© 2001 American Heart Association, Inc.
Molecular Medicine |
Endothelium-Derived Hyperpolarizing Factor Synthase (Cytochrome P450 2C9) Is a Functionally Significant Source of Reactive Oxygen Species in Coronary Arteries
From the Institut für Kardiovaskuläre Physiologie (I.F., U.R.M., D.B., B.F., R.P.B., R.B.) and Institut für Anatomie II (F.D.), Klinikum der J.W.G.-Universität, Frankfurt, Germany.
Correspondence to Ingrid Fleming, PhD, Institut für Kardiovaskuläre Physiologie, Klinikum der J.W.G.-Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany. E-mail fleming{at}em.uni-frankfurt.de
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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 (O2-) scavengers Tiron and nordihydroguaretic acid also induced a leftward shift in the NO-mediated concentration-relaxation curve to bradykinin. CYP activity and O2- 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 • cytochrome P450 • endothelium-derived hyperpolarizing factor • NADPH oxidase • reactive oxygen species
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Introduction |
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The bioavailability of nitric oxide (NO) within the vascular wall and, as a consequence, NO-mediated relaxation is attenuated by an elevation in superoxide anion (O2-) 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 reactions3 and completely inhibits the activity of all isoforms of NOS,3 4 5 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 O2--producing enzymes to the overall ROS production remains to be elucidated.
In the coronary system, NO inhibits the activity of the cytochrome P450 (CYP)-like endothelium-derived hyperpolarizing factor (EDHF) synthase. A decrease in the bioavailability of NO, as demonstrated in various states associated with endothelial dysfunction, alleviates this intrinsic inhibition10 so that the activity of the EDHF synthase and the production of vasodilator epoxyeicosatrienoic acids (EETs) are increased. As a consequence of this interaction, vascular responsiveness is thought to be at least partially maintained despite the apparent loss of NO.
Interest in the consequences of vascular CYP expression has focused on the vascular effects of EET production, and little attention has been paid to the fact that O2-, hydrogen peroxide, and hydroxyl radicals can also be generated during the CYP reaction cycle when the electrons for the reduction of the central heme iron are transferred on the activated bound oxygen molecule.11 12 13 Therefore, it is conceivable that CYP epoxygenases, which have recently been detected in coronary endothelial cells,14 15 16 17 may contribute to the generation of oxygen-derived free radicals within the vascular wall. The aim of the present investigation was to determine whether the putative coronary EDHF synthase CYP 2C915 is a physiologically relevant source of ROS.
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Materials and Methods |
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Materials
OxyBURST green H2HFF BSA (oxyBURST) and 2',7' dichlorodihydrofluorescein diacetate (H2DCF-DA) were from Molecular Probes, DMEM-F12 and M-199 medium from GIBCO, and dibenzylfluorescein from NatuTec. Diphenyleneiodonium was from Alexis, and 11,12-EET was purchased from Cayman Chemical. All other chemicals were obtained from Sigma.
Preparation and Transfection of Porcine
Coronary Arteries
Porcine epicardial artery segments (
40 mm length;
mean external diameter 2.4 to 2.8 mm) were excised, side branches were
sealed with surgical clips, and the segment was cannulated at both ends
and placed into vessel chambers. After equilibration, MEM containing
10% FCS, 2.5 µg/mL FITC-labeled antisense or sense oligonucleotides,
and 20 µL/mL transfection reagent (Superfect, QIAGEN) were applied
luminally and left in contact for 3 hours under a maintained transmural
pressure of 90 mm Hg. Thereafter, coronary arteries were perfused (5
mL/h, 37°C) with MEM containing 2% FCS for 18 to 20 hours. After
incubation, segments were cut into rings for organ chamber studies and
precontracted with U46619 (0.1 to 1 µmol/L), and the relaxation to
bradykinin was determined in the presence of diclofenac (10 mmol/L), as
described elsewhere.10
Antisense oligonucleotides derived from the cDNA sequences of human CYP
2C8 and 2C9 (5'-GAGGAGTGGGGCCAGGAGGGAG-3') were used to modulate the
expression of 2C protein, as described
elsewhere.15
Measurement of CYP Activity
The activity of CYP 2C8 and 2C9 was determined by the
dealkylation of the CYP 2C8/9 substrate dibenzylfluorescein (DBF) to
fluorescein by supersomes isolated from baculovirus-infected insect
cells overexpressing the respective enzyme together with the P450
reductase and cytochrome b5 (Gentest
Corporation). Microsomes (1 pmol/mL P450) were incubated in
KH2PO4/K2HPO4
(50 mmol/L, pH 7.4), containing MgCl2 (1
mmol/L), NADPH (1 mmol/L), isocitrate (10 mmol/L), isocitrate
dehydrogenase (0.2 U/mL), and DBF (2 µmol/L) for 30 minutes at
37°C. After terminating the reaction by the addition of NaOH (500
mmol/L final concentration), the amount of fluorescein generated was
assessed using a microplate fluorescence reader
(Victor2, Wallac
Distribution GmbH).
