© 1995 American Heart Association, Inc.
Articles |
Fluid Shear Stress Stimulates Mitogen-Activated Protein Kinase in Endothelial Cells
From the Department of Physiology, Emory University, Atlanta, Ga (H.T.), and the Department of Medicine (Cardiology Division), University of Washington, Seattle.
Correspondence to Bradford C. Berk, MD, PhD, University of Washington, Cardiology Division, Box 357710, Seattle, WA 98195. E-mail beberk@u.washington.edu.
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Abstract Local alterations in the hemodynamic environment regulate endothelial cell function, but the signal-transduction mechanisms involved in this process remain unclear. Because mitogen-activated protein (MAP) kinases have been shown to be activated by physical forces, we measured the phosphorylation and enzyme activity of MAP kinase to identify the signal events involved in the endothelial cell response to fluid shear stress. Flow at physiological shear stress (3.5 to 117 dynes/cm2) activated 42-kD and 44-kD MAP kinases present in cultured bovine aortic endothelial cells, with maximal effect at 12 dynes/cm2. Activation of a G protein was necessary, as demonstrated by complete inhibition by the nonhydrolyzable GDP analog GDP-ßS. Activation of protein kinase C (PKC) was required, as shown by inhibiting PKC with staurosporine or downregulating PKC with phorbol 12,13-dibutyrate. Both Ca2+-dependent and -independent PKC activity, measured by translocation and substrate phosphorylation, increased in response to flow. However, MAP kinase activation was not dependent on Ca2+ mobilization, since Ca2+ chelation had no inhibitory effect. On the basis of these findings, it is proposed that flow activates two signal-transduction pathways in endothelial cells. One pathway is Ca2+ dependent and involves activation of phospholipase C and increases in intracellular Ca2+. A new pathway, described in the present study, is Ca2+ independent and involves a G protein and increases in PKC and MAP kinase activity.
Key Words: endothelium • signal transduction • MAP kinase • protein kinase C • shear stress
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fluid shear stress is an important hemodynamic force recognized by endothelial cells that modulates vessel function and structure. Changes in shear stress stimulate the secretion of several factors that regulate vessel tone, including vasodilators, such as nitric oxide1 and prostacyclin,2 and vasoconstrictors, such as endothelin-1.3 Changes in shear stress also cause long-term alterations in vessel function4 through regulation of protein3 and gene expression.5 6 7 The importance of shear stress in the pathogenesis of vascular disease is evidenced by the fact that areas of low shear stress, such as the carotid sinus, have an increased predisposition for developing atherosclerosis.8 In contrast, areas exposed to high shear stress, such as flow dividers, exhibit the least lipid accumulation.8
It has become clear that force-transduction mechanisms in
anchorage-dependent cells are due to a combination of force
transmission via cytoskeletal elements and transduction of the physical
forces to biochemical signals at mechanotransducer sites.9
Transmission of mechanical forces likely involves F-actin
microfilaments and changes in their interactions with linker proteins
such as 
-actin, talin, and vinculin that communicate with
transmembrane proteins such as the integrins.10 Important
sites of transduction in endothelial cells are likely
to include stretch-activated (and inactivated)
ion channels, focal adhesions, cell-to-cell contacts, and
cytoskeletal interactions with plasma membrane and nuclear membrane
structures.9 Many signal-transduction events have been
demonstrated to occur at these sites, including
hyperpolarization (and depolarization), stimulation
of phospholipid turnover, and activation of kinases. Recently, the MAP
kinases,1 members of a well-characterized protein
kinase system, have been shown to mediate cell responses to physical
forces such as osmotic stress11 and
stretch.12 For example, cyclic stretch in cardiac myocytes
stimulates several kinases, including tyrosine kinases, MAP kinase,
pp90RSK, and PKC.13
Stretch-mediated activation of MAP kinase in cardiac myocytes was
found to be dependent on PKC, indicating that a kinase cascade typical
of membrane receptors was activated.12
Although the pathways leading from growth factor–receptor activation to stimulation of MAP kinase have been well characterized,14 the pathways leading to activation of MAP kinase by physical forces are not well understood. Activation of MAP kinase by mitogens14 15 involves a kinase cascade initiated by tyrosine phosphorylation followed by stimulation of upstream kinases such as Raf kinase and MAP kinase kinase. Fluid shear stress activates receptor-like events in endothelial cells. In particular, fluid shear stress stimulates phospholipase C,16 with increases in inositol 1,4,5-trisphosphate formation16 and intracellular Ca2+ concentration.17 18 Fluid shear stress should also stimulate PKC activity, because phospholipase C activation will generate diacylglycerol as well as increase intracellular Ca2+.3 5 6 Thus, one pathway proposed for signal transduction by fluid shear stress in endothelial cells is Ca2+ dependent and involves activation of phospholipase C, increases in intracellular Ca2+, and activation of PKC.9 17 18 In the present study we report stimulation of MAP kinase by fluid shear stress in a time- and force-dependent manner, suggesting an important role in the cell response to physical forces. In addition, we define a new pathway for the endothelial cell response to fluid shear stress that is Ca2+ independent and involves a G protein and increases in PKC and MAP kinase activity.
