1 Vascular Bioengineering
Laboratory, The shear-induced intracellular signal transduction pathway in
vascular endothelial cells involves tyrosine phosphorylation and
activation of mitogen-activated protein (MAP) kinase, which may be
responsible for the sustained release of nitric oxide. MAP kinase is
known to be activated by reactive oxygen species (ROS), such as
H2O2,
in several cell types. ROS production in ligand-stimulated
nonphagocytic cells appears to require the participation of a
Ras-related small GTP-binding protein, Rac1. We hypothesized that Rac1
might serve as a mediator for the effect of shear stress on MAP kinase
activation. Exposure of bovine aortic endothelial cells to laminar
shear stress of 20 dyn/cm2 for
5-30 min stimulated total cellular and cytosolic tyrosine phosphorylation as well as tyrosine phosphorylation of MAP kinase. Treating endothelial cells with the antioxidants
N-acetylcysteine and pyrrolidine
dithiocarbamate inhibited in a dose-dependent manner the
shear-stimulated increase in total cytosolic and, specifically, MAP
kinase tyrosine phosphorylation. Hence, the onset of shear stress
caused an enhanced generation of intracellular ROS, as evidenced by an
oxidized protein detection kit, which were required for the
shear-induced total cellular and MAP kinase tyrosine phosphorylation. Total cellular and MAP kinase tyrosine phosphorylation was completely blocked in sheared bovine aortic endothelial cells expressing a
dominant negative Rac1 gene product (N17rac1). We concluded that the
GTPase Rac1 mediates the shear-induced tyrosine phosphorylation of MAP
kinase via regulation of the flow-dependent redox changes in
endothelial cells in physiological and pathological circumstances.
endothelium; signal transduction; shear stress; oxidative stress; mitogen-activated protein kinase; reactive oxygen species
VASCULAR ENDOTHELIAL CELLS (ECs) are constantly exposed
to flow-induced shear stress. ECs respond to fluid shear stress by rapid release of bioactive compounds, such as the vasodilators prostacyclin and nitric oxide (NO) (21, 49), and by changes in the
synthesis of proteins, such as the vasoconstrictor endothelin-1 (52).
Specifically, for NO, exposure of cultured human umbilical vein ECs
(HUVECs) to laminar shear stress increased NO release in a biphasic
manner. The initial burst was dependent on
Ca2+-calmodulin, whereas the
sustained NO release was Ca2+
independent (39, 40). Hence, activation of EC signal transduction by
shear stress is believed to involve two pathways: a
Ca2+-dependent and a
Ca2+-independent pathway (6). The
latter involves the
Ca2+-independent tyrosine
phosphorylation and activation of the 42- and 44-kDa
mitogen-activated protein (MAP) kinases through small GTP-binding
proteins, such as p21ras (Ras),
and Ca2+-independent protein
kinase C isozymes (46, 60). Tyrosine kinase inhibitors abolished the
Ca2+-independent phase of
shear-induced NO release, suggesting that shear stress activates the
endothelial constitutive NO synthase (ecNOS) via a mechanotransduction
that involves tyrosine kinases (3). It was found that the focal
adhesion-associated tyrosine kinases
p60src and
p125FAK are upstream to the
Ras-MAP kinase pathway (33, 45). Involvement of the tyrosine kinases in
focal adhesion sites is expected, since focal adhesions reorganize
during EC exposure to flow (15) and activated integrins elicit signals
by inducing tyrosine phosphorylation of intracellular proteins (22).
MAP kinase phosphorylates transcription factors, such as
c-fos and AP-1, known to be activated
by flow (29, 41), phospholipases, and other kinases (34). MAP kinase is
a serine/threonine kinase, and ecNOS is phosphorylated on serine and
threonine residues (13). Thus MAP kinase seems to be a suitable candidate for regulation of the sustained phase of shear-induced NO
release (6).
It was shown that oxygen-derived free radicals, such as superoxide
(O Laurindo et al. (43) discovered that increases in blood flow triggered
free radical release in vivo and in isolated perfused rabbit aortas.
ROS, in particular
H2O2,
were produced by cultured porcine aortic ECs subjected to a form of
mechanical deformation, i.e., cyclic strain (27). Cyclic strain-induced
ROS in HUVECs were involved in monocyte chemotactic protein (MCP)-1
gene expression (62) and plasminogen activator inhibitor (PAI)-1
release (10). More recently, intracellular
O Potential sources of free radicals in cultured cells are the enzymes of
the mitochondrial electron transport chain: xanthine oxidase (XO),
cytochrome P-450, cyclooxygenase,
lipoxygenase, and the superoxide-generating NADPH oxidase. On
stimulation of phagocytic cells, such as neutrophils and monocytes,
Rac1, a small GTP-binding protein of the Ras superfamily, enhances the
activity of the enzyme NADPH oxidase, resulting in production of
O By employing chemical antioxidants, we demonstrated that the
shear-induced increase in cellular tyrosine phosphorylation and, specifically, in tyrosine phosphorylation of MAP kinase is, at least
partly, mediated by ROS. The effect of shear-induced ROS generation on
cellular and MAP kinase tyrosine phosphorylation was completely blocked
in cells infected with an adenovirus that encodes a dominant negative
Rac1 (N17rac1), but not with a control virus, suggesting that the
Ras-related small GTP-binding proteins may function as regulators of
the intracellular redox status in sheared ECs.
Materials.
DMEM, fetal bovine serum (FBS),
L-glutamine,
penicillin-streptomycin, collagenase, sodium pyruvate, nonessential
amino acids, amphotericin B (Fungizone), trypsin-EDTA, and ATP-free
medium 199 (M199) were purchased from GIBCO BRL (Gaithersburg, MD). The protease inhibitors 4-(2-aminoethyl)benzenesulfonyl fluoride, chymostatin, leupeptin, aprotinin, and pepstatin, the phosphatase inhibitor sodium orthovanadate
(Na3VO4),
and
H2O2,
NAC, pyrrolidine dithiocarbamate (PDTC), DMSO, and Triton X-100 were
purchased from Sigma Chemical (St. Louis, MO). The OxyBlot oxidized
protein detection kit was obtained from Oncor (Gaithersburg, MD).
Cell culture.