Measurement of Oxygen-Derived Free Radical
Production
Measurement of oxygen-derived free radical production
by supersomes isolated from cells overexpressing CYP 2C8 and 2C9 by a
purified preparation of xanthine oxidase and by isolated human
leukocytes, which contain the NADPH oxidase, was determined by
chemiluminescence18 using
the recently described Cypridina
hilgendorfii luciferin analogue derivative
6-(4-methoxy-phenylethyny)-2-methyl-7H-imidazo[1,2-a]pyrazin-3-one
(compound 5, 5 µmol/L)19
(provided by Dr O. Shimomura, Woods Hole, Mass).
ROS production by CYP 2C8/9-containing supersomes was
assessed in a spectrofluorometer under the same conditions as described
for ROS measurement using oxyBURST (
Ex: 498, Em: 528
nm).20
ROS production in isolated porcine coronary artery endothelial cells was determined by means of dichlorofluorescein (DCF) fluorescence in cells loaded with (H2DCF-DA (5 µmol/L) using a confocal microscope (Zeiss). Changes in DCF fluorescence were quantified as changes in pixel intensity and normalized relative to the signal obtained under basal (unstimulated) conditions.
Oxidative Fluorescent Microtopography
The redox-sensitive fluorophore hydroethidine was
used to evaluate the production of
O2- in situ. Rings
of porcine coronary artery were frozen in OTC Tissue Tek (Sakura), cut
into 20-µm-thick sections, and placed on a glass slide. Hydroethidine
(10 µmol/L) was applied topically to each section before sealing with
a coverslip as described.21
After a 30-minute incubation period, during which hydroethidine was
oxidized to the fluophore ethidium, images were obtained using an
imaging system ( Ex: 520, Em: 605 nm;
Attofluor).
Transfection of Cultured Endothelial
Cells
Porcine coronary artery endothelial cells were
prepared as described
elsewhere,22 and human
umbilical vein endothelial cells for transfection were purchased from
Cell Systems/Clonetics. Subconfluent cells (80% confluent) were
exposed for 4 hours to 2.63 µg/mL plasmid DNA (pcDNA3.1) containing
the coding region of CYP 2C9 under the control of a
cytomegalovirus promoter. Thereafter, cells were maintained in
medium containing 4% FCS for an additional 48 hours. The expression of
CYP 2C9 and activity of nuclear factor-B (NF-B) were assessed as
described.16 23
Immunofluorescence Experiments
Coronary artery segments were fixed with formaldehyde
(2% in PBS). After extensive washing, the segments were permeabilized
with Triton X-100 (0.2%) and incubated in glycine (100 mmol/L) for 10
minutes. After extensive washing, segments were coincubated with a
specific polyclonal CYP 2C antibody (kindly provided by Dr E. Morgan,
Atlanta, Ga) and a monoclonal actin antibody (Sigma) for 2 hours
followed by fluorescein-conjugated and Texas Red–conjugated secondary
antibodies (Dianova) for 60 minutes. Human endothelial cells were fixed
in formaldehyde, permeabilized, and coincubated with a CYP 2C antibody
and a monoclonal vascular cell adhesion molecule-1 (VCAM-1) antibody
(R&D Systems). After incubation with fluorescein and Texas Red–coupled
secondary antibodies, the preparations were mounted and viewed using a
confocal microscope.
Statistics
Data are expressed as mean±SEM, and statistical
evaluation was performed using Student’s
t test for paired or unpaired
data, one-way ANOVA followed by a Bonferroni
t test, or ANOVA for repeated
measures where appropriate. Values of
P<0.05 were considered
statistically significant. pD2 (-log
EC50) values were calculated by nonlinear
regression of the concentration-relaxation curves to
bradykinin.