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Materials and Methods |
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Cell Culture
Bovine aortic endothelial cells were harvested from fetal calf aortas by using collagenase, as previously described.19 Cells were grown in M199 (GIBCO) medium supplemented with 10% fetal calf serum and characterized by the uptake of fluorescein-labeled acetylated low-density lipoprotein. Cells used in experiments were passage
6, as MAP kinase activation decreased in later passages. For some
experiments, endothelial cells were obtained from
weanling calf aortas, weanling pig aortas, or human umbilical veins by
use of the collagenase isolation procedure.
Shear Stress Experiments
Cells were grown on 2-cmx4-cm slides of tissue-culture
plastic cut from the bottom of tissue-culture dishes. Upon reaching
95% confluence, cells were rinsed free of culture media with HBSS
(containing, in mmol/L, NaCl 130, KCl 5, CaCl2 1.5,
MgCl2 1.0, HEPES 20, pH 7.4), with 10 mmol/L glucose added,
and either maintained in static condition or exposed to fluid shear
stress (3.5, 12, 35, and 117 dynes/cm2) in a
parallel-plate chamber7 at 37°C. After varying times
of exposure to fluid shear stress, cells were washed gently with
ice-cold PBS (composition, in mmol/L, NaCl 137, KCl 2.7,
Na2HPO4 4.3, KH2PO4
1.4, pH 7.3), and MAP kinase activation was determined. For experiments
in which shear stress was varied at constant flow velocity, dextran-70
(Hyskon, from Medisan Pharmaceuticals, which is a 32% dextran-70
solution with a viscosity of 2.2 poise) was added to increase the
viscosity. The shear stress was calculated as previously
described,17 on the basis of 
=(6
µQ)/(h2b), where is wall shear stress (2 to 40
dynes/cm2), µ is viscosity of the medium in poise
(0.006915 for HBSS to 
0.14 for HBSS plus Hyskon), Q is flow rate
(0.05 mL/s), b is chamber width (1.7 cm), and h is chamber height
(0.025 cm).
Western Blot Analysis for MAP Kinase
Activation
Lysates were prepared by rapid freezing and slow thawing of
endothelial cells at 4°C in lysis buffer (50 mmol/L
NaCl, 50 mmol/L NaF, 50 mmol/L sodium pyrophosphate, 5 mmol/L EDTA, 5
mmol/L EGTA, 2 mmol/L Na3VO4, 0.01%
Triton X-100, 0.5 mmol/L PMSF, 10 µg/mL leupeptin, 10 mmol/L HEPES,
pH 7.4), followed by scraping, sonication, and
centrifugation (30 minutes, 4°C, 14 000 rpm in
microfuge). Sample protein concentrations were determined by the
Bradford technique.20 Laemmli buffer (2') was added to
equal amounts of soluble protein in the supernatant, and the lysates
were analyzed by SDS-PAGE and transferred to nitrocellulose
filters (Hybond, Amersham). To ensure quantitative transfer of
proteins, the gels were routinely stained with Coomassie and, as
needed, the filters were stained with Ponceau S. Specific proteins were
detected by immunoblotting with primary antibody (MAP
kinase antibodies were from Santa Cruz Biological and PKC antibodies
from GIBCO-BRL) and a horseradish peroxidase–conjugated secondary
antibody (Fisher and Amersham). Detection was by chemiluminescence
(Amersham ECL).
Myelin Basic Protein Phosphorylation and In-Gel
Kinase Assay for MAP Kinase Activity
Lysates were prepared as described for Western blotting and size
fractionated (15 to 30 µg protein) by 10% SDS-PAGE in a gel
containing 0.4 mg/mL myelin basic protein. In-gel kinase assays
were then performed according to Chao et al,21 as modified
by our group.22 After washing to remove SDS and
renaturation in 6 mol/L guanidinium, the gel was incubated with
[
32P]ATP (7.5 µCi/mL) at 30°C for 1 hour, washed
in sodium pyrophosphate/trichloroacetic acid buffer, and dried.