Fresh bovine aortas were obtained from a local slaughterhouse and
filled with an ice-cold solution of 99% PBS-1%
penicillin-streptomycin. The fat surrounding the aorta was cut off, and
the intercostal and other branches were sealed. The aorta was filled
with the same solution, to which 1 mg/ml collagenase was
added, and incubated with the collagenase solution for 1 h at room
temperature. Then it was filled with complete culture medium (DMEM
supplemented with 10% FBS, 1% penicillin-streptomycin, 2% sodium
pyruvate, 1% nonessential amino acids, 2 mM
L-glutamine, and 25 mM HEPES) and massaged gently to detach ECs. The cell suspension was distributed into culture flasks and incubated at 37°C and 5%
CO2. When confluency was reached,
culture purity was verified by uptake of fluorescently labeled
DiI-Ac-LDL (Biomedical Technologies, Stoughton, MA). ECs were passaged
1:4 twice and then frozen in 60% complete culture medium-10%
DMSO-30% FBS in a liquid nitrogen tank. Some experiments were
performed using bovine aortic ECs (BAECs) purchased from Clonetics (San
Diego, CA). Primary cells were plated in tissue culture flasks at a
cell density of 5,000 cells/cm2
and grown in EGM-2 medium (Clonetics). Media were replaced every other
day, and cells were subcultured on confluency. Cells in passages 3-10 were used in the
present experiments.
Shear stress experiments.
Cells
(~104/cm2)
were seeded on glass slides (75 × 38 mm; Corning, Corning, NY)
that had been dipped into 100% ethanol, air-dried, and coated with
0.2% gelatin (Sigma Chemical). Seeded slides were placed in 100-mm
culture dishes with 10 ml of complete culture medium and incubated at
37°C and 5% CO2. Four days
after the seeding, ECs were confluent
(~106 cells/slide). Confluent
ECs were serum starved overnight in DMEM supplemented with 0.5% FBS,
1% penicillin-streptomycin, 2% sodium pyruvate, 1% nonessential
amino acids, 2 mM L-glutamine,
and 25 mM HEPES. Some cells were treated with 9 or 90 µM
H2O2
for 15 min before shear exposure. Other cells were incubated with 5 or 20 mM NAC for 4 h before shear exposure, or they were incubated with
100 µM PDTC for 4 h before shear exposure. Each slide comprised of
one side of a parallel-plate flow chamber, and the chamber was
connected at both ends to a reservoir forming a flow-loop system (19).
A hydrostatic pressure-drop system was used to control the flow through
the chamber. The wall shear stress on the cell monolayer was calculated
using the following equation: Western blot for cellular tyrosine phosphorylation.
At the end of the shearing period, the cells were scraped off the
slides in ice-cold PBS, and cell suspensions were centrifuged at 5,000 rpm for 5 min. The cell pellets were resuspended in ice-cold Triton
X-100 lysis buffer consisting of 145 mM NaCl, 0.1 mM
MgCl2, 15 mM HEPES, 10 mM EGTA,
1% Triton X-100, 1 mM
Na3VO4,
and protease inhibitors: 4-(2-aminoethyl)benzenesulfonyl fluoride
(25 g/l), leupeptin (50 mg/l), and chymostatin, aprotinin, and
pepstatin (25 mg/l each). The lysates were sonicated, and their protein content was measured using the bicinchoninic acid protein assay (Pierce, Rockford, IL) and normalized with 2× nonreducing sample buffer (0.5 M Tris · HCl, pH 6.8, 20% glycerol, 10%
SDS, and 0.1% bromphenol blue). To study tyrosine phosphorylation in
cytosolic fractions, cell lysates were sonicated and centrifuged at
14,000 rpm for 10 min at 4°C. The cytosolic protein content
(supernatant) was measured and normalized with sample buffer, as for
the whole cell lysates. Before electrophoresis, all samples were heated in a 95°C water bath for 5 min.
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
2·), and other reactive oxygen species (ROS), such as
H2O2,
induced MAP kinase tyrosine phosphorylation and activation in several
cell types, e.g., O
2· activated
MAP kinase in vascular smooth muscle cells (VSMCs) (4). H2O2
activated MAP kinase in NIH/3T3 cells (54) and also induced cellular
tyrosine phosphorylation in bovine pulmonary artery ECs (9, 61). The
role of ROS as intracellular second messengers was demonstrated by
their requirement in VSMC signaling in response to platelet-derived
growth factor (PDGF): increasing the intracellular concentration of the
peroxide-scavenging enzyme catalase or treating cells with the
antioxidant N-acetylcysteine (NAC)
blocked the PDGF-induced cellular tyrosine phosphorylation and tyrosine
phosphorylation of MAP kinase (57).
2· production in HUVECs was found
to be elevated within minutes from the onset of laminar shear stress
and was maintained at an elevated level as flow continued for 6 h (11, 16). Shear-induced ROS mediated the gene expression of
intercellular adhesion molecule (ICAM)-1 (11) and
c-fos (28). In this study we
hypothesized that ROS are involved in several aspects of the signal
transduction of shear stress in ECs:
1) in shear-induced cellular
tyrosine phosphorylation and, specifically,
2) in tyrosine phosphorylation of
MAP kinase, since MAP kinase is known to be activated by shear stress
(60) or oxidative stress (4, 54, 57).
2· (1). Sundaresan et al. (58)
first provided evidence that the pathway by which ligand stimulation of
ROS occurs in nonphagocytic cells involves the small GTP-binding
proteins Ras and Rac1. Expression of activated Ras or activated Rac1
isoforms resulted in increased generation of
O
2·, which was subsequently dismutated to
H2O2
(32). Expression of a dominant negative Rac1 gene product (N17rac1) was
shown to inhibit the intracellular burst of ROS in HUVECs after
hypoxia-reoxygenation, suggesting that Rac1-dependent pathways may
regulate the intracellular ROS levels during
ischemia-reperfusion (38).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
= (6µQ)/(h2w),
where
is the wall shear stress
(dyn/cm2), µ is the viscosity
of the medium (0.01 dyn · s · cm
2),
Q is the flow rate (cm3/s),
h is the channel height (0.025 cm),
and w is the channel width (2.5 cm)
(2, 7). The flow circuit was primed with 20 ml of DMEM (with 25 mM
HEPES) or ATP-free M199. In some experiments, cells were not serum
starved before shear exposure and were subsequently sheared in ATP-free
M199 containing 100 µM
Na3VO4.
Each EC monolayer was exposed to steady laminar shear stress of 20 dyn/cm2, typical of the arterial
circulation, in recirculating flow systems maintained at 37°C and
in the presence of 5% CO2 for
5-30 min. Static controls were maintained in the incubator for
5-30 min in a medium that was the same as the perfusion medium.
Immunodetection of oxidatively modified intracellular proteins. OxyBlot provides the methodology for immunodetection of carbonyl groups that are introduced into intracellular proteins by oxidative reactions (44). Protein carbonyl content in BAECs exposed to 75 µM H2O2 was shown to triple within 10 min and reached a plateau by 15 min (12). According to the instructions, the carbonyl groups of the protein side chains in cell lysates were derivatized to 2,4-dinitrophenyl (DNP)-hydrazone by reaction with DNP-hydrazine. The DNP-derivatized protein samples were separated by SDS-PAGE, then subjected to Western blotting, as described above. The membranes were incubated with primary antibody specific to the DNP moiety of the proteins, incubated with a horseradish peroxidase-conjugated goat anti-rabbit IgG, and then treated with chemiluminescent reagents, as described above.