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Results |
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Effects of CYP 2C Downregulation and Inhibition on NO-Mediated Relaxation
Antisense oligonucleotides derived from the cDNA sequences of human CYP 2C8 and 2C9, which have previously been shown to attenuate CYP 2C protein expression and EDHF-mediated responses,15 24 were used to downregulate CYP 2C expression.
Treatment of coronary arteries with the transfection reagent
alone or sense or scrambled oligonucleotides failed to affect
NO-mediated relaxation, as described
previously.15 However,
antisense oligonucleotide treatment, which decreased CYP expression
(see Figures 4C
and 4D), induced a marked leftward shift in the
concentration-relaxation curve to bradykinin
(Figure 1A). Although bradykinin-induced relaxation was
detectable in rings maintained for up to 60 hours, EDHF-mediated
relaxation decreased in a time-dependent
manner.15 On the other hand,
NO-mediated relaxation increased, inducing a leftward shift in the
relaxation-response curve, accounting for the slight difference in the
relaxation of solvent-treated rings in
Figures 1A and 1B.
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In freshly isolated arteries, the selective CYP 2C9
inhibitor sulfaphenazole induced a pronounced leftward shift in the
concentration-relaxation curve to bradykinin
(Figure 1B
; pD2 values being
7.87±0.06 and 8.54±0.03 in the presence of solvent and
sulfaphenazole, respectively,
P<0.001, n=12). An improvement
in the bradykinin-induced NO-mediated relaxation of coronary artery
rings was also observed in the presence of the
O2- scavenger Tiron
or the dual CYP and lipoxygenase inhibitor nordihydroguaretic acid
(pD2 values being 8.26±0.09, 8.70±0.08, and
8.77±0.08 in the presence of solvent, Tiron, and nordihydroguaretic
acid, respectively, P<0.001,
n=8). None of the antioxidants investigated exerted a significant
effect on the contraction to U46619.
Effect of Sulfaphenazole on ROS Production and
Substrate Conversion by CYP 2C8 and 2C9
To determine whether the putative coronary EDHF
synthase generates ROS, supersomes isolated from cells overexpressing
either CYP 2C8 or 2C9 were incubated with the oxidative-sensitive
fluorogenic reagent oxyBURST. ROS generation was observed using both
CYP 2C8- and 2C9-containing supersomes. The generation of ROS by both
CYP enzymes was attenuated by 17-ODYA and miconazole (data not shown)
and abolished by DPI
(Figure 2A). However, sulfaphenazole selectively inhibited
ROS generation by CYP 2C9
(Figure 2A). The IC50 value for
sulfaphenazole on ROS generation by CYP 2C9–containing microsomes was
1.72±0.35 µmol/L, whereas 100 µmol/L sulfaphenazole inhibited CYP
2C8–derived ROS generation by only 70%.
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To determine the selectivity of sulfaphenazole for CYP 2C9,
CYP activity was assessed as the conversion of DBF to fluorescein. The
activity of both CYP 2C8 and 2C9 was attenuated by 17-ODYA (by 36±4%
and 63±4%, respectively) and miconazole (by 26±7% and 87±3%,
respectively, n=3) and almost abolished by DPI
(Figure 2B). Sulfaphenazole markedly attenuated the activity
of CYP 2C9 but was without effect on CYP 2C8
(Figure 2B) or CYP 3A4 (data not
shown).
Sensitivity of Superoxide-Generating
Enzymes to Sulfaphenazole
To ensure that sulfaphenazole did not affect ROS
generation by the NADPH oxidase or xanthine oxidase, the generation of
ROS by human leukocytes and a purified preparation of xanthine oxidase
was assessed using compound 5–enhanced chemiluminescence and compared
with that of CYP 2C9–containing supersomes. In isolated human
leukocytes, neither bradykinin (100 nmol/L) nor 11,12-EET (up to 10
µmol/L) was able to activate the NADPH oxidase (data not shown), but
phorbol 12-myristate 13-acetate (PMA, 1 µmol/L) induced a significant
(20-fold) increase in ROS production. Sulfaphenazole failed to affect
ROS generation under either basal conditions (data not shown) or after
stimulation with PMA
(Figure 3). Similarly, sulfaphenazole was without effect on
ROS production by xanthine oxidase, which was abolished by oxypurinol
(100 µmol/L, 95.7±2.0% inhibition, n=3), but inhibited CYP
2C9–induced compound 5-chemiluminescence
(Figure 3). ROS generation by PMA-stimulated leukocytes,
xanthine oxidase, and CYP 2C9 was completely abolished by DPI or the
addition of superoxide dismutase (100 U/mL).