Autoradiography of the gel was performed, followed
by densitometry (LaCie scanner) in the linear range of film exposure to
quantify phosphorylation of myelin basic protein (by
using NIH Image 1.49). Comparison of densitometric results with
phosphoimager results showed a highly significant correlation between
the two techniques (R2=.95). To confirm the
specificity of the in-gel kinase assay, MAP kinase activity was
simultaneously determined by an immune complex
assay.15 Lysates were incubated with antibody to MAP
kinase (BioDesign) in buffer (50 mmol/L NaCl, 50 mmol/L NaF, 30 mmol/L
sodium pyrophosphate, 5 mmol/L EDTA, 100 µmol/L
Na3VO4, 5 mmol/L PMSF, 1% Triton X-100,
0.1% BSA, 0.1% azide, 10 mmol/L Tris-HCl, pH 7.6), followed by
incubation with goat anti-mouse IgG (Alpha Quest). The immune
complexes were aggregated with protein-A Sepharose (Pharmacia) and
pelleted, and MAP kinase activity was determined by both the in-gel
kinase assay and phosphorylation of myelin basic
protein in solution. For this assay, immunoprecipitated proteins were
incubated in kinase buffer (5 mmol/L ß-glycerophosphate, 10
mmol/L MgCl2, 2 mmol/L dithiothreitol, 100 µmol/L
Na3VO4, 0.02% Triton X-100, 50 µmol/L
ATP, 1 mg/mL myelin basic protein, 20 mmol/L HEPES, pH 7.4) with
[-32P]ATP (0.5 µCi/µL) at 30°C for 20 minutes.
The reaction was terminated by addition of 25% trichloroacetic acid.
The mixture was spotted onto 2-cmx2-cm Whatman P-81 phosphocellulose
paper. The paper was washed free of unincorporated
[-32P]ATP in 30 mmol/L phosphoric acid and washed once
in 95% ethanol. The bound 32P–myelin basic protein was
quantified by liquid scintillation counting. Comparison of
32P incorporation into soluble myelin basic protein with
densitometry of in-gel kinase assay autoradiograms
(normalized to incorporation in unstimulated cells) showed a highly
significant correlation between the two techniques
(R2=.92). In addition, the immunodepleted
lysates were analyzed by the in-gel kinase assay; more than
95% of the autoradiographic signal at 42 and 44 kD was
eliminated (data not shown). On the basis of these findings, all
experiments were performed by using the in-gel kinase assay because
of its rapidity, simplicity, and reproducibility (which was better than
the immune complex assay).
Scrape Loading GDP-ßS
Bovine aortic endothelial cells were scrape
loaded with GDP-ßS or 2% BSA, as previously
described.23 After 6 hours, cells began to return to their
normal "cobblestoned" appearance, but basal levels of MAP kinase
activation did not return to baseline until 48 hours (data not shown).
After 48 hours of recovery, the cells were washed with HBSS and
maintained in static condition or exposed to fluid shear stress.
Ca2+ Chelation and Measurement of Intracellular
Ca2+
Bovine aortic endothelial cells were pretreated
with 75 µmol/L BAPTA-AM for 30 minutes at 37°C in
Ca2+-free HBSS (HBSS with 1 mmol/L CaCl2
replaced by 1 mmol/L NaCl and 10 mmol/L EDTA). Cells were washed with
Ca2+-free HBSS and then exposed to agonists, as described
for each experimental protocol. To measure intracellular
Ca2+, cells were simultaneously loaded
with 3 µmol/L fura 2-AM and 75 µmol/L BAPTA-AM, as described above.
Cells were then detached with Versene (GIBCO), and fluorescent
measurements were performed in an SLM DMX-1000 spectrofluorometer
equipped with a beam splitter, two excitation monochromators, and a
dual chopping mechanism to allow rapid alternating excitation of fura 2
at 340 and 380 nm, as previously described.24 The emission
ratio of the fluorescence signals at 510 nm was used to
determine Ca2+ after calibration using 30 mmol/L digitonin
to permeabilize cells and addition of 10 mmol/L EGTA to
chelate Ca2+. Measurements of intracellular
Ca2+ in fetal calf aortic endothelial cells
in response to fluid shear stress were performed exactly as described
previously.17 25 For these experiments, changes in
intracellular Ca2+ are reported as the observed fura 2
ratio at time t (F) divided by the fura 2 ratio at time 0
(Fo; before flow initiation). A 1-unit change in fura 2
ratio corresponds to 600 nmol/L change in intracellular
Ca2+.