Immunoprecipitation and Western blot for MAP kinase activation.
After their protein content was measured, all cytosolic samples were
normalized to the sample with the lowest protein content. Each sample
was rotated end-over at 4°C for 4 h with 20 µl of an
agarose-conjugated antiphosphotyrosine slurry (UBI) and centrifuged at
10,000 rpm for 30 s to separate the antiphosphotyrosine-agarose beads
from the cell lysates. The supernatant was discarded. The beads were
washed three times in PBS, resuspended in 50 µl of 2× reducing
sample buffer (0.5 M Tris · HCl, pH 6.8, 20%
glycerol, 10% SDS, 0.1% bromphenol blue, and 5% -mercaptoethanol;
Novex), and then heated in a 95°C water bath for 5 min to
dissociate the proteins from the beads. Immunoprecipitated proteins
were separated by SDS-PAGE, as described above. Western blot analysis
was performed using a rabbit anti-MAP kinase polyclonal antibody
(erk1-CT; UBI) diluted 1:1,000 in blocking buffer followed by a
biotinylated goat anti-rabbit antibody (Tropix) diluted 1:4,000 in
blocking buffer. All other reagents were identical to those used in the antiphosphotyrosine Western blots.
Cell infection with recombinant adenoviruses.
The replication-deficient adenovirus encoding the epitope-tagged
dominant-negative Rac1 cDNA (Ad.N17rac1) was constructed by homologous
recombination in 293 cells with use of the adenovirus-based plasmid
JM17, as previously described (56). The replication-deficient adenovirus Ad.gal containing the Escherichia coli
Lac Z gene has also been previously described (57). All
viruses were amplified and titered in 293 cells and purified on CsCl
gradients (23). Infections were done overnight in 80% confluent BAECs.
Western blot analysis of Rac1 expression used an antibody directed at the myc-epitope tag (9E10; Santa Cruz
Biotech, Santa Cruz, CA), which identified the N17rac1 gene product, as
described elsewhere (56). X-gal staining of BAECs infected with the
Ad.
gal at a multiplicity of infection of 100 showed >90%
transfection efficiency.
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RESULTS |
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Shear stress effects on cellular tyrosine phosphorylation.
Exposure of serum-starved (quiescent) BAECs to a calculated shear
stress of 20 dyn/cm2 without
Na3VO4
in the perfusion medium caused a rapid increase in the phosphotyrosine
content of whole cell lysates that peaked within 15 min after the onset
of shear stress and was still above baseline at 30 min (Fig.
1A).
When nonquiescent BAECS were exposed to shear stress for 5, 20, or 30 min in the presence of the tyrosine phosphatase inhibitor (100 µM),
there was much more pronounced cellular tyrosine phosphorylation in
response to shear stress (Fig. 1B).
There was a time dependency between shear stress and tyrosine
phosphorylation with saturation of the total cellular tyrosine
phosphorylation at 30 min after initiation of shear (~100% increase
over the static control; Fig. 1B).
Experiments were repeated at least three times, and a representative
experiment is shown. All corresponding static controls were incubated
for 30 min in a medium that was the same as the perfusion medium
(except for lane 2 in Fig.
1B). The addition of 100 µM
Na3VO4
for 30 min into the culture medium of static cells did not cause any
significant changes at the cellular phosphotyrosine levels compared
with static cells that were not exposed to
Na3VO4
(cf. lanes 2 and
3 in Fig. 1B). The static control cells in
Fig. 1A were different from those in
Fig. 1B, because all cells in Fig.
1A were made quiescent before treatment and analysis.
|
H2O2 effects
on cellular tyrosine phosphorylation.
Static nonquiescent BAECs were exposed to 9 or 90 µM
H2O2
for 15 min, and cell lysates were probed with an antiphosphotyrosine antibody. In the experiment shown in Fig.
2A,
representative of two identical experiments,
H2O2
enhanced the total cellular tyrosine phosphorylation in a
dose-dependent manner compared with static controls that were not
exposed to
H2O2.
When static cells were exposed to 90 µM
H2O2,
the increase in cellular tyrosine phosphorylation was ~200% (Fig.
2A). When nonquiescent cells were
incubated with 9 µM
H2O2
for 15 min before 20 min of shear exposure (in the presence of 100 µM
Na3VO4),
the increase in total cellular tyrosine phosphorylation was even
greater than in sheared cells that were not preincubated with
H2O2
(Fig. 2B). Static control cells were
exposed to a medium that was the same as the perfusion medium for 20 min (Fig. 2B). Therefore, exposure
to
H2O2
and application of fluid shear stress may have additive effects on
cellular tyrosine phosphorylation.
|
Shear stress effects on the intracellular redox status.
Exposure of quiescent BAECs to 5 min of arterial shear stress (20 dyn/cm2) without
Na3VO4
caused a marked increase in the amount of oxidatively modified proteins
in whole cell lysates, as measured by the OxyBlot oxidized protein
detection kit (Fig. 3). Within 15 min of
exposure to shear stress, there was a drop in the levels of protein
oxidation, possibly because of counteraction of ROS by the
intracellular antioxidant defense systems. When ECs were incubated with
the membrane-permeant antioxidant NAC for 4 h before 15 min of shear exposure (without
Na3VO4),
it was shown that 20 mM NAC partly inhibited the shear-induced cellular
protein oxidation (Fig. 3), whereas 5 mM NAC had no effect (not shown).
NAC at 20 mM had no effect on protein oxidation of static controls not
exposed to H2O2
(not shown). This experiment was repeated twice with similar results.
|
Effects of antioxidants on shear-induced cytosolic tyrosine
phosphorylation.
To investigate whether ROS are involved in the signal transduction
caused by shear stress, nonquiescent BAECs were incubated with 5 or 20 mM NAC for 4 h before 30 min of shear exposure in the presence of
Na3VO4.
Static controls were incubated for 30 min in a medium that was the same
as the perfusion medium. When cytosolic proteins were probed for
antiphosphotyrosine, it was found that NAC effectively inhibited
tyrosine phosphorylation in the cell cytosolic fraction in a
dose-dependent manner (Fig. 4). Incubation
with micromolar concentrations of a thiol antioxidant, PDTC, for 4 h
before shear exposure also inhibited the shear-induced cytosolic
tyrosine phosphorylation (Fig. 4). Neither of the antioxidants had any
effect on the cytosolic tyrosine phosphorylation of static controls
(not shown). This experiment was repeated at least twice. In the
experiment shown, 30 min of shear stress caused a 50% increase in
cytosolic tyrosine phosphorylation compared with static control, whereas previous exposure to NAC (20 mM) or PDTC (100 µM) followed by
shear resulted in only a 10% and a 5% increase in cytosolic tyrosine
phosphorylation, respectively, compared with static control. NAC also
inhibited the increase in cytosolic tyrosine phosphorylation in
quiescent BAECs that were sheared in the absence of
Na3VO4 (not shown).
|
Effects of antioxidants on shear-induced MAP kinase tyrosine
phosphorylation.