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Generation of ROS in Porcine Coronary
Endothelial Cells
In sections of porcine coronary artery stained with
dihydroethidine, a fluorescent signal was detected in both endothelial
and smooth muscle cells. However, after treatment of arterial segments
with CYP 2C antisense oligonucleotides, CYP 2C protein levels were
decreased and the fluorescent ethidium signal in the endothelium was
markedly attenuated
(Figures 4A through 4D). CYP 2C antisense oligonucleotides did
not affect the fluorescent signal in the vascular smooth muscle.
Incubation of arterial rings with sulfaphenazole also selectively
attenuated the ethidium fluorescent signal detected in endothelial
cells without affecting the signal in either the media or adventitia
(Figures 4E and 4F).
To determine whether the endothelial agonist bradykinin,
which elicits EDHF-mediated responses, elevated the production of ROS
in coronary artery endothelial cells, experiments were performed using
H2DCF-DA–loaded endothelial cells. Coronary
artery endothelial cells were isolated and maintained in culture over 2
days. These cells, which express CYP 2C
protein,15 16
exhibited a basal production of ROS, which was attenuated (by 24±4%,
P<0.01, n=7) in the presence
of sulfaphenazole
(Figure 5A).
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Stimulation of
N
-nitro-l-arginine–pretreated
coronary endothelial cells with bradykinin resulted in a time-dependent
increase in DCF fluorescence, which was markedly attenuated by
sulfaphenazole
(Figure 5B). A sulfaphenazole-sensitive increase in DCF
fluorescence was also observed in bradykinin-stimulated cells in the
absence of
N-nitro-l-arginine,
although it is impossible to exclude that part of this signal reflects
the generation of peroxynitrite (data not shown). No increase in ROS
production was observed in coronary endothelial cells stimulated with
PMA (data not shown).
Effect of CYP Induction and CYP 2C9
Overexpression on NF-B and VCAM-1
11,12-EET, which is the major epoxygenase product
detectable in ß-naphthoflavone–stimulated porcine coronary artery
endothelial cells,15 exerts
anti-inflammatory effects on tumor necrosis factor-
(TNF-)–stimulated endothelial cells by preventing the activation of
NF-B.17 Therefore, we
determined the effects of CYP induction and CYP 2C9 overexpression on
the activity of NF-B.
In confluent primary cultures of human endothelial cells,
11,12-EET had a biphasic effect on NF-B, consisting of a transient
increase followed by a decrease in DNA binding. Pretreatment of
endothelial cells with 11,12-EET attenuated the activation of NF-B
by TNF-
(Figure 6A). Enhancing CYP expression by incubating
endothelial cells with the Ca2+ antagonist
nifedipine (0.1 µmol/L, 18
hours)16 increased both the
basal and TNF-–induced increase in NF-B DNA binding
(Figure 6B). Overexpression of CYP 2C9 increased endothelial
generation of 11,12-EET by 453±40% (n=4) and ROS production by
283±22% (n=3) compared with nontransfected endothelial cells and
markedly enhanced the binding of DNA by NF-B
(Figure 6C). The addition of anti-p65 antibody to the assay
caused a supershift in the gel mobility of the NF-B protein-DNA
complex
(Figure 6C).
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The expression of VCAM-1 is controlled by
NF-B25 and is reported to
be increased by ROS26 but
decreased by 11,12-EET.17 To
determine which of these influences predominates in CYP-expressing
cells, we assessed the expression of VCAM-1 in human endothelial cells
transfected with CYP 2C9. VCAM-1 was not detected in unstimulated
endothelial cells but was clearly evident 10 hours after transfection
of endothelial cells with CYP 2C9, an effect not observed in
transfected cells treated with sulfaphenazole
(Figure 7). There seemed to be no paracrine effect of CYP
products on VCAM-1 expression in neighboring endothelial cells, because
the only cells that stained positively for VCAM-1 were those expressing
CYP 2C9.