PKC Assay
Cells from four plastic flow slides were scraped into 0.4 mL of
lysis buffer (20 mmol/L Tris-HCl, pH 7.4, 10 mmol/L EDTA, 5 mmol/L
EGTA, 5 mmol/L 2-mercaptoethanol, 10 mmol/L benzamidine, 1 mg/mL
leupeptin, 50 µg/mL PMSF, 0.1 mg/mL ovalbumin, and 0.1
µg/mL aprotinin) in 60-mmol/L dishes on ice. After incubation for 5
minutes, cells were disrupted with a Dounce homogenizer
(20 strokes), and centrifugation was performed
(100 000g for 40 minutes). The supernatant was saved as the
cytosolic fraction. The pellet (particulate fraction) was washed once
with lysis buffer and resuspended in 0.1 mL of lysis buffer by
sonication. Protein was determined and fractions were stored at
-80°C. Prior to assay, the particulate fraction was incubated with
25 µL of 2% NP-40 in lysis buffer for 1 hour on ice and
centrifuged (100 000g for 30 minutes). The
supernatant was assayed as "soluble" particulate fraction. For
assay of PKC, partially purified fractions (15 µg cytosolic
protein and 4 µg particulate protein) were incubated for 3 minutes
in a final volume of 35 µL containing 50 mmol/L HEPES (pH 7.4), 10
mmol/L magnesium acetate, 25 µmol/L [32P]ATP, 5.5
µmol/L [ser25]PKC (19-31) peptide substrate (LC
Laboratories), and either Ca2+-phospholipids (0.7 mmol/L
CaCl2, 240 µg/mL
phosphatidylserine, and 16 µg/mL diolein) or 1.0
mmol/L EGTA. The sequence of the synthetic peptide substrate
[ser25]PKC (19-31), which has high affinity for PKC, is
H-RFARKGSLRQKNV-OH and contains only a single serine
residue.26 The reaction was stopped by spotting 20 µL of
sample onto Whatman P-81 filter paper and washing six times (10 minutes
each) with 75 mmol/L phosphoric acid, followed by one wash with 70%
ethanol. Incorporated 32P was determined by liquid
scintillation.
Statistical Analysis
All experiments were performed at least three times, and data
are presented as mean±SEM. Significant differences were
determined by Student’s t test (P<.05).
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Results |
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p42 and p44 MAP Kinases Are Activated by Flow
Stimulation of MAP kinase by fluid shear stress was measured by several techniques. MAP kinase phosphorylation was determined by Western blotting with anti–MAP kinase antibodies that detect phosphorylated MAP kinase by virtue of its retarded protein mobility ("band shift") on SDS-PAGE. Compared with static control, fluid shear stress activated the 42-kD ERK2 isoform of MAP kinase, with peak activation at 5 minutes and return to baseline by 60 minutes (Fig 1
, top). The
antibodies reproducibly detected phosphorylated 42-kD
MAP kinase better than phosphorylated 44-kD MAP kinase.
MAP kinase phosphorylation was dependent on shear
stress in the physiologic range (3.5 to 35
dynes/cm2, 5 minutes, 37°C), with a maximum at 12
to 35 dynes/cm2 (Fig 1, bottom). Flow was a powerful
activator of MAP kinase, with increases in
phosphorylation equivalent to 10% serum and 200 nmol/L
PMA.
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Because the Western blot technique (Fig 1
) fails to detect
phosphorylated 44-kD (p44) MAP kinase as efficiently as
phosphorylated 42-kD (p42) MAP kinase, we performed an
in-gel kinase assay21 22 on the basis of
phosphorylation of myelin basic protein. After
stimulation by fluid shear stress, cell extracts were size fractionated
by SDS-PAGE in a myelin basic protein–containing gel. As shown in
Fig 2A and 2B, the time course and force dependence of
p42 and p44 MAP kinase activity were similar to that demonstrated in
Fig 1, although the time for peak activation was 10 minutes. The
difference in the time for peak activation by Western "band
shift" (Fig 1) compared with in-gel kinase assay (Fig 2) likely
reflects differences among preparations of endothelial
cells. The time for peak activation ranged from 2 to 10 minutes (n=8,
with peak at 2 minutes in two experiments and peaks at 5 and 10 minutes
in three experiments). In addition, both p42 and p44 MAP kinases were
equally activated by flow (maximum at 12 dynes/cm2
for 10 minutes, Fig 2C and 2D). To verify that the activation of MAP
kinase by flow was a characteristic feature of
endothelial cells, these experiments were repeated with
cells from several sources, including weanling pig and calf aortic
endothelial cells and human umbilical vein
endothelial cells. The time course and dependence on
shear stress were similar for endothelial cells from
all sources, except that peak activation for the human umbilical vein
endothelial cells was 10 to 30 minutes (data not
shown). Activation of MAP kinase was dependent upon the shear stress
rather than pressure or flow velocity. Increasing the pressure from 0
to 50 mm Hg caused no "band shift" of MAP kinase (data not
shown). In addition, increasing the shear stress (by increasing the
viscosity with dextran-70) at constant flow velocity caused activation
of MAP kinase, with the same shear stress dependence as that observed
by increasing the flow velocity at constant viscosity (onset at 2 to 5
dynes/cm2, maximum at 12 to 40
dynes/cm2).
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The 42- and 44-kD proteins were the myelin basic protein kinases most strongly activated by flow, with maximum increases of 10.6±2.2-fold and 11.2±1.8-fold, respectively (12 dynes/cm2 for 10 minutes, n=3). Additional myelin basic protein kinases were detected at molecular weights of 66 kD, 95 kD, and 180 kD. However, these kinases in sum accounted for less than 10% of the total myelin basic protein phosphorylation stimulated by flow (data not shown). The identity of these kinases is currently unknown.