Because tyrosine phosphorylation is necessary for MAP kinase
activation, we investigated the ability of antioxidants to inhibit the
shear-induced tyrosine phosphorylation of MAP kinase. In Fig. 5A,
quiescent cells were sheared in the absence of
Na3VO4;
in Fig. 5B, nonquiescent cells were
sheared in the presence of
Na3VO4. In either case, cytosolic fractions were immunoprecipitated with an
antibody to phosphotyrosine and immunoprecipitates were probed with an
antibody (erk1-CT) to the phosphorylated 42- and 44-kDa isoforms of MAP
kinase (NIH/3T3 cell lysates were included as a positive control).
There was a time-dependent relationship between shear stress and MAP
kinase tyrosine phosphorylation, with peak phosphorylation occurring at
5 min after exposure to shear in Fig.
5A and at 10 min after exposure to
shear in Fig. 5B (longer times are not
shown). As was the case with cellular tyrosine phosphorylation, NAC
blocked the shear-induced increase in tyrosine phosphorylation of MAP
kinase in a dose-dependent manner (Fig.
5B). These experiments were repeated
twice with similar results: BAEC incubation with 5 mM NAC followed by
exposure to 5 min of shear stress did not cause any change in the
tyrosine phosphorylation levels of MAP kinase compared with cells that
were exposed to 5 min of shear without the NAC preincubation (Fig.
5B). However, BAEC incubation with
20 mM NAC followed by exposure to shear stress caused the shear-induced
tyrosine phosphorylation of MAP kinase to remain slightly above the
baseline levels (in the case of treatment with 20 mM NAC followed by 5 min of flow; Fig. 5B) or at the
baseline levels (in the case of treatment with 20 mM NAC followed by 10 min of flow; Fig. 5).
|
Role of Rac1-mediated ROS production in cellular and MAP kinase
tyrosine phosphorylation.
Because the GTPase Rac1 is an integral part of the NADPH oxidase
complex and regulates ROS production in phagocytic and nonphagocytic cells (1, 38, 58), we asked whether the flow-induced cellular tyrosine
phosphorylation was dependent on Rac1-mediated ROS production. Adenovirus-mediated expression of the dominant-negative Rac1, N17rac1,
in BAECs markedly attenuated the flow-mediated increase in cellular
tyrosine phosphorylation (Fig.
6A;
nonquiescent BAECs that were sheared in the presence of
Na3VO4).
Static control cells infected with Ad.gal displayed a profile of
tyrosine-phosphorylated proteins that was slightly different from that
of noninfected cells (cf. lane 3 of
Fig. 1B with lane
1 of Fig. 6A).
However, Ad.
gal-infected cells displayed a tyrosine phosphorylation
response to flow that was similar to that of noninfected cells (cf.
lane 6 of Fig.
1B with lane
2 of Fig. 6A). When
quiescent Ad.
gal- or Ad.N17rac1-infected BAECs were sheared in the
absence of
Na3VO4 and cytosolic fractions were analyzed for MAP kinase tyrosine phosphorylation, expression of N17rac1, and not of
gal, completely blocked the shear-induced tyrosine phosphorylation of MAP kinase (Fig.
6B). Each of these experiments was
repeated twice with identical results.
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DISCUSSION |
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The present study suggests that ROS act as signal-transducing molecules
in ECs exposed to fluid shear stress or, more appropriately, to a step
change in fluid shear stress. ROS have already been implicated in the
signal transduction of PDGF in VSMCs (57). ROS were also involved in
cyclic strain-induced cellular responses in ECs, specifically, the
MCP-1 gene expression (62) and release of PAI-1 (10). Recent studies
demonstrated that shear flow to ECs can induce intracellular ROS
generation, which results in increased ICAM-1 (11) and
c-fos gene expression (28). Shear stress-mediated ROS production may be partly responsible for the activation by shear stress of the redox-sensitive transcription factors
nuclear factor-B and AP-1 (5, 41) and for activation of Ras, the
small GTP-binding protein upstream of MAP kinase, which is a signaling
target of ROS (42) and is also activated by shear stress (46). Recent
findings on gene induction of Cu-Zn superoxide dismutase (SOD) and
mitochondrial Mn SOD by shear stress suggest the activation of cellular
defense mechanisms against oxidative stress caused by EC exposure to
shear stress (31, 59).
Our findings on the effects of arterial shear stress on cellular tyrosine phosphorylation agree with those of other investigators. Specifically, Ayajiki et al. (3) showed that 30 min of arterial shear stress markedly increased the cellular tyrosine phosphorylation of 103- and 114-kDa proteins in cultured HUVECs. This range of proteins corresponds to the major band observed in cell lysates of quiescent BAECs that were sheared in the absence of the tyrosine phosphatase inhibitor (Fig. 1A). In agreement with our findings on shear-induced MAP kinase tyrosine phosphorylation (Fig. 5A), Tseng et al. (60) showed that arterial shear stress induced tyrosine phosphorylation and activation of MAP kinase in BAECs, with a peak activation time of 2-10 min after the onset of shear stress.
In this study we demonstrated the importance of intracellular ROS as modulators for shear-induced cellular tyrosine phosphorylation and, in particular, tyrosine phosphorylation of MAP kinase, by providing the first evidence that the shear-stimulated tyrosine phosphorylation response does require ROS. Thus the antioxidant NAC at 20 mM, which acts as a scavenger for ROS intermediates and as a precursor for glutathione (48, 53), inhibited the shear-induced cytosolic and MAP kinase tyrosine phosphorylation (Figs. 4 and 5). Reducing thiol agents, such as PDTC, affect the expression of thioredoxin, an oxidoreductase with antioxidant functions (50). PDTC at 100 µM was effective in inhibiting tyrosine phosphorylation in cytosolic fractions of sheared BAECs (Fig. 4). NAC at 20 mM was also shown to inhibit the shear-induced intracellular protein oxidation that preceded in time the cellular tyrosine phosphorylation signal but coincided with the MAP kinase tyrosine phosphorylation signal (Fig. 3). The importance of intracellular ROS in shear-induced MAP kinase activation agrees with findings by Sundaresan et al. (57), who demonstrated that NAC caused a concentration-dependent reduction of MAP kinase activation in VSMCs stimulated by PDGF.
The pathway by which ROS are generated is best characterized in
phagocytic cells. Activation of phagocytic cells leads to the assembly
of the NADPH oxidase enzymatic complex, which transfers electrons from
NADPH to molecular O2 with the
subsequent generation of O2· and
appears to be regulated by the GTPase Rac1 (1, 8). Many, but not all,
of the components of the NADPH oxidase complex have been shown to be
expressed in a variety of nonphagocytic cell types, including ECs (35).