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Stimulation of CYP 2C9–transfected endothelial cells with
TNF- did not affect the level of CYP 2C9 protein but markedly
increased (14-fold) the expression of VCAM-1 in the entire cell
population. However, VCAM-1 expression was greater in cells
overexpressing CYP 2C9, and the inclusion of sulfaphenazole reduced
VCAM-1 expression to the level of TNF-–stimulated cells transfected
with the empty vector
(Figure 7).
In native porcine coronary endothelial cells, which
express CYP 2C9 and stain ethidium positive, VCAM-1 was also detected
(Figure 8). Treatment with CYP 2C9 antisense oligonucleotides
markedly attenuated the basal expression of VCAM-1 and exerted a
moderate effect on the expression of VCAM-1 after stimulation of
segments with TNF-
(Figure 8).
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CYP 2C9 has previously been reported to generate 11,12-EET in coronary endothelial cells and plays a crucial role in EDHF-mediated hyperpolarization and relaxation. In the present study, we have demonstrated that, in both cultured and native porcine coronary endothelial cells, CYP 2C9 is also a physiologically relevant source of ROS. Overexpression of CYP 2C9 in coronary artery endothelial cells markedly increases 11,12- and 8,9-EET16 generation as well as that of ROS. The consequences of superoxide anion or hydrogen peroxide production by CYP 2C9 range from the impairment of NO-mediated relaxation to a chronic elevation in the activity of the redox-sensitive transcription factor NF-
B and the expression of
VCAM-1. Although accepted to play a role in the pathophysiology of hypertension, atherosclerosis, and heart failure, it is not generally appreciated that ROS, such as O2- and hydrogen peroxide, are intracellular signaling molecules that are involved in the regulation of vascular tone under normal conditions.27 In the pulmonary artery, for example, so much O2- is generated in response to an increase in fluid shear stress that the production of NO can only be demonstrated in the presence of high concentrations of superoxide dismutase.28 The most frequently studied sources of ROS in endothelial cells are the NADPH oxidase, xanthine oxidase, cyclooxygenase, and eNOS.9 It is only relatively recently that CYP enzymes, some of which may generate ROS,11 13 29 have been identified in the vasculature, both in endothelial14 15 17 30 and vascular smooth muscle cells.31 In the porcine coronary artery, the putative EDHF synthase may be the dominant source of endothelial O2- in vivo, as it can be continuously activated by the rhythmic vessel distension that occurs during the cardiac cycle.32 Given that many constitutively expressed enzymes are able to generate ROS, it is difficult to identify the enzymatic source of vascular O2-.17 Most studies have relied on pharmacological agents, such as l-arginine analogues, oxypurinol, or, in the present study, sulfaphenazole, to suggest a physiological role for ROS generation by NOS,5 xanthine oxidase,33 34 and CYP 2C9, respectively. The most convincing evidence obtained in support of our hypothesis that CYP 2C is a physiologically relevant source of ROS was provided using an antisense approach. Indeed, after treatment with CYP 2C antisense oligonucleotides, free radical generation in the endothelium of porcine coronary arteries was decreased and NO-mediated relaxations were significantly enhanced. Although it is not strictly correct to compare results obtained in 2 different models, it is interesting to note that the extent of the shift in the concentration-relaxation curve to an endothelial agonist in CYP 2C antisense-treated arteries was much more pronounced than that resulting from knockout of the gp91phox component of the endothelial NADPH oxidase in the mouse aorta.35
Homology among the different CYP 2C isoforms is exceedingly high,36 and using the antisense approach and antibodies used in the present study, it is not possible to differentiate between the expression of CYP 2C8 and 2C9. However, the finding that sulfaphenazole, a selective inhibitor of CYP 2C9,37 38 inhibits EDHF-mediated responses15 and potentiates NO-mediated relaxation in the porcine coronary artery suggests that the CYP isoform responsible for the generation of EDHF/EET and ROS is a porcine equivalent of CYP 2C9. A possible interaction between CYP 2C–derived EETs and the NADPH oxidase could also be discounted, because a CYP inhibitor–sensitive production of ROS could be demonstrated in coronary artery endothelial cells treated with antisense oligonucleotides against p22phox (authors’ unpublished data, August 2000), an approach that inhibits O2- production in vascular smooth muscle cells.39 Moreover, in in vitro assays, NADPH oxidase, xanthine oxidase, CYP 2C8, and CYP 3A4 were not sensitive to sulfaphenazole, leaving CYP 2C9 as the most probable candidate for the sulfaphenazole-sensitive formation of ROS in endothelial cells.