To verify that the 42- and 44-kD proteins identified by Western blot
and in-gel kinase assays were MAP kinases, cell lysates were
immunoprecipitated with anti–MAP kinase antibodies and subjected to an
in-gel kinase assay (Fig 3). Proteins of the
appropriate molecular weights were immunoprecipitated and showed a
similar shear stress dependence for activity as demonstrated in Fig 2.
In addition, the cell lysates that had been "immunodepleted" were
then used for an in-gel kinase assay. More than 95% of the
autoradiographic signal at 42 and 44 kD was abolished
(not shown), indicating that the MAP kinases are the dominant myelin
basic protein kinases in endothelial cells at 42 and 44
kD. Thus, fluid shear stress stimulates a force-dependent
activation of MAP kinase in cultured bovine aortic
endothelial cells, as demonstrated by Western blot
"band shift," in-gel kinase activity, and increased myelin
basic protein phosphorylation of MAP kinase
immunoreactive proteins (Fig 3).
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MAP Kinase Activation by Flow Is G Protein Dependent
MAP kinase has been shown to be activated by both tyrosine
kinase–coupled receptors and G protein–coupled
receptors.14 To determine the role of G proteins in MAP
kinase activation by fluid shear stress, endothelial
cells were loaded with GDP-ßS, a nonhydrolyzable analogue of GDP, to
inhibit G protein activity. Because GDP-ßS is highly charged, it was
scrape loaded into the cells,23 and the cells were allowed
to recover for 48 hours. The cells were then exposed to fluid shear
stress (12 dynes/cm2 for 5 minutes) or -thrombin (10
U/mL for 5 minutes) or maintained in static condition. Because
-thrombin stimulates MAP kinase through a G
protein–dependent mechanism in CCL39 fibroblasts,27
MAP kinase activation by -thrombin should be inhibited by
GDP-ßS. As shown in Fig 4, MAP kinase activity
stimulated by -thrombin was inhibited in a
concentration-dependent manner by GDP-ßS. Activation of MAP
kinase by fluid shear stress was also significantly inhibited by
GDP-ßS at concentrations as low as 30 µmol/L (Fig 4).
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P<.10 vs control.
To identify the G protein mediating flow activation of MAP kinase,
pertussis toxin and cholera toxin were used to inhibit G protein
function. Pertussis toxin alone (100 ng/mL, 24 hours) had no effect on
MAP kinase activity (Fig 5) and failed to inhibit the
stimulation of MAP kinase by fluid shear stress (12
dynes/cm2, 10 minutes), indicating that
Gi was not involved in flow-mediated activation. As a
positive control for ADP ribosylation, the ability of pertussis toxin
to block lysophosphatidic acid–stimulated MAP kinase activity was
determined. As previously described,28 pertussis toxin
completely inhibited MAP kinase activation by lysophosphatidic acid
(not shown). Similar experiments with 1 µg/mL cholera toxin showed
that it also had no effect on flow-mediated MAP kinase activation
(not shown).
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MAP Kinase Activation by Flow Is Independent of
Ca2+ Mobilization
The rapid increase in intracellular Ca2+ stimulated by
flow in endothelial cells17 18 has been
proposed to be essential for many flow-mediated physiologic
responses, such as the increase in nitric oxide
production.29 30 It has been shown that the
increase in intracellular Ca2+ is due to phospholipase
C–mediated phosphatidylinositol 4,5-bisphosphate hydrolysis generating
inositol 1,4,5-trisphosphate.16 17 To determine whether
the flow-mediated increase in intracellular Ca2+ was
essential for fluid shear stress–stimulated MAP kinase activation,
intracellular Ca2+ was chelated with the
membrane-permeant form of BAPTA (75 µmol/L, 30 minutes, 37°C).
This treatment completely inhibited the increase in intracellular
Ca2+ stimulated by 1 mmol/L ATP without reducing baseline
intracellular Ca2+ concentration (Fig 6). To
prevent Ca2+ influx from increasing intracellular
Ca2+, fluid shear stress was performed in a
nominally Ca2+-free balanced salt solution, supplemented
with EDTA (10 µmol/L). Under these conditions the flow-mediated
increase in intracellular Ca2+ was completely inhibited
(Fig 7). In the presence of extracellular
Ca2+, flow (shear stress, 12 dynes/cm2)
increased the fura 2 ratio by 20% (F:Fo=1.2, a change
in intracellular Ca2+ of about 100 nmol/L), while in the
absence of extracellular Ca2+ there was no significant
change in intracellular Ca2+ (fura 2 ratio,
F:Fo=1.0). MAP kinase activity in response to flow (12
dynes/cm2 for 5 or 10 minutes) was then measured by
phosphorylation of myelin basic protein in solution and
in-gel kinase assay. Both assays showed no significant inhibition
of fluid shear stress–stimulated MAP kinase activity by
Ca2+ chelation (Fig 8). Specifically, myelin
basic protein phosphorylation was increased over static
control cells by 2.8-fold under both conditions, while in-gel
kinase activity increased by 7.3-fold in the presence of BAPTA and
8.5-fold in the presence of Ca2+. Therefore, flow
activates MAP kinase by a mechanism independent of increases in
intracellular Ca2+.
