 6C).
To further assess the role of CYP epoxygenases in hypoxiainduced angiogenesis, we determined the effects of 14,15EEZE on endothelial cell tube formation under normoxic and
hypoxic conditions. Human endothelial cells were cultured in
the absence or presence of 14,15-EEZE (24 hours) before being
seeded on Matrigel and cultured for an additional 14 hours
either under normoxic or hypoxic conditions. Although tubelike structures were evident after 14 hours in CYP-expressing
endothelial cells treated with solvent (0.01% DMSO), 14,15EEZE completely prevented tube formation under hypoxic
conditions. The EET antagonist, which did not promote

CYP 2C9 in angiogenesis

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Journal of Cell Science

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 mol/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.

endothelial cell apoptosis or compromise cell viability (data

not shown), did not influence tube formation in cells
maintained under normoxic conditions (Fig. 7).
To demonstrate that the hypoxia-induced increase in CYP
2C expression and CYP-2C-dependent formation of
endothelial cell tubes was not a phenomenon restricted to
human umbilical vein endothelial cells, additional experiments
were performed using porcine coronary artery endothelial
cells. Hypoxia also induced a time-dependent increase in CYP
2C protein in porcine endothelial cells (Fig. 8A), which was
associated with an increase in endothelial cell migration
through Transwell filters (Fig. 8B). The latter effect was
attributed to an increase in CYP activity as the effect was
prevented in the presence of 14,15-EEZE and MS-PPOH.
Hypoxia also potentiated the formation of tube-like structures
by porcine endothelial cells and again this effect was abrogated
by the EET antagonist and the epoxygenase inhibitor (Fig. 8C).
Effect of 14,15-EEZE on hypoxia-induced angiogenesis
in vivo
The CAM assay was used to assess the effect of 14,15-EEZE
on hypoxia-induced angiogenesis in vivo. CAMs were treated
with discs containing either solvent or 14,15-EEZE and
incubated under normoxic or hypoxic
(14% O2) conditions. Hypoxia increased
CAM vessel density compared with that
recorded in eggs maintained in
normoxia and was sensitive to the EETantagonist 14,15-EEZE (Fig. 9).

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 timedependent 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 mol/l) or MS-PPOH (MS, 10 mol/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 means.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.

Discussion
The results of the present investigation
demonstrate that in two different types
of endothelial cells CYP 2C8/9
expression is upregulated by hypoxia
and that the subsequent endothelial cell
migration and tube formation are
inhibited by an EET antagonist and a
CYP 2C inhibitor as well as by
downregulating CYP 2C8/9 expression
using antisense oligonucleotides. The
EET antagonist also abolished hypoxiainduced angiogenesis in the chick
chorioallantoic membrane in vivo. As
such, these data provide the first
indication that CYP 2C8/9 is involved in
the process of hypoxia-induced
angiogenesis. Indeed, CYP-2C9-derived
EETs induce endothelial cell migration,
activate metalloproteases and initiate the
degradation of the extracellular matrix.

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Journal of Cell Science

CYP 2C8/9 epoxygenases are expressed in native

endothelial cells but RNA and protein levels decrease rapidly
following cell isolation. Although the expression of CYP
2C8/9 in cultured cells can be restored by different stimuli, the
mechanisms involved in its regulation are not well understood
(for a review, see Fleming, 2004). The expression of several
CYP enzymes is modulated by changes in oxygen tension, for
example, hypoxia downregulates CYP 2J2 (Marden et al.,
2003) whereas transient cerebral ischemia elicits the
upregulation of CYP 2C11 in rats (Alkayed et al., 2002).
Furthermore, the exposure of rats to hypoxia for 48 hours
induces the expression of a sulfaphenazole-sensitive CYP
epoxygenase, most probably CYP 2C11 (Earley et al., 2003).
As the promoter regions of several CYP 2C genes contain
multiple HIF 1 sites, we analyzed the effect of hypoxia on
CYP 2C8 and 2C9 expression in cultured endothelial cells. We
found that hypoxia increased the activity of the CYP 2C9

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 mol/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 (means.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.