To date, when considering the consequences of vascular CYP
activity, most attention has been focused on the generation of
vasoactive arachidonic acid metabolites by CYP epoxygenases and
-hydroxylases. In addition to activating
Ca2+-dependent potassium channels and
tyrosine
kinases,40 41
11,12-EET, for example, has been reported to exert an anti-inflammatory
effect in endothelial cells by inhibiting the activation of NF-B and
decreasing the cytokine-induced expression of
VCAM-1.17 However, there is
indirect evidence suggesting that another CYP-derived product exerts
distinctly different effects on endothelial signaling. For example,
overexpression of the EET-generating epoxygenase CYP 2J2 in bovine
endothelial cells attenuated the TNF-–induced activation of NF-B
but to a lesser extent than the exogenously applied
EET.17 In the present study,
elevating CYP 2C protein levels by incubating endothelial cells with
nifedipine or by overexpressing CYP 2C9 markedly enhanced the basal
activity of NF-B. A direct relationship between CYP expression and
NF-B could be demonstrated in that the CYP-induced increase in
NF-B activity could be prevented by sulfaphenazole both under basal
conditions and after cell stimulation with TNF-. Moreover, the
VCAM-1 expression elicited by CYP overexpression under basal conditions
was completely abolished by sulfaphenazole, whereas the expression of
VCAM-1 induced by TNF- in CYP-expressing cells was only partially
sensitive to the CYP inhibitor. Because NF-B is a redox-sensitive
transcription factor, and 11,12-EET alone attenuates its
activation,17 CYP-derived
ROS seem to be responsible for the increase in NF-B activity and the
subsequent induction of VCAM-1 in CYP 2C9-expressing endothelial
cells.
Taken together, the results of the present study suggest that CYP 2C9, expressed in coronary endothelial cells, is not only crucial for the generation of the potent vasorelaxant 11,12-EET but is a potential major source of ROS within the coronary wall. Thus, whereas the anti-inflammatory CYP product EDHF/EET may be the dominant endothelium-derived vasoactive autacoid in states associated with a manifest endothelial dysfunction, the enhanced activation of the CYP-like EDHF synthase may eventually be detrimental to vascular homeostasis as a consequence of the simultaneous generation of EETs and ROS.
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Acknowledgments |
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This study was supported by the Heinrich and Fritz Riese-Stiftung, Deutsche Forschungsgemeinschaft (FI 830/1-1), and the Institut de Recherches Internationales Servier. The authors are indebted to Isabel Winter and Stergiani Hauk for expert technical assistance and to Dr David Harrison (Atlanta, Ga) for helpful suggestions during the preparation of this manuscript.
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Footnotes |
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Original received September 1, 2000; resubmission received October 31, 2000; accepted November 14, 2000.