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Ca2+-Dependent and -Independent PKC Isozymes Are
Activated by Flow
PKC activity should be stimulated by fluid shear stress, because
flow activation of phospholipase C generates diacylglycerol and
inositol 1,4,5-trisphosphate.16 These two second
messengers should increase PKC activity directly and by increasing
intracellular Ca2+ concentration.17 18 To
assess the role of PKC in fluid shear stress–stimulated MAP kinase
activation, the effects of PKC downregulation (24-hour pretreatment
with 2 µmol/L PDBU) and pharmacological inhibition (2 nmol/L
staurosporine) were studied. Both treatments significantly
inhibited PMA and fluid shear stress–stimulated MAP kinase
activation (12 dynes/cm2 for 5 minutes), as assayed by
Western blot analysis (Fig 9A). The inhibition
of flow-stimulated MAP kinase activation by PDBU treatment and
staurosporine was approximately equal: 73% and 90%,
respectively. Because staurosporine is a relatively
nonspecific protein kinase inhibitor, this experiment was
repeated with 1 µmol/L chelerythrine and analyzed by the
in-gel kinase assay (Fig 9B). PDBU treatment and
staurosporine caused approximately equal inhibition of PMA
(87% and 81%, respectively) and fluid shear stress–stimulated
MAP kinase activation (84% and 77%, respectively). In contrast,
chelerythrine inhibited PMA-mediated MAP kinase activation by 64% but
fluid shear stress–mediated activation by only 23%. This
differential sensitivity to chelerythrine suggested that specific PKC
isozymes, relatively unaffected by chelerythrine,31 32 may
be involved in the fluid shear stress–mediated response.
Alternatively, differences in the relative affinity of chelerythrine
for PKC under flow conditions may be important.
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To identify the PKC isozymes activated by flow, both Western
blot and PKC activity analyses were performed. The major PKC
isozymes present in endothelial cells, as
determined by Western blot, were PKC-, PKC-
, and PKC-
(Fig 10), similar to previously reported
findings.33 34 35 No PKC-ß, -, or -
were detected in
these endothelial cells, using the GIBCO-BRL
antibodies, despite their presence in brain tissue similarly studied
(data not shown). As shown in Fig 10, significant amounts of PKC-
and PKC- were in the particulate fraction in unstimulated
conditions, as previously reported.34 35 36 Because PKC-
and PKC- were present in the particulate fraction, it was
difficult to measure the extent of translocation of these isozymes by
Western blot (data not shown).
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Therefore, to quantify changes in PKC activity stimulated by PMA and
flow, cell extracts were prepared, cytosolic and particulate fractions
were purified, and phosphorylation of the PKC
pseudosubstrate [ser25]PKC-(19-31)26 was
measured. In control, unstimulated cells (Fig 11) there
was approximately equal total PKC activity toward this substrate in
cytosolic and particulate fractions. As a positive control, a maximal
concentration of PMA (1 µmol/L, 10 minutes) was utilized. PMA
stimulated a 13-fold increase in PKC activity, measured as the increase
in particulate fraction 32P incorporation in the presence
of Ca2+ and phospholipids (Fig 11). Fluid shear stress (12
dynes/cm2, 10 minutes) stimulated a 6.4-fold
increase in PKC activity (Fig 11). The increase in particulate PKC
activity in response to both PMA and flow was significantly greater
than the decrease in cytosolic PKC activity. This may be due to
activation of PKC isozymes already present in the particulate
fraction (eg, PKC- and PKC-). To determine whether PKC was
activated under conditions in which Ca2+
mobilization was blocked, endothelial cells were
treated with 75 µmol/L BAPTA-AM and stimulated with flow (12
dynes/cm2, 10 minutes), and PKC activity was
measured (Fig 11). Under these conditions, PKC activity was stimulated
significantly (4.7-fold increase over control), to nearly the same
extent as in cells lacking BAPTA (6.4-fold increase). There was no
significant effect of BAPTA treatment on PKC activity in unstimulated
cells (not shown). These results suggest that flow stimulates a PKC
isozyme that does not require increases in Ca2+ for
translocation and activation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The two major findings of this study are that physiological levels of fluid shear stress stimulate MAP kinase activity in bovine aortic endothelial cells, and the signal events involve a novel pathway that is dependent on a G protein and PKC activity but is independent of Ca2+ mobilization. The time course for MAP kinase activation indicates that this is one of the early signal-transduction events stimulated by fluid shear stress. Activation of MAP kinase occurs in a force-dependent manner and has a time course very similar to agonist-stimulated MAP kinase activation.15 These two features, along with previously demonstrated fluid shear stress–stimulated activation of phospholipase C,16 increases in inositol 1,4,5-trisphosphate formation,16 and intracellular Ca2+ concentration,17 18 suggest that fluid shear stress activates a receptor-like process in endothelial cells.