In this report we provide the first evidence that the small GTP-binding protein Rac1 functions as a regulator of the shear-induced ROS production. This agrees with the results by Sundaresan et al. (58), who
demonstrated that Rac1, as well as Ras, regulates the increase of
intracellular ROS in NIH/3T3 cells on stimulation with growth factors,
the receptors of which have intrinsic tyrosine kinase activity, or
cytokines. Several other second-messenger responses to shear stress,
such as changes in phosphatidylinositol lipid metabolism, intracellular
free Ca2+ levels, and prostacyclin
and NO release, are similar to responses resulting from
agonist-receptor coupling, suggesting that they may share signal
transduction pathways. In addition, Kim et al. (38) recently
demonstrated a requirement for Rac1, but not for Ras proteins, in the
generation of intracellular ROS after reoxygenation of hypoxic HUVECs.
All our shear experiments were repeated in the presence of the tyrosine phosphatase inhibitor Na3VO4 (100 µM) in the perfusion medium to amplify the shear-induced tyrosine phosphorylation signal. Use of Na3VO4 during the shear exposure is controversial, because 1) vanadate has been demonstrated to stimulate NADPH oxidation (47), 2) vanadate is shown to increase the tyrosine kinase activity of the endogenous insulin receptor kinase in adipocytes (36), 3) vanadate is known to act synergistically with compounds that activate protein kinase C to form ROS, which enhance protein tyrosine phosphorylation in macrophages (20), and 4) vanadate is an inhibitor of Na+-K+-ATPase (37). In our case, however, it seems unlikely that Na3VO4 by itself increases tyrosine kinase activity, since addition of 100 µM Na3VO4 into the medium of static control cells, for a maximum of 30 min, did not cause any significant changes in total cellular tyrosine phosphorylation (Fig. 1B). Only with exposure to shear stress did Na3VO4 greatly enhance the total cellular tyrosine phosphorylation in BAECs (Fig. 1). In contrast, it had no visible additive effect on the shear-induced tyrosine phosphorylation of MAP kinase (Fig. 5). The fact that the effect of ROS on cellular tyrosine phosphorylation was potentiated by added vanadate is due to the combined activation of protein tyrosine kinases and the inactivation of protein tyrosine phosphatases (25, 51). Although orthovanadate could be working on its own, it is more likely that it combines with H2O2 to form pervanadate, a more potent inhibitor of protein tyrosine phosphatases (30). Similarly, Na3VO4 alone, at micromolar concentrations, produced no increase in DNA binding of the transcription factor AP-1, whereas the same concentrations of Na3VO4 greatly enhanced the H2O2-mediated activation of AP-1 in porcine aortic ECs (5).
Laurindo et al. (43) were the first to demonstrate free radical
generation, in vivo and ex vivo, due to step increases in shear stress.
Their techniques for measuring ROS, electron paramagnetic resonance
spectroscopy and measurements of ascorbic radicals in plasma, allowed
them to detect mainly extracellular
O2· release. This was also
suggested by the fact that the measured shear-induced increase in ROS
was abolished by addition of SOD, a superoxide scavenger that does not
cross intact EC membranes or intercellular junctions. However,
different cellular enzymatic systems have been recognized as sources
for ROS production in ECs exposed to reoxygenation after hypoxia or
anoxia, depending on whether the technique employed measures
intracellular or extracellular ROS. Specifically, with use of electron
paramagnetic resonance spectroscopy, it was found that BAECs subjected
to anoxia followed by reoxygenation generate oxygen free radicals, and
the signal was partially inhibited by allopurinol, an inhibitor of XO
(65). XO reduces molecular O2 and
generates intracellular O
2·, which
then dismutates to form
H2O2.
With use of a fluorometric assay that measured the extracellular
H2O2
release by bovine pulmonary artery ECs on reoxygenation, it was shown
that allopurinol had no effect and only the flavoprotein inhibitor
diphenylene iodonium (DPI) reduced the
H2O2
release (64). DPI inhibits the membrane-bound enzyme NADPH oxidase,
which reduces molecular O2 to
O
2·, and other flavoproteins, such
as XO, ecNOS, and NADH dehydrogenase (18, 24, 55). With use of a
fluorometric assay sensitive to intracellular
H2O2
and peroxynitrite, it was found that allopurinol and
NG-methyl-L-arginine,
an inhibitor of ecNOS, reduced intracellular ROS, suggesting that XO
generates O
2·, which partly
dismutates to
H2O2
and partly reacts with NO to form peroxynitrite (63). Although the ROS
sources in cultured ECs have been studied extensively for
hypoxia-reoxygenation, the shear-induced ROS production was only
recently studied. Intracellular
O
2· levels in sheared HUVECs were
measured by lucigenin-amplified chemiluminescence (11),
2',7'-dichlorofluorescin fluorescence (28), and ethidium
fluorescence (16), and, in each case, steady laminar shear stress was
found to increase the EC intracellular ROS. An elevated level was
measured by 15 min, remained elevated as flow continued for up to 6 h,
but returned to baseline at 24 h.
Because ECs are a rich source of XO, XO is a suitable candidate enzymatic system for flow-triggered intracellular ROS generation, with possible contributions by ecNOS, cyclooxygenase, phospholipase A2 (PLA2), and the mitochondrial (NADH dehydrogenase and cytochrome oxidase) and microsomal (cytochrome P-450) electron transport chains. However, our study proved that Rac1 is a major component in generating ROS in sheared ECs, indicating the possible involvement of the plasma membrane-bound NADPH oxidase complex. Indeed, NADPH oxidase activity in cultured HUVECs was found to be induced by steady laminar shear stress at 1 and 5 h, a transient response that returned to baseline at 24 h (no shorter times were tested) (16). There are plenty of data available that suggest that exposure to shear stress might activate the NADPH oxidase enzyme. 1) MAP kinase is activated by shear stress, PLA2 is activated by MAP kinase (34), and products of PLA2 (largely arachidonic acid) are known to mediate activation of NADPH oxidase (14). 2) Superoxide production in NIH/3T3 fibroblasts stably transfected with a constitutively active isoform of Ras was found to be inhibited by DPI (32). Because Ras is upstream of MAP kinase and is activated by shear stress (46), it is expected that NADPH oxidase and other flavoproteins might be a source for shear-induced ROS. 3) The activity of NADPH oxidase was increased in cyclically strained porcine aortic ECs (27), suggesting that NADPH oxidase might play a role in the generation of oxidative stress in the mechanically deformed vessel wall.
Because shear-induced ROS production mediates the tyrosine phosphorylation and, presumably, activation of MAP kinase, which may be one of the kinases that phosphorylate ecNOS, leading to the sustained release of NO, our results suggest that EC free radical production may exert an autocrine role in the control of vascular tone by shear stress. It is also known that shear stress-mediated NO formation inhibits apoptosis of cultured HUVECs (17, 26). If ROS, through MAP kinase, regulate the sustained release of NO, then shear-induced ROS generation may indirectly interfere with cell death signal transduction and contribute to EC integrity. Finally, flow-triggered oxidative stress may also play a role in hemodynamic force-induced pathological conditions, such as the endothelial dysfunction associated with atherosclerosis, the endothelial dysfunction after hypoxia and reperfusion, and the superoxide-dependent vasoconstriction that occurs after balloon angioplasty.