This manuscript was sent to Donald D. Heistad, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
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R. P. Brandes A Radical Adventure: The Quest for Specific Functions and Inhibitors of Vascular NAPDH Oxidases Circ. Res., April 4, 2003; 92(6): 583 - 585. [Full Text] [PDF]
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R. Dechend, C. Viedt, D. N. Muller, B. Ugele, R. P. Brandes, G. Wallukat, J.-K. Park, J. Janke, P. Barta, J. Theuer, et al. AT1 Receptor Agonistic Antibodies From Preeclamptic Patients Stimulate NADPH Oxidase Circulation, April 1, 2003; 107(12): 1632 - 1639. [Abstract] [Full Text] [PDF]
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S. Spiekermann, U. Landmesser, S. Dikalov, M. Bredt, G. Gamez, H. Tatge, N. Reepschlager, B. Hornig, H. Drexler, and D. G. Harrison Electron Spin Resonance Characterization of Vascular Xanthine and NAD(P)H Oxidase Activity in Patients With Coronary Artery Disease: Relation to Endothelium-Dependent Vasodilation Circulation, March 18, 2003; 107(10): 1383 - 1389. [Abstract] [Full Text] [PDF]
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H. Miura, J. J. Bosnjak, G. Ning, T. Saito, M. Miura, and D. D. Gutterman Role for Hydrogen Peroxide in Flow-Induced Dilation of Human Coronary Arterioles Circ. Res., February 7, 2003; 92 (2): e31 - e40. [Abstract] [Full Text] [PDF]
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M. Sasaki, D. Ostanin, J. W. Elrod, T. Oshima, P. Jordan, M. Itoh, T. Joh, A. Minagar, and J. S. Alexander TNF-alpha -induced endothelial cell adhesion molecule expression is cytochrome P-450 monooxygenase dependent Am J Physiol Cell Physiol, February 1, 2003; 284(2): C422 - C428. [Abstract] [Full Text] [PDF]
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M. R. Sayen, A. B. Gustafsson, M. A. Sussman, J. D. Molkentin, and R. A. Gottlieb Calcineurin transgenic mice have mitochondrial dysfunction and elevated superoxide production Am J Physiol Cell Physiol, February 1, 2003; 284(2): C562 - C570. [Abstract] [Full Text] [PDF]
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A. W. Miller, P. V. G. Katakam, H.-C. Lee, C. D. Tulbert, D. W. Busija, and N. L. Weintraub Arachidonic Acid-Induced Vasodilation of Rat Small Mesenteric Arteries Is Lipoxygenase-Dependent J. Pharmacol. Exp. Ther., January 1, 2003; 304(1): 139 - 144. [Abstract] [Full Text] [PDF]
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 T. Hillig, P. Krustrup, I. Fleming, T. Osada, B. Saltin, and Y. Hellsten Cytochrome P450 2C9 plays an important role in the regulation of exercise-induced skeletal muscle blood flow and oxygen uptake in humans J. Physiol., January 1, 2003; 546(1): 307 - 314. [Abstract] [Full Text] [PDF]
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M. Medhora, J. Daniels, K. Mundey, B. Fisslthaler, R. Busse, E. R. Jacobs, and D. R. Harder Epoxygenase-driven angiogenesis in human lung microvascular endothelial cells Am J Physiol Heart Circ Physiol, January 1, 2003; 284(1): H215 - H224. [Abstract] [Full Text] [PDF]
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S. Shigematsu, S. Ishida, D. C. Gute, and R. J. Korthuis Bradykinin-induced proinflammatory signaling mechanisms Am J Physiol Heart Circ Physiol, December 1, 2002; 283(6): H2676 - H2686. [Abstract] [Full Text] [PDF]
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 D. N. Muller, E. Shagdarsuren, J.-K. Park, R. Dechend, E. Mervaala, F. Hampich, A. Fiebeler, X. Ju, P. Finckenberg, J. Theuer, et al. Immunosuppressive Treatment Protects Against Angiotensin II-Induced Renal Damage Am. J. Pathol., November 1, 2002; 161(5): 1679 - 1693. [Abstract] [Full Text] [PDF]
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Q. Hu, Z.-X. Yu, V. J. Ferrans, K. Takeda, K. Irani, and R. C. Ziegelstein Critical Role of NADPH Oxidase-derived Reactive Oxygen Species in Generating Ca2+ Oscillations in Human Aortic Endothelial Cells Stimulated by Histamine J. Biol. Chem., August 30, 2002; 277(36): 32546 - 32551. [Abstract] [Full Text] [PDF]
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C. Urbich, E. Dernbach, A. Aicher, A. M. Zeiher, and S. Dimmeler CD40 Ligand Inhibits Endothelial Cell Migration by Increasing Production of Endothelial Reactive Oxygen Species Circulation, August 20, 2002; 106(8): 981 - 986. [Abstract] [Full Text] [PDF]