The present study is the first to document that a
Ca2+-independent signal-transduction pathway (MAP
kinase activation) is stimulated by flow in endothelial
cells. At present we have not correlated this pathway with
activation of any physiological response. However,
it is of interest that the maximal effect occurs in a range of shear
stress that relates well to the normalized wall shear stresses found
throughout the vascular tree. Previous investigators have shown that
flow stimulates a rapid increase in intracellular Ca2+ in
endothelial cells.17 18 Several events
stimulated by flow in endothelial cells, including
increases in nitric oxide,29 37
c-fos,5 and prostacyclin,2 have
been shown to be Ca2+-dependent processes. However, fluid
shear stress activation of MAP kinase is not dependent on increases in
intracellular Ca2+, because MAP kinase activity,
measured by two assays (Fig 8), was not inhibited by Ca2+
chelation. Recently, Kuchan and Frangos30 demonstrated
that the sustained increase in nitric oxide production by
endothelial cells stimulated by flow was
Ca2+ independent. Thus, MAP kinase activation by fluid
shear stress may be a component of a Ca2+-independent
signaling pathway in endothelial cells.
Fluid shear stress stimulation of phospholipase C results in increases
in intracellular Ca2+ and diacylglycerol,2 16
which should activate PKC. The importance of PKC in the
endothelial cell response to flow is indicated by the
findings that release of endothelin-1,3 as well as
increased c-fos5 and platelet-derived
growth factor gene expression,5 have been found to be
dependent on PKC. Fluid shear stress–stimulated MAP kinase
activation appears to be dependent on PKC as well, as shown in the
present study by inhibition with staurosporine and
down-regulation with PDBU (Fig 9A). The finding that MAP kinase
activation is dependent on PKC but mainly independent of increases in
intracellular Ca2+ (Fig 8) suggests that the PKC isozyme
activated by flow is a Ca2+-independent isozyme.
The importance of a Ca2+-independent isozyme is also
supported by the greater inhibition of fluid shear stress–mediated
MAP kinase activation by staurosporine compared with
chelerythrine (Fig 9A and 9B). Balboa and colleagues38
reported that chelerythrine inhibited phospholipase D activation in a
manner similar to antisense PKC- oligonucleotides,
indicating that chelerythrine inhibited Ca2+-dependent
isozymes such as PKC-. Several investigators have reported
differential PKC inhibition by chelerythrine and
staurosporine,31 32 suggesting that these
compounds have effects on different PKC isozymes. Previous
investigators agree that endothelial cells express
PKC- and PKC-.33 34 35 Several authors have also found
PKC- in endothelial cells, as in the present
study.33 34 Therefore, it appears that the
Ca2+-independent isozyme activated by flow is most
likely PKC- and/or PKC-, on the basis of Western blot and
activity assays (although newer isozymes such as PKC-
and PKC-
were not studied). The fact that PKC- does not translocate or
downregulate in response to PMA,36 yet MAP kinase
activation by flow is inhibited by PDBU pretreatment (Fig 9A and 9B),
argues that PKC- is less likely than PKC- to be the PKC isozyme
activated by flow. To prove conclusively which isozyme is
involved, experiments with isozyme-specific antisense
oligonucleotides or pseudosubstrate
inhibitors will likely be required,38 as
translocation of PKC- and PKC- is an inadequate measurement of
their activities.34 35 36 To our knowledge the present
study is the first demonstration of specific PKC isozyme activation by
mechanical forces.
The present findings provide several insights into the nature of
the membrane proteins that may act as the fluid shear stress receptors
in endothelial cells and as sensors of physical forces
in other cell types. Our data indicate that stimulation of MAP kinase
by this receptor requires activation of a G protein and a specific PKC
isozyme. The use of GDP-ßS in the present study does not allow us
to differentiate between a heterotrimeric (ß-type) and a
small-molecular-weight G protein (eg, ras, rac, or rho). A
previous study that used pertussis toxin to inhibit fluid shear
stress–stimulated increases in cGMP implicated a heterotrimeric G
protein.37 However, we found that pertussis toxin failed
to block activation of MAP kinase by flow, indicating that if a
heterotrimeric G protein is involved it is not ADP-ribosylated by
pertussis toxin. It appears more likely that the G protein inhibited by
GDP-ßS in endothelial cells is a ras- or rho-like
protein, since these proteins interact with the
cytoskeleton,24 39 which appears essential to
endothelial cell responses to fluid shear
stress.9 10 40 41 42 43 The endothelial cell
response to fluid shear stress is similar to cellular responses to
physical forces such as hyperosmolar stress11 and
stretching,12 which have been shown to activate
members of the MAP kinase and PKC families. However, while MAP kinase
activation by stretching of cardiac myocytes was partially dependent on
Ca2+ influx,12 the endothelial
response to fluid shear stress appears unique in that it is independent
of Ca2+ mobilization. In addition, the
endothelial cell response was completely dependent on
PKC and appeared to require a Ca2+-independent isoform of
PKC. A possible unifying mechanism for cellular responses to physical
forces (including shear stress) would be via integrin-mediated
events, because integrins have been shown to activate all the
signal events demonstrated in the present study, including MAP
kinase,44 PKC,45 Ca2+
mobilization,46 and G proteins.47 Future
studies should define the integrins that are involved in the
endothelial response to fluid shear stress.