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ACKNOWLEDGEMENTS |
---|
We acknowledge the expert technical assistance of S. J. Gips, C. C. Wilhide, and T. C. Huang during the different phases of the study. We
thank A. Hall for the N17rac1 cDNA and R. G. Crystal for the Ad.gal.
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FOOTNOTES |
---|
B. R. Alevriadou was supported in this work by National Heart, Lung, and Blood Institute Grant HL-54089, a Whitaker Biomedical Engineering research grant, and a grant from the Center for Alternatives to Animal Testing. K. Irani was supported by the Johns Hopkins Clinician Scientist Award, the Bernard Foundation, and an endowment from Mr. and Mrs. Abraham Weiss.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: B. R. Alevriadou, The Johns Hopkins University School of Medicine, BME Dept., Traylor Bldg., Rm. 619, 720 Rutland Ave., Baltimore, MD 21205 (E-mail: ralevria{at}bme.jhu.edu).
Received 23 April 1998; accepted in final form 13 January 1999.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abo, A.,
E. Pick,
A. Hall,
N. Totty,
C. G. Teahan,
and
A. W. Segal.
Activation of the NADPH oxidase involves the small GTP-binding protein p21rac1.
Nature
353:
668-670,
1991[Medline].
2.
Alevriadou, B. R.,
and
L. V. McIntire.
Rheology.
In: Thrombosis and Hemorrhage, edited by J. Loscalzo,
and A. I. Schafer. Cambridge, MA: Blackwell, 1994, p. 369-381.
3.
Ayajiki, K.,
M. Kindermann,
M. Hecker,
I. Fleming,
and
R. Busse.
Intracellular pH and tyrosine phosphorylation but not calcium determine shear stress-induced nitric oxide production in native endothelial cells.
Circ. Res.
78:
750-758,
1996
4.
Baas, A. S.,
and
B. C. Berk.
Differential activation of mitogen-activated protein kinases by H2O2 and O2 in vascular smooth muscle cells.
Circ. Res.
77:
29-36,
1995
5.
Barchowsky, A.,
S. R. Munro,
S. J. Morana,
M. P. Vincenti,
and
M. Treadwell.
Oxidant-sensitive and phosphorylation-dependent activation of NF-B and AP-1 in endothelial cells.
Am. J. Physiol.
269 (Lung Cell. Mol. Physiol. 13):
L829-L836,
1995
6.
Berk, B. C.,
M. A. Corson,
T. E. Peterson,
and
H. Tseng.
Protein kinases as mediators of fluid shear stress stimulated signal transduction in endothelial cells: a hypothesis for calcium-dependent and calcium-independent events activated by flow.
J. Biomech.
28:
1439-1450,
1995[Medline].
7.
Bird, R. B.,
W. E. Stewart,
and
E. N. Lightfoot.
Transport Phenomena. New York: Wiley, 1960.
8.
Bokoch, G. M.
Regulation of the human neutrophil NADPH oxidase by the Rac GTP-binding proteins.
Curr. Opin. Cell Biol.
6:
212-218,
1994[Medline].
9.
Carbajal, J. M.,
and
R. C. Schaeffer, Jr.
H2O2 and genistein differentially modulate protein tyrosine phosphorylation, endothelial morphology, and monolayer barrier function.
Biochem. Biophys. Res. Commun.
249:
461-466,
1998[Medline].
10.
Cheng, J. J.,
Y. J. Chao,
B. S. Wung,
and
D. L. Wang.
Cyclic strain-induced plasminogen activator inhibitor-1 (PAI-1) release from endothelial cells involves reactive oxygen species.
Biochem. Biophys. Res. Commun.
225:
100-105,
1996[Medline].
11.
Chiu, J. J.,
B. S. Wung,
J. Y. Shyy,
H. J. Hsieh,
and
D. L. Wang.
Reactive oxygen species are involved in shear stress-induced intercellular adhesion molecule-1 expression in endothelial cells.
Arterioscler. Thromb. Vasc. Biol.
17:
3570-3577,
1997
12.
Ciolino, H. P.,
and
R. L. Levine.
Modification of proteins in endothelial cell death during oxidative stress.
Free Radic. Biol. Med.
22:
1277-1282,
1997[Medline].
13.
Corson, M. A.,
N. L. James,
S. E. Latta,
R. M. Nerem,
B. C. Berk,
and
D. G. Harrison.
Phosphorylation of endothelial nitric oxide synthase in response to fluid shear stress.
Circ. Res.
79:
984-991,
1996
14.
Dana, R.,
H. L. Malech,
and
R. Levy.
The requirement for phospholipase A2 for activation of the assembled NADPH oxidase in human neutrophils.
Biochem. J.
297:
217-223,
1994[Medline]. [Corrigenda. Biochem. J. 298: 759, 1994.]
15.
Davies, P. F.,
A. Robotewskyj,
and
M. L. Griem.
Quantitative studies of endothelial cell adhesion. Directional remodeling of focal adhesion sites in response to flow forces.
J. Clin. Invest.
93:
2031-2038,
1994[Medline].
16.
De Keulenaer, G. W.,
D. C. Chappell,
N. Ishizaka,
R. M. Nerem,
R. W. Alexander,
and
K. K. Griendling.
Oscillatory and steady laminar shear stress differentially affect human endothelial redox state. Role of a superoxide-producing NADH oxidase.
Circ. Res.
82:
1094-1101,
1998
17.
Dimmeler, S.,
J. Haendeler,
M. Nehls,
and
A. M. Zeiher.
Suppression of apoptosis by nitric oxide via inhibition of interleukin-1-converting enzyme (ICE)-like and cysteine protease protein (CPP)-32-like proteases.
J. Exp. Med.
185:
601-607,
1997
18.
Doussiere, J.,
and
P. V. Vignais.
Diphenylene iodonium as an inhibitor of the NADPH oxidase complex of bovine neutrophils. Factors controlling the inhibitory potency of diphenylene iodonium in a cell-free system of oxidase activation.
Eur. J. Biochem.
208:
61-71,
1992[Abstract].
19.
Frangos, J. A.,
S. G. Eskin,
L. V. McIntire,
and
C. L. Ives.
Flow effects on prostacyclin production in cultured human endothelial cells.
Science
227:
1477-1479,
1985[Medline].
20.
Goldman, R.,
and
U. Zor.
Activation of macrophage PtdIns-PLC by phorbol ester and vanadate: involvement of reactive oxygen species and tyrosine phosphorylation.
Biochem. Biophys. Res. Commun.