promoter and enhanced the expression of CYP 2C8/9 mRNA

and protein in human and porcine endothelial cells. Not only
was CYP 2C8/9 expression increased by hypoxia but an EET
antagonist prevented tube formation in endothelial cells
maintained under hypoxic conditions, while leaving cells
cultured in a normoxic environment, which express negligible
amounts of CYP 2C8/9, completely unaffected.
CYP-derived EETs are now recognized as intracellular
signal transduction molecules exerting different effects on a
variety of cell types including the activation of kinase cascades
as well as the modulation of gene expression (Fleming, 2001).
In endothelial cells, CYP 2C9 overexpression and 11,12-EET
application induce proliferation and angiogenesis (Michaelis et
al., 2003) and an angiogenic effect of EETs derived from
astrocytes has also been described (Munzenmaier and Harder,
2000; Zhang and Harder, 2002). As the process of angiogenesis
involves endothelial cell migration and the degradation of the
extracellular matrix in addition to cell proliferation, we
assessed the effects of CYP 2C upregulation on these
processes. Endothelial cell migration under control conditions
(i.e. uninfected and cultured under normoxic conditions) was
insensitive to interference with EET generator or effector
pathways. However, the migration of hypoxia-treated cells was
attenuated by an epoxygenase inhibitor and an EET antagonist.
Moreover, treatment of these cells with CYP 2C antisense
oligonucleotides to prevent the hypoxia-induced upregulation
of CYP 2C8/9 expression also markedly attenuated endothelial
cell migration, indicating that CYP 2C-derived EETs are able
to modulate endothelial cell migration. To exclude other effects
elicited by hypoxia, we repeated the same experiments using
endothelial cells infected with an adenoviral CYP 2C9
expression vector and found that the overexpression of CYP
2C9 was associated with a marked increase in endothelial cell
migration. Our results contrast with the effects of EETs on
smooth muscle cell migration because 11,12-EET has been
reported to inhibit the PDGF-induced migration of rat aortic
smooth muscle cells (Sun et al., 2002). However as EETs as
well as an inhibitor of the soluble epoxide hydrolase (which
increases EET levels) effectively prevented the PDGFstimulated proliferation and expression of cyclin D1 in human
vascular smooth muscle cells (Davis et al., 2002), it seems that
EETs exert opposing effects on endothelial cell and vascular
smooth muscle cell proliferation and migration.
Endothelial cell migration is a complex process during
which the cells need to dissolve old contacts with the
subcellular matrix at the same time as forming new contacts at
the leading edge of the cell. GTPases of the Rho family play
an important role in the regulation of the restructuring of the
actin cytoskeleton and in the process of migration (Ridley,
2001). The results of the present investigation show that CYP2C9-induced endothelial cell migration is accompanied by the
activation of Rac and its association with the downstream
effector kinase PAK-1. The latter serine/threonine kinase has
previously been associated with disassembly of focal adhesions
and actin stress fibers (Frost et al., 1998). Other simultaneously
activated processes are also implicated in endothelial cell
migration. We previously reported that CYP-2C9-induced
endothelial cell proliferation is accompanied by the attenuated
expression of p27Kip 1 (Potente et al., 2003). However, in
addition to its role in cell cycle regulation, this cyclindependent kinase inhibitor possesses cell-cycle-independent

Journal of Cell Science

CYP 2C9 in angiogenesis

functions and inhibits cellular changes that normally occur
during cell locomotion (e.g. lamellipodia formation and
reorganization of actin filaments and focal adhesions) most
probably by binding to RhoA, thereby inhibiting its activation
(Diez-Juan and Andres, 2003; Besson et al., 2004). It is
therefore possible that the downregulation of p27Kip 1 also
contributes to the migratory effect of CYP 2C9 reported here.
To migrate, cells must also be able to degrade the
extracellular matrix, a process that involves a number of
different metalloproteases (Chang and Werb, 2001). In the
present study, we have shown that metalloprotease activity is
enhanced in CYP-2C9-overexpressing cells as well as in cells
treated with the CYP 2C9 product, 11,12-EET. Moreover,
CYP-2C9-overexpressing cells migrated through a Matrigelcoated filter faster than CYP-deficient cells. Although we did
not identify the metalloprotease activated by 11,12-EET, it is
probably a member of the ADAM (a disintegrin and
metalloprotease) family, because different ADAMs have been
reported to degrade the MBP used as a substrate in the
zymography experiments (Moss et al., 1997) and elicit the
release of HB-EGF from endothelial cells (Asakura et al.,
2002; Hinkle et al., 2004), which is another consequence of
enhanced CYP 2C9 activity (Michaelis et al., 2003).
Since the first proposal that CYP-derived metabolites of
arachidonic acid may be linked to vascular smooth muscle
hyperpolarisation and relaxation, it has become clear that the
arachidonic acid epoxides are important intracellular signaling
molecules that modulate much more than membrane potential.
Indeed it is likely that the importance of EETs in vascular
homeostasis has been largely underestimated because of the
labile nature of the EET-forming enzymes in cell culture and
the lack of appropriate experimental models and tools. This
also means that the contribution of CYP-derived products in
the vast majority of the experimental models based on cell
culture systems to address topics related to vascular
signaling/homeostasis and angiogenesis has been overlooked.
In addition to the data obtained showing that CYP-derived
EETs enhance endothelial cell migration and MMP activity in
an overexpression system, our results showing that hypoxia
increases CYP 2C8/9 expression in cultured endothelial cells
and that hypoxia-induced endothelial tube formation in vitro
and angiogenesis in vivo can be prevented by an EET
antagonist highlight the importance of EETs as signaling
molecules in the angiogenic process.
The authors are indebted to Isabel Winter for expert technical
assistance. This work was partially supported by Philip Morris, the
Deutsche Forschungsgemeinschaft (FI 830/2-2), the National Institute
of Health (NIH GM31278, to J.R.F.), by a Young Investigators Grant
(to U.R.M.) from the Medical Faculty of the Johann-WolfgangGoethe-Universitt and the Marie Christine Held und Erika Hecker
Stiftung.

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