Activation of MAP kinase by fluid shear stress is a novel pathway for the regulation of endothelial cell function. The stimulation of MAP kinase by fluid shear stress demonstrated here was transient. This finding suggests that a potentially important role for MAP kinase may be regulation of rapid alterations in response to changes in blood flow, such as during exercise-induced vasodilatation. However, stimulation of MAP kinase in the present study occurred when cells under static conditions were suddenly exposed to shear stress. Future studies that investigate changes in MAP kinase activity when cells already exposed to shear stress are subjected to higher levels of shear stress should be very informative regarding the normal physiological response. In addition, there may also be an important role for MAP kinase in chronic changes in endothelial cell gene expression, since MAP kinase is an activator of transcription factors, such as p62TCF.48 In summary, on the basis of our findings, we propose that flow activates dual signal-transduction pathways in endothelial cells via a receptor-like mechanism. One pathway is Ca2+ dependent and involves activation of phospholipase C and increases in intracellular Ca2+. A new pathway, described in the present study, is Ca2+ independent and involves a G protein and increases in PKC and MAP kinase activity.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by grants from the American Heart Association, the Emory-Georgia Tech Biomedical Technology Research Center, and the National Institutes of Health. Dr Berk is an Established Investigator of the American Heart Association. Dr. Tseng was supported by the Medical Scientist Training Program of the National Institutes of Health.
Received June 20, 1995; accepted August 10, 1995.
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References |
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 T. Di, J. A. Sullivan, R. R. Magness, L. Zhang, and I. M. Bird Pregnancy-Specific Enhancement of Agonist-Stimulated ERK-1/2 Signaling in Uterine Artery Endothelial Cells Increases Ca2+ Sensitivity of Endothelial Nitric Oxide Synthase as well as Cytosolic Phospholipase A2 Endocrinology, July 1, 2001; 142(7): 3014 - 3026. [Abstract] [Full Text] [PDF]
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X. Bao, C. Lu, and J. A. Frangos Mechanism of temporal gradients in shear-induced ERK1/2 activation and proliferation in endothelial cells Am J Physiol Heart Circ Physiol, July 1, 2001; 281(1): H22 - H29. [Abstract] [Full Text] [PDF]
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N. Azuma, N. Akasaka, H. Kito, M. Ikeda, V. Gahtan, T. Sasajima, and B. E. Sumpio Role of p38 MAP kinase in endothelial cell alignment induced by fluid shear stress Am J Physiol Heart Circ Physiol, January 1, 2001; 280(1): H189 - H197. [Abstract] [Full Text] [PDF]
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 H. Kito, E. L. Chen, X. Wang, M. Ikeda, N. Azuma, N. Nakajima, V. Gahtan, and B. E. Sumpio Role of mitogen-activated protein kinases in pulmonary endothelial cells exposed to cyclic strain J Appl Physiol, December 1, 2000; 89(6): 2391 - 2400. [Abstract] [Full Text] [PDF]
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S. Lehoux, B. Esposito, R. Merval, L. Loufrani, and A. Tedgui Pulsatile Stretch-Induced Extracellular Signal-Regulated Kinase 1/2 Activation in Organ Culture of Rabbit Aorta Involves Reactive Oxygen Species Arterioscler Thromb Vasc Biol, November 1, 2000; 20(11): 2366 - 2372. [Abstract] [Full Text] [PDF]
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I. S. Wittstein, W. Qiu, R. C. Ziegelstein, Q. Hu, and D. A. Kass Opposite Effects of Pressurized Steady Versus Pulsatile Perfusion on Vascular Endothelial Cell Cytosolic pH : Role of Tyrosine Kinase and Mitogen-Activated Protein Kinase Signaling Circ. Res., June 23, 2000; 86(12): 1230 - 1236. [Abstract] [Full Text] [PDF]
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K. S. Russell, M. P. Haynes, D. Sinha, E. Clerisme, and J. R. Bender Human vascular endothelial cells contain membrane binding sites for estradiol, which mediate rapid intracellular signaling PNAS, May 23, 2000; 97(11): 5930 - 5935. [Abstract] [Full Text] [PDF