199:
334-338,
1994[Medline].
21.
Grabowski, E. F.,
E. A. Jaffe,
and
B. B. Weksler.
Prostacyclin production by cultured endothelial cell monolayers exposed to step increases in shear stress.
J. Lab. Clin. Med.
103:
36-43,
1985.
22.
Guan, J. L.,
and
D. Shalloway.
Regulation of focal adhesion-associated protein tyrosine kinase by both cellular adhesion and oncogenic transformation.
Nature
358:
690-692,
1992[Medline].
23.
Guzman, R. J.,
E. A. Hirschowitz,
S. L. Brody,
R. G. Crystal,
S. E. Epstein,
and
T. Finkel.
In vivo suppression of injury-induced vascular smooth muscle cell accumulation using adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene.
Proc. Natl. Acad. Sci. USA
91:
10732-10736,
1994
24.
Hancock, J. T.,
and
O. T. Jones.
The inhibition by diphenyleneiodonium and its analogues of superoxide generation by macrophages.
Biochem. J.
242:
103-107,
1987[Medline].
25.
Hecht, D.,
and
Y. Zick.
Selective inhibition of protein tyrosine phosphatase activities by H2O2 and vanadate in vitro.
Biochem. Biophys. Res. Commun.
188:
773-779,
1992[Medline].
26.
Hermann, C.,
A. M. Zeiher,
and
S. Dimmeler.
Shear stress inhibits H2O2-induced apoptosis of human endothelial cells by modulation of the glutathione redox cycle and nitric oxide synthase.
Arterioscler. Thromb. Vasc. Biol.
17:
3588-3592,
1997
27.
Howard, A. B.,
R. W. Alexander,
R. M. Nerem,
K. K. Griendling,
and
W. R. Taylor.
Cyclic strain induces an oxidative stress in endothelial cells.
Am. J. Physiol.
272 (Cell Physiol. 41):
C421-C427,
1997
28.
Hsieh, H. J.,
C. C. Cheng,
S. T. Wu,
J. J. Chiu,
B. S. Wung,
and
D. L. Wang.
Increase of reactive oxygen species (ROS) in endothelial cells by shear flow and involvement of ROS in shear-induced c-fos expression.
J. Cell. Physiol.
175:
156-162,
1998[Medline].
29.
Hsieh, H. J.,
N. Q. Li,
and
J. A. Frangos.
Pulsatile and steady flow induces c-fos expression in human endothelial cells.
J. Cell. Physiol.
154:
143-151,
1993[Medline].
30.
Huyer, G.,
S. Liu,
J. Kelly,
J. Moffat,
P. Payette,
B. Kennedy,
G. Tsaprailis,
M. J. Gresser,
and
C. Ramachandran.
Mechanism of inhibition of protein-tyrosine phosphatases by vanadate and pervanadate.
J. Biol. Chem.
272:
843-851,
1997
31.
Inoue, N.,
S. Ramasamy,
T. Fukai,
R. M. Nerem,
and
D. G. Harrison.
Shear stress modulates expression of Cu/Zn superoxide dismutase in human aortic endothelial cells.
Circ. Res.
79:
32-37,
1996
32.
Irani, K.,
Y. Xia,
J. L. Zweier,
S. J. Sollott,
C. J. Der,
E. R. Fearon,
M. Sundaresan,
T. Finkel,
and
P. J. Goldschmidt-Clermont.
Mitogenic signaling mediated by oxidants in Ras-transformed fibroblasts.
Science
275:
1649-1652,
1997
33.
Jalali, S.,
Y. S. Li,
M. Sotoudeh,
S. Yuan,
S. Li,
S. Chien,
and
J. Y. Shyy.
Shear stress activates p60src-Ras-MAPK signaling pathways in vascular endothelial cells.
Arterioscler. Thromb. Vasc. Biol.
18:
227-234,
1998
34.
Johnson, G. L.,
and
R. R. Vaillancourt.
Sequential protein kinase reactions controlling cell growth and differentiation.
Curr. Opin. Cell Biol.
6:
230-238,
1994[Medline].
35.
Jones, S. A.,
V. B. O'Donnell,
J. D. Wood,
J. P. Broughton,
E. J. Hughes,
and
O. T. Jones.
Expression of phagocyte NADPH oxidase components in human endothelial cells.
Am. J. Physiol.
271 (Heart Circ. Physiol. 40):
H1626-H1634,
1996
36.
Kadota, S.,
I. G. Fantus,
G. Deragon,
H. J. Guyda,
and
B. I. Posner.
Stimulation of insulin-like growth factor II receptor binding and insulin receptor kinase activity in rat adipocytes. Effects of vanadate and H2O2.
J. Biol. Chem.
262:
8252-8256,
1987
37.
Karlish, S. J.,
L. A. Beauge,
and
I. M. Glynn.
Vanadate inhibits (Na+-K+)ATPase by blocking a conformational change of the unphosphorylated form.
Nature
282:
333-335,
1979[Medline].
38.
Kim, K. S.,
K. Takeda,
R. Sethi,
J. B. Pracyk,
K. Tanaka,
Y. F. Zhou,
Z. X. Yu,
V. J. Ferrans,
J. T. Bruder,
I. Kovesdi,
K. Irani,
P. Goldschmidt-Clermont,
and
T. Finkel.
Protection from reoxygenation injury by inhibition of rac1.
J. Clin. Invest.
101:
1821-1826,
1998
39.
Kuchan, M. J.,
and
J. A. Frangos.
Role of calcium and calmodulin in flow-induced nitric oxide production in endothelial cells.
Am. J. Physiol.
266 (Cell Physiol. 35):
C628-C636,
1994
40.
Kuchan, M. J.,
H. Jo,
and
J. A. Frangos.
Role of G proteins in shear stress-mediated nitric oxide production by endothelial cells.
Am. J. Physiol.
267 (Cell Physiol. 36):
C753-C758,
1994
41.
Lan, Q.,
K. O. Mercurius,
and
P. F. Davies.
Stimulation of transcription factors NFB and AP1 in endothelial cells subjected to shear stress.
Biochem. Biophys. Res. Commun.
201:
950-956,
1994[Medline].
42.
Lander, H. M.,
J. S. Ogiste,
K. K. Teng,
and
A. Novogrodsky.
p21ras as a common signaling target of reactive free radicals and cellular redox stress.
J. Biol. Chem.
270:
21195-21198,
1995
43.
Laurindo, F. R.,
A. M. Pedro,
H. V. Barbeiro,
F. Pileggi,
M. H. Carvalho,
O. Augusto,
and
P. L. da Luz.
Vascular free radical release. Ex vivo and in vivo evidence for a flow-dependent endothelial mechanism.
Circ. Res.
74:
700-709,
1994[Abstract].
44.
Levine, R. L.,
J. A. Williams,
E. R. Stadtman,
and
E. Shacter.
Carbonyl assays for determination of oxidatively modified proteins.
Methods Enzymol.
233:
346-357,
1994[Medline].
45.
Li, S.,
M. Kim,
Y. L. Hu,
S. Jalali,
D. D. Schlaepfer,
T. Hunter,
S. Chien,
and
J. Y. Shyy.
Fluid shear stress activation of focal adhesion kinase. Linking to mitogen-activated protein kinases.
J. Biol. Chem.
272:
30455-30462,
1997
46.
Li, Y. S.,
J. Y. Shyy,
S. Li,
J. Lee,
B. Su,
M. Karin,
and
S. Chien.
The Ras-JNK pathway is involved in shear-induced gene expression.
Mol. Cell. Biol.
16:
5947-5954,
1996[Abstract].
47.
Liochev, S. I.,
and
I. Fridovich.
Superoxide generated by glutathione reductase initiates a vanadate-dependent free radical chain oxidation of NADH.
Arch. Biochem. Biophys.
294:
403-406,
1992[Medline].
48.
Phelps, D. T.,
S. M. Deneke,
D. L. Daley,
and
B. L. Fanburg.
Elevation of glutathione levels in bovine pulmonary artery endothelial cells by N-acetylcysteine.
Am. J. Respir. Cell. Mol. Biol.
7:
293-299,
1992[Medline].
49.
Rubanyi, G. M.,
J. C. Romero,
and
P. M. Vanhoutte.
Flow-induced release of endothelium-derived relaxing factor.
Am. J. Physiol.
250 (Heart Circ. Physiol. 19):
H1145-H1149,
1986
50.
Schenk, H.,
M. Klein,
W. Erdbrugger,
W. Droge,
and
K. Schulze-Osthoff.
Distinct effects of thioredoxin and antioxidants on the activation of transcription factors NF-B and AP-1.
Proc. Natl. Acad. Sci. USA
91:
1672-1676,
1994[Abstract].
51.
Schieven, G. L.,
J. M. Kirihara,
D. E. Myers,
J. A. Ledbetter,
and
F. M. Uckun.
Reactive oxygen intermediates activate NF-B in a tyrosine kinase-dependent mechanism and in combination with vanadate activate the p56lck and p59fyn tyrosine kinases in human lymphocytes.
Blood
82:
1212-1220,
1993[Abstract].
52.
Sharefkin, J. B.,
S. L. Diamond,
S. G. Eskin,
L. V. McIntire,
and
C. W. Dieffenbach.
Fluid flow decreases preproendothelin mRNA levels and suppresses endothelin-1 peptide release in cultured human endothelial cells.
J. Vasc. Surg.
14:
1-9,
1991[Medline].
53.
Staal, F. J.,
M. Roederer,
L. A. Herzenberg,
and
L. A. Herzenberg.
Intracellular thiols regulate activation of nuclear factor B and transcription of human immunodeficiency virus.
Proc. Natl. Acad. Sci. USA
87:
9943-9947,
1990[Abstract].
54.
Stevenson, M. A.,
S. S. Pollock,
C. N. Coleman,
and
S. K. Calderwood.
X-irradiation, phorbol esters, and H2O2 stimulate mitogen-activated protein kinase activity in NIH-3T3 cells through the formation of reactive oxygen intermediates.
Cancer Res.
54:
12-15,
1994[Abstract].
55.
Stuehr, D. J.,
O. A. Fasehun,
N. S. Kwon,
S. S. Gross,
J. A. Gonzalez,
R. Levi,
and
C. F. Nathan.
Inhibition of macrophage and endothelial cell nitric oxide synthase by diphenyleneiodonium and its analogs.
FASEB J.
5:
98-103,
1991
56.
Sulciner, D. J.,
K. Irani,
Z. X. Yu,
V. J. Ferrans,
P. Goldschmidt-Clermont,
and
T. Finkel.
rac1 regulates a cytokine-stimulated, redox-dependent pathway necessary for NF-B activation.
Mol. Cell. Biol.
16:
7115-7121,
1996[Abstract].
57.
Sundaresan, M.,
Z. X. Yu,
V. J. Ferrans,
K. Irani,
and
T. Finkel.
Requirement for generation of H2O2 for platelet-derived growth factor signal transduction.
Science
270:
296-299,
1995[Abstract].
58.
Sundaresan, M.,
Z. X. Yu,
V. J. Ferrans,
D. J. Sulciner,
J. S. Gutkind,
K. Irani,
P. J. Goldschmidt-Clermont,
and
T. Finkel.
Regulation of reactive-oxygen-species generation in fibroblasts by Rac1.
Biochem. J.
318:
379-382,
1996[Medline].
59.
Topper, J. N.,
J. Cai,
D. Falb,
and
M. A. Gimbrone, Jr.
Identification of vascular endothelial genes differentially responsive to fluid mechanical stimuli: cyclooxygenase-2, manganese superoxide dismutase, and endothelial cell nitric oxide synthase are selectively up-regulated by steady laminar shear stress.
Proc. Natl. Acad. Sci. USA
93:
10417-10422,
1996
60.
Tseng, H.,
T. E. Peterson,
and
B. C. Berk.
Fluid shear stress stimulates mitogen-activated protein kinase in endothelial cells.
Circ. Res.
77:
869-878,
1995
61.
Vepa, S.,
W. M. Scribner,
and
V. Natarajan.
Activation of protein phosphorylation by oxidants in vascular endothelial cells: identification of tyrosine phosphorylation of caveolin.
Free Radic. Biol. Med.
22:
25-35,
1997[Medline].
62.
Wung, B. S.,
J. J. Cheng,
H. J. Hsieh,
Y. J. Shyy,
and
D. L. Wang.
Cyclic strain-induced monocyte chemotactic protein-1 gene expression in endothelial cells involves reactive oxygen species activation of activator protein 1.
Circ. Res.
81:
1-7,
1997
63.
Zulueta, J. J.,
R. Sawhney,
F. S. Yu,
C. C. Cote,
and
P. M. Hassoun.
Intracellular generation of reactive oxygen species in endothelial cells exposed to anoxia-reoxygenation.
Am. J. Physiol.
272 (Lung Cell. Mol. Physiol. 16):
L897-L902,
1997
64.
Zulueta, J. J.,
F. S. Yu,
I. A. Hertig,
V. J. Thannickal,
and
P. M. Hassoun.
Release of hydrogen peroxide in response to hypoxia-reoxygenation: role of an NAD(P)H oxidase-like enzyme in endothelial cell plasma membrane.
Am. J. Respir. Cell. Mol. Biol.
12:
41-49,
1995[Abstract].
65.
Zweier, J. L.,
P. Kuppusamy,
and
G. A. Lutty.
Measurement of endothelial cell free radical generation: evidence for a central mechanism of free radical injury in postischemic tissues.
Proc. Natl. Acad. Sci. USA
85:
4046-4050,
1988[Abstract].