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J. Bauersachs, M. Christ, G. Ertl, U.R. Michaelis, B. Fisslthaler, R. Busse, and I. Fleming Cytochrome P450 2C expression and EDHF-mediated relaxation in porcine coronary arteries is increased by cortisol Cardiovasc Res, June 1, 2002; 54(3): 669 - 675. [Abstract] [Full Text] [PDF]
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I. Fleming To Move or Not To Move?: Cytochrome P450 Products and Cell Migration Circ. Res., May 17, 2002; 90(9): 936 - 938. [Full Text] [PDF]
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J. Sun, X. Sui, J. A. Bradbury, D. C. Zeldin, M. S. Conte, and J. K. Liao Inhibition of Vascular Smooth Muscle Cell Migration by Cytochrome P450 Epoxygenase-Derived Eicosanoids Circ. Res., May 17, 2002; 90(9): 1020 - 1027. [Abstract] [Full Text] [PDF]
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M. Potente, U. R. Michaelis, B. Fisslthaler, R. Busse, and I. Fleming Cytochrome P450 2C9-induced Endothelial Cell Proliferation Involves Induction of Mitogen-activated Protein (MAP) Kinase Phosphatase-1, Inhibition of the c-Jun N-terminal Kinase, and Up-regulation of Cyclin D1 J. Biol. Chem., May 3, 2002; 277(18): 15671 - 15676. [Abstract] [Full Text] [PDF]
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R. Popp, R. P. Brandes, G. Ott, R. Busse, and I. Fleming Dynamic Modulation of Interendothelial Gap Junctional Communication by 11,12-Epoxyeicosatrienoic Acid Circ. Res., April 19, 2002; 90(7): 800 - 806. [Abstract] [Full Text] [PDF]
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R. Locher, R. P. Brandes, W. Vetter, and M. Barton Native LDL Induces Proliferation of Human Vascular Smooth Muscle Cells via Redox-Mediated Activation of ERK 1/2 Mitogen-Activated Protein Kinases Hypertension, February 1, 2002; 39(2): 645 - 650. [Abstract] [Full Text] [PDF]
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K. Szocs, B. Lassegue, D. Sorescu, L. L. Hilenski, L. Valppu, T. L. Couse, J. N. Wilcox, M. T. Quinn, J.D. Lambeth, and K. K. Griendling Upregulation of Nox-Based NAD(P)H Oxidases in Restenosis After Carotid Injury Arterioscler Thromb Vasc Biol, January 1, 2002; 22(1): 21 - 27. [Abstract] [Full Text] [PDF]
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 R. J. Roman P-450 Metabolites of Arachidonic Acid in the Control of Cardiovascular Function Physiol Rev, January 1, 2002; 82(1): 131 - 185. [Abstract] [Full Text] [PDF]
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B. Fisslthaler, R. Popp, U. R. Michaelis, L. Kiss, I. Fleming, and R. Busse Cyclic Stretch Enhances the Expression and Activity of Coronary Endothelium-Derived Hyperpolarizing Factor Synthase Hypertension, December 1, 2001; 38(6): 1427 - 1432. [Abstract] [Full Text] [PDF]
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J.-M. Li and A. M. Shah Differential NADPH- versus NADH-dependent superoxide production by phagocyte-type endothelial cell NADPH oxidase Cardiovasc Res, December 1, 2001; 52(3): 477 - 486. [Abstract] [Full Text] [PDF]
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I. Fleming Cytochrome P450 and Vascular Homeostasis Circ. Res., October 26, 2001; 89(9): 753 - 762. [Abstract] [Full Text] [PDF]
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 A. H. WAGNER, M. R. SCHROETER, and M. HECKER 17{beta}-Estradiol inhibition of NADPH oxidase expression in human endothelial cells FASEB J, October 1, 2001; 15(12): 2121 - 2130. [Abstract] [Full Text] [PDF]
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 I. Fleming and R. Busse Vascular cytochrome P450 in the regulation of renal function and vascular tone: EDHF, superoxide anions and blood pressure Nephrol. Dial. Transplant., July 1, 2001; 16(7): 1309 - 1311. [Full Text] [PDF]
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W. B. Campbell and D. R. Harder Prologue: EDHF-what is it? Am J Physiol Heart Circ Physiol, June 1, 2001; 280(6): H2413 - H2416. [Full Text] [PDF]
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V. W. M. van Hinsbergh NO or H2O2 for Endothelium-Dependent Vasorelaxation : Tetrahydrobiopterin Makes the Difference Arterioscler Thromb Vasc Biol, May 1, 2001; 21(5): 719 - 721. [Full Text] [PDF]
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R. Popp, R. P. Brandes, G. Ott, R. Busse, and I. Fleming Dynamic Modulation of Interendothelial Gap Junctional Communication by 11,12-Epoxyeicosatrienoic Acid Circ. Res., April 19, 2002; 90(7): 800 - 806. [Abstract] [Full Text] [PDF]
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