Busse Beate Fisslthaler, Rüdiger Popp, U. Ruth ... - Hypertension

Cyclic Stretch Enhances the Expression and Activity of Coronary Endothelium-Derived
Hyperpolarizing Factor Synthase
Beate Fisslthaler, Rüdiger Popp, U. Ruth Michaelis, Ladislau Kiss, Ingrid Fleming and Rudi
Busse
Hypertension. 2001;38:1427-1432
doi: 10.1161/hy1201.096532
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Cyclic Stretch Enhances the Expression and Activity
of Coronary Endothelium-Derived Hyperpolarizing
Factor Synthase
Beate Fisslthaler, Rüdiger Popp, U. Ruth Michaelis, Ladislau Kiss, Ingrid Fleming, Rudi Busse
Abstract—Endothelium-derived hyperpolarizing factor (EDHF) mediates NO/prostacyclin-independent relaxation in the
coronary circulation. Because hemodynamic stimuli modulate endothelial gene expression and because coronary arteries
are subjected to pronounced variations in vessel distension, we determined the effects of cyclic stretch on the expression
and activity of the coronary EDHF synthase/cytochrome P450 (CYP) 2C8/9. In cultured porcine coronary and human
umbilical vein endothelial cells, acute application of cyclic stretch (6%, 1 Hz, 10 minutes) elicited the generation of
8,9-epoxyeicosatrienoic acid (EET), 11,12-EET, and 14,15-EET. Prolonged stretch (4 to 36 hours) increased the
expression of CYP 2C mRNA and protein 5- to 10-fold and was accompanied by a 4- to 8-fold increase in EET
generation. A corresponding increase in CYP 2C mRNA and protein was also observed in pressurized segments of
porcine coronary artery perfused under pulsatile conditions (8%, 1 Hz) for 6 hours. Although in cultured endothelial
cells, cyclic stretch elicited the rapid activation of tyrosine kinases as well as Akt and the p38 mitogen-activated protein
kinase, the mechanism by which cyclic stretch induces the expression of CYP 2C could not be elucidated, because
inhibitors of these pathways induced CYP 2C expression in cells maintained under static conditions. These results have
identified coronary EDHF synthase/CYP 2C as a novel mechanosensitive gene product in native and cultured
endothelial cells. Because this enzyme generates both EETs and superoxide anions, this finding has wide-reaching
implications for vascular homeostasis in conditions of manifest endothelial dysfunction. (Hypertension. 2001;38:1427-
1432.)
Key Words: endothelium-derived factor � cytochrome P450 � arteries � inositol � protein kinases
Humoral stimuli acutely modulate local blood flow by
eliciting the production and release of vasoactive autacoids
from the endothelium, such as NO, prostacyclin (PGI2), � endothelin-1, prostaglandin H2, and superoxide anions (O2 ).
However, hemodynamic stimuli such as fluid shear stress and
pulsatile stretch, to which the endothelial lining of blood
vessels is continually subjected, are the physiologically most
important determinants of the continuous production of these
autacoids in vivo. 1 In several vascular beds, an endotheliumderived
hyperpolarizing factor (EDHF) represents a third
endothelial vasodilator that mediates NO/PGI2-independent relaxation. Several different EDHFs are now known to exist
in different species, but the hyperpolarizing factor produced
by coronary and renal arteries from humans, pigs, cows, dogs,
rats, and rabbits displays characteristics similar to those of an
epoxyeicosatrienoic acid (EET) generated from arachidonic
acid by a cytochrome P450 (CYP) epoxygenase. 2
Pulsatile changes in transmural pressure, causing simultaneous
vessel distension, enhance the production of EDHFs/
EETs by native porcine coronary endothelial cells, and with
the use of a patch-clamp bioassay system, it could be
demonstrated that such rhythmic changes in arterial diameter
evoke the generation of a CYP inhibitor–sensitive EDHF. 3
Because cyclic stretch elicits the production of a hyperpolarizing
factor from porcine coronary arteries 3 and because a
CYP 2C epoxygenase homologous to CYP 2C8/9 was recently
identified as a putative coronary EDHF synthase, 4 we
sought to determine whether this hemodynamic stimulus
modulates the expression and activity of CYP 2C.
Methods
Cell Culture
Porcine coronary artery endothelial cells and human umbilical vein
endothelial cells were prepared as described 5 and seeded on flexiblebottomed
6-well culture plates coated with pronectin (BioFlex,
Flexcell International Corp). After they reached confluence, the cells
were mounted onto loading plates in a FlexerCell FX-3000 strain
unit (Flexcell) and placed in an incubator. Cells were stretched with
an average strain of 6% at a rate of 1 Hz, and static control
experiments were performed on cells on stretch plates not exposed to
cyclic strain. After stimulation, endothelial cells were harvested, and
CYP-derived eicosanoids were determined as described. 4
Received April 28, 2001; first decision June 18, 2001; revision accepted June 26, 2001.
From the Institut für Kardiovaskuläre Physiologie, Klinikum der J.W.G.-Universität (B.F., R.P., U.R.M., I.F., R.B.), Frankfurt am Main, Germany; and
Zentrum der Inneren Medizin, Justus Liebig Universität Gießen (L.K.), Gießen, Germany.
Correspondence to Dr Ingrid Fleming, Institut für Kardiovaskuläre Physiologie, Klinikum der J.W.G.-Universität, Theodor-Stern-Kai 7, D-60590
Frankfurt am Main, Germany. E-mail fleming@em.uni-frankfurt.de
© 2001 American Heart Association, Inc.
Hypertension is available at http://www.hypertensionaha.org
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1428 Hypertension December 2001
Cyclic Stretch of Porcine Coronary
Artery Segments
Porcine epicardial artery segments (length �40 mm, mean external
diameter 2.4 to 2.8 mm) were excised and cut into 2 segments of
equal length. The segments were cannulated at both ends and placed
into vessel chambers in which perfusion pressure was continuously
monitored. Vessels were pressurized to 40 to 60 mm Hg and
perfused (1 mL/min) with minimum essential medium. After stabilization,
sinusoidal pressure changes (30 to 40 mm Hg, 1 Hz), were
applied to 1 of the coronary artery pairs. The diameter changes
induced corresponded to a calculated strain of between 6% and 8%.
Isolation of RNA and RT-PCR
Total RNA was isolated from cultured coronary endothelial cells or
coronary arterial segments by intraluminal incubation with either
dispase (2.4 U/mL) or guanidine isothiocyanate. Random hexanucleotide
primers were used for reverse transcription (RT) of equal
amounts of RNA, and the oligonucleotides used for polymerase
chain reaction (PCR) were derived from a porcine CYP 2C34
sequence that exhibits a high homology to the human 2C8/9
sequence, as described. 6 In some experiments, the expression of CYP
3A, CYP 2J, and the endothelial NO synthase (eNOS) was determined.
Elongation factor-2 (EF-2) served as an internal control for
cultured endothelial cells, whereas platelet and endothelial cell
adhesion molecule-1 (DECAM-1) was used as a control for porcine
coronary arteries (for primers, see online data supplement available
at http://www.hypertensionaha.org). For the verification of the DNA
fragment, the PCR products were transferred to nylon membranes
and hybridized with 32 P-labeled DNA fragments derived from a
plasmid containing the coding sequence of CYP 2C8.
Immunofluorescence Experiments
In some experiments, cells or arterial segments were fixed with
formaldehyde and permeabilized with Triton X-100 (0.2% [vol/
vol]), and the segments were incubated with a specific CYP 2C
antibody (kindly provided by Dr E. Morgan, Emory University,
Atlanta, Ga) or antibodies recognizing actin (Sigma), Akt/protein
kinase B (PKB) (Cell Signaling), or phosphotyrosine (Transduction
Laboratories), as described. 7
Statistical Analysis
Data are expressed as mean�SEM, and statistical evaluation was
performed by using the Student’s t test for paired or unpaired data,
1-way ANOVA followed by a Bonferroni t test, or ANOVA for
repeated measures, where appropriate. Values of P�0.05 were
considered statistically significant.
Results
Acute Effect of Cyclic Stretch on the Generation
of EETs by Coronary Endothelial Cells
In unstimulated endothelial cells, free arachidonic acid
(13.5�1.8 ng per well) and small amounts of 5,6-, 8,9-,
11,12-, and 14,15-EET were detected. Cyclic strain (6%, 1
Hz, 10 minutes) slightly enhanced the liberation of arachidonic
acid (to 17.6�1.6 ng per well) as well as the generation
of EETs (Figure 1). Although stretch enhanced the production
of all the regioisomers detected, the most pronounced effects
were on the generation of 11,12- and 14,15-EET. To enhance
endothelial CYP expression, some cell batches were incubated
with the Ca2� antagonist nifedipine. As previously
reported, 6 nifedipine (0.1 �mol/L, 18 hours) enhanced the
expression of CYP 2C protein by �3-fold (data not shown).
In these cells, the amount of free arachidonic acid was
virtually unchanged (23.1�8.1 versus 38.3�8.7 ng per well,
n�3, P�NS), whereas the basal generation of EETs was
Figure 1. Effect of cyclic strain (6%, 1 Hz, 10 minutes; hatched
bars) on the acute generation of the 4 regioisomers of EET (5,6-
EET, 8,9-EET, 11,12-EET, and 14,15-EET) by coronary artery
endothelial cells. Open bars indicate the static condition. Experiments
were performed in cells preincubated with either solvent
(culture medium) or nifedipine (0.1 �mol/L, 18 hours). The
results are expressed either as the median of 3 or the
mean�SEM of 4 separate experiments. *P�0.05 vs static.
greater than that detected in solvent-treated cells. In contrast
to solvent-treated cells, nifedipine-treated cells produced
greater amounts of 5,6- and 8,9-EET, findings that may
indicate that in addition to CYP 2C, nifedipine enhances the
expression of at least 1 additional CYP enzyme, with a profile
of EET generation slightly different from that of CYP 2C8/9.
In nifedipine-treated cells, cyclic stretch enhanced only the
production of 11,12- and 14,15-EET, although the effect was
no longer statistically significant (Figure 1).
Effect of Cyclic Stretch on the Expression of CYP
2C in Cultured Endothelial Cells
Low levels of CYP 2C mRNA were expressed in porcine
coronary endothelial cells cultured under static conditions.
Cyclic stretch time-dependently increased the expression of
CYP 2C, an effect that was first evident after 4 to 8 hours and
was highly significant after 18 to 24 hours (Figure 2). CYP
2C mRNA was maintained at an elevated level (�10-fold
greater than in unstimulated endothelial cells) as long as the
stimulus was applied (maximally, 36 hours). In the same
experiments, cyclic stretch did not significantly affect the
expression of either CYP 3A, CYP 2J, or eNOS mRNA
(Figure 2).
In primary cultures of porcine coronary endothelial cells,
immunofluorescence experiments revealed that not all endothelial
cells expressed CYP 2C. When present, the enzyme
was generally localized to a perinuclear region (Figure 3).
Exposing these cells to cyclic stretch increased the overall
intensity of the signal as well as the number of positively
stained cells.
Human umbilical vein endothelial cells express markedly
lower levels of CYP 2C protein than coronary endothelial
cells, but the expression of CYP mRNA and protein was also
significantly increased by exposure to cyclic stretch over 9 to
18 hours (Figure 4).
Chronic Effect of Cyclic Stretch on the Generation
of EETs by Coronary Endothelial Cells
The increased expression of CYP 2C was accompanied by an
increase in endothelial EET production (Figure 5). Stimulation
of endothelial cells with ionomycin to elevate intracel-
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Figure 2. Effect of stretch on the expression of CYP 2C mRNA
in cultured endothelial cells. Cultured porcine coronary artery
endothelial cells were either incubated under static conditions or
exposed to cyclic stretch (6%, 1 Hz) for up to 24 hours. Total
RNA was isolated for RT-PCR, and CYP 2C, CYP 2J, CYP 3A,
eNOS, and EF-2 PCR products were detected as described in
the text. The bar graph provides a statistical summary of the
data obtained in 4 separate experiments. *P�0.05 vs control.
lular Ca 2� and activate phospholipase A 2 enhanced the production
of these 3 regioisomers 2- to 3-fold (Figure 5). Exposure to
cyclic stretch (6%, 1 Hz) for 18 hours increased the generation
Figure 3. Effect of prolonged cyclic stretch on the expression of
CYP 2C protein in cultured porcine endothelial cells. A, Immunohistochemical
staining of CYP 2C in porcine coronary endothelial
cells maintained under static conditions and after exposure
to cyclic stretch (6%, 1 Hz). The results presented are
representative of data obtained in 3 further experiments, and the
bar represents 20 �m. B, The bar graph shows the changes in
CYP 2C staining between static cultures and cells stretched for
18 hours as pixel intensity/mm 2 after nomalization of the control
group to 100%. *P�0.05 vs control.
Fisslthaler et al Cyclic Stretch and Endothelial CYP 2C Expression 1429
Figure 4. Effect of prolonged cyclic stretch on the expression of
CYP 2C protein and mRNA in cultured human endothelial cells.
A, Immunohistochemical staining of CYP 2C in human umbilical
vein endothelial cells after incubation under static conditions
and after exposure to cyclic stretch (6%, 1 Hz). The results presented
are representative of data obtained in 3 further experiments,
and the bar represents 20 �m. B, Confluent cultures of
human endothelial cells were either maintained under static conditions
(time 0) or exposed to cyclic stretch (6%, 1 Hz). Thereafter,
total RNA was isolated for RT-PCR as described in the text,
and in each experiment, a negative control (nc, no reverse transcriptase
in the RT reaction) was included. The results presented
are representative of data obtained in 2 further experiments.
of 11,12-EET and 14,15-EET by 4-fold and induced an 8-fold
increase in 8,9-EET production. Subsequent treatment of these
stretch-conditioned cells with ionomycin did not significantly
increase EET production (Figure 5).
Effect of Cyclic Stretch on the Expression of CYP
2C in Native Endothelial Cells
In porcine coronary artery segments, CYP 2C9 is expressed
exclusively in the endothelial cell layer (Figure 6). No CYP
2C immunostaining was detected in either smooth muscle
cells or in the adventitia. Exposure of coronary segments to
cyclic stretch (�8%, 1 Hz) for 6 hours markedly increased
the CYP 2C fluorescent signal compared with that observed
in vessels that were perfused under nonpulsatile conditions
Figure 5. Effect of prolonged cyclic stretch on the generation of
the EET regioisomers by cultured endothelial cells. Porcine coronary
endothelial cells were either maintained under static conditions
or exposed to cyclic stretch (6%, 1 Hz) for 18 hours.
After stimulation, cells were either harvested directly or exposed
to ionomycin (0.1 �mol/L, 5 minutes). The results are expressed
as the mean�SEM of 3 or 4 separate experiments. *P�0.05 vs
control.
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1430 Hypertension December 2001
Figure 6. Effect of cyclic stretch on the expression of CYP 2C in
native endothelial cells. A, Immunohistochemical staining of
CYP 2C (green) and actin (red) in sections of the same porcine
coronary artery after 6-hour incubation under either nonpulsatile
conditions (left) or cyclic stretch (�8%, 1 Hz; right). The results
presented are representative of data obtained in 2 further experiments.
The bar represents 100 �m. B, Paired segments of porcine
coronary artery were perfused under nonpulsatile (static) or
pulsatile conditions (8%, 1 Hz, 6 hours, stretch). Thereafter, total
RNA was isolated for RT-PCR as described in the text, and a
positive control (pc, human liver) and negative control (nc, no
reverse transcriptase in the RT reaction) were included in each
experiment. PECAM-1 indicates platelet and endothelial cell
adhesion molecule-1. The bar graph provides a statistical summary
of the data obtained in 4 separate experiments after normalization
of the control group to 100. *P�0.05 vs control.
(Figure 6). Similarly, CYP 2C mRNA expression was increased
by �5-fold in samples prepared from intact porcine
coronary arteries (Figure 6).
Effect of Kinase Inhibitors on Stretch-Induced
Expression of CYP 2C
In unstimulated endothelial cells, Akt/PKB was distributed
throughout the cytoplasm and in the nucleus. The application
of cyclic stretch induced the time-dependent translocation of
Akt/PKB, so that after 60 minutes of stimulation, the kinase
was exclusively localized to the nucleus (Figure 7). However,
the stretch-induced activation and translocation of Akt/PKB
was unrelated to the increase in CYP 2C expression, inasmuch
as neither wortmannin, a phosphatidylinositol 3-kinase
(PI 3-K) inhibitor, nor rapamycin, an inhibitor of the Akt/
PKB substrate the mammalian target of rapamycin (mTOR),
affected the stretch-induced CYP expression (Figure 7).
Rapamycin also failed to affect the increase in EET generation
induced by cyclic stretch (6%, 1 Hz, 18 hours) (data not
shown).
In unstimulated endothelial cells, immunostaining with a
phosphotyrosine antibody revealed a punctate signal mostly
localized to the cell boundary. In response to cyclic stretch,
the pattern rapidly altered so that the cell-cell boundary was
less intensely labeled, whereas the phosphotyrosine signal in
the nuclear region was markedly increased. The tyrosine
kinase inhibitor herbimycin and the Src inhibitor 4-amino-
5(4-methylphenyl)-7-(t-butyl)phyrazolo[3,4-d]pyrimidine
(PP1) both attenuated the stretch-induced alterations in phosphotyrosine
staining. To determine the effects of tyrosine
kinase inhibition on CYP expression, endothelial cells were
exposed to cyclic stretch in the absence and presence of
herbimycin. As described before, cyclic stretch increased the
expression of CYP 2C protein and mRNA, but herbimycin,
on its own, elicited a much more pronounced effect (Figure
8B and 8C). The signal obtained in response to the combination
of herbimycin and stretch was not different from that
observed with stretch alone.
An additional signaling molecule rapidly activated after the
application of cyclic stretch was the p38 mitogen-activated
protein (MAP) kinase (data not shown). However, it was not
possible to link the activation of this kinase to the stretchinduced
increase in CYP 2C expression, because 2 specific
inhibitors, SB203580 and SB220025, increased CYP 2C
expression under static conditions (by 513�20% and
461�30%, respectively; P�0.01; n�5). None of the kinase
inhibitors used directly affected the activity of isolated
microsomes containing CYP 2C9 (data not shown).
Discussion
In the present study, we have identified an epoxygenase
homologous to CYP 2C8/9 as a novel stretch-inducible
Figure 7. Effect of interfering with the PI 3-K/Akt/mTOR signaling
pathway on the stretch-induced expression of CYP 2C. A,
Immunohistochemical staining of Akt/PKB in porcine coronary
endothelial cells after incubation under static conditions and
after exposure to cyclic stretch (6%, 1 Hz). The results presented
are representative of data obtained in 2 further experiments,
and the bar represents 20 �m. B, Cultured coronary endothelial
cells were either maintained under static conditions (S) or
exposed to cyclic stretch (C; 6%, 1 Hz, 18 hours) in the
absence and presence of wortmannin (Wort, 20 nmol/L) and
rapamycin (Rapa, 100 nmol/L). Thereafter, RNA was isolated for
RT-PCR as described, and CYP 2C PCR products were
detected by Southern blotting and hybridization with isoformspecific
DNA fragments. To ensure that equal amounts of cDNA
were amplified per PCR reaction, PCR for EF-2 was performed.
The results presented are representative of data obtained in 2
further experiments.
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Figure 8. Effect of tyrosine kinase inhibitors on the stretchinduced
tyrosine phosphorylation of endothelial cells and the
expression of CYP 2C. A and B, Immunohistochemical staining
of tyrosine-phosphorylated proteins (A) and CYP 2C (B) in porcine
coronary endothelial cells maintained under static conditions
and after exposure to cyclic stretch (6%, 1 Hz) in the
absence and presence of herbimycin (Herbi, 5 �mol/L) and PP1
(10 �mol/L). The results presented are representative of data
obtained in 2 further experiments, and the bar represents 20
�m. C, Cultured coronary endothelial cells were either maintained
under static conditions (S) or exposed to cyclic stretch
(C; 6%, 1 Hz, 18 hours) in the absence and presence of Herbi (5
�mol/L). Thereafter, RNA was isolated for RT-PCR as described.
The results presented are representative of data obtained in 2
further experiments.
protein in native and cultured endothelial cells. The effects of
stretch on CYP 2C appear to be 2-fold, inasmuch as the acute
application of moderate levels of cyclic stretch enhanced
enzyme activity and EET production, whereas chronic stretch
enhanced the expression of CYP 2C mRNA and protein.
Native and cultured endothelial cells express several CYP
isozymes, including the hydroxylases CYP 3A and CYP 2B
and the arachidonic acid epoxygenases CYP 2J and CYP
Fisslthaler et al Cyclic Stretch and Endothelial CYP 2C Expression 1431
2C. 4,6,8,9 Of these isozymes, cultured endothelial cells constitutively
express CYP 3A and CYP 2J, whereas CYP 2C
levels are highly variable, and often no signal is detectable
with the use of RT-PCR. For example, although native
porcine coronary artery endothelial cells clearly express CYP
2C protein, CYP 2C mRNA and protein are barely detectable
in primary cultures of these cells when maintained under
static conditions. 4,6 Because pulsatile stretch has been reported
to activate the EDHF synthase/CYP 2C in porcine
coronary arteries, 3 we postulated that cyclic stretch may
regulate the activity and expression of CYP 2C in a manner
analogous to the regulation of eNOS activity and expression
by fluid shear stress. The results obtained clearly indicate that
cyclic stretch regulates the expression of CYP 2C mRNA and
protein in both native and cultured endothelial cells. Thus, it
is tempting to speculate that cyclic stretch maintains the
expression of EDHF synthase in vivo and that its decreased
expression in vitro is attributable to the lack of this continuous
hemodynamic stimulus. Although endotheliumdependent
hyperpolarization of coronary artery smooth muscle
cells was not investigated in the present study, we have
previously demonstrated that the generation of EETs by
endothelial cells pretreated with either nifedipine 6 or
�-naphthoflavone, 4 which were similar to those measured in
stretch-stimulated endothelial cells, was sufficient to potentiate
the endothelium-dependent hyperpolarization of porcine
coronary artery smooth muscle cells.
The application of cyclic stretch to a variety of different
cells is reported to activate numerous signal transduction
cascades, including the Janus kinase/signal transducer(s) and
activator(s) of transcription 10 and Ras/Raf/extracellular signal–regulated
kinase 1/2 pathways, 11 as well as the epidermal
growth factor receptor tyrosine kinase, 12 the c-Jun NH 2terminal,
11 and p38 MAP kinases. 12 The mechanism by which
pulsatile stretch increases CYP expression in endothelial cells
remains to be fully elucidated. In the present study, we
observed that the PI 3-K/Akt/mTOR signaling pathway was
rapidly activated in stretched endothelial cells, but neither
wortmannin nor rapamycin was found to significantly inhibit
the induction of CYP 2C protein or the stretch-induced
generation of EETs. Additional signaling molecules rapidly
activated after the application of cyclic stretch were tyrosine
kinases and p38 MAP kinase (authors’ unpublished data,
2001). However, it was not possible to link either of these
kinases to the stretch-induced increase in CYP 2C expression.
Part of the difficulty in elucidating the pathway determining
the stretch-induced expression of CYP 2C was due to the fact
that SB203580 and SB220025, 2 specific inhibitors of p38
MAP kinase, and the tyrosine kinase inhibitor herbimycin
enhanced the basal expression of CYP 2C in endothelial cells
maintained under static conditions. However, each of these
inhibitors prevented the stretch-induced activation of the
respective kinases in coronary endothelial cells. Because
many CYP substrates increase the expression of the enzymes
responsible for their metabolism, 13 it is likely that the
pharmacological inhibitors may be at least partially metabolized
by CYP 2C. Indeed, our previous observation, ie, that
the Ca 2� antagonist nifedipine enhances the expression and
the activity of the EDHF synthase/CYP 2C in native and
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1432 Hypertension December 2001
cultured endothelial cells, 6 may be attributable to a similar
phenomenon.
The functional consequences of endothelial CYP 2C/
EDHF synthase regulation by cyclic stretch are potentially
wide reaching, because in addition to activating Ca 2� -
dependent K � channels and modulating arterial tone, the
EETs regulate multiple endothelial and smooth muscle signaling
pathways and may promote both endothelial cell
proliferation 14 and angiogenesis. 15 Moreover, EDHF synthase
has recently been shown to generate physiologically relevant
amounts of free radicals, including O 2 �7 ; thus, the activation
of CYP 2C in endothelial cells may participate in the
stretch-induced generation of O 2 � , which has, until now, been
attributed to the activation of NADPH oxidase. 16
Acknowledgments
This work was supported by the Deutsche Forschungsgemeinschaft
(FI 830/1-1) and Institut de Recherches Internationales Servier. The
authors are indebted to Isabel Winter, Stergiani Hauk, and Sina Bätz
for expert technical assistance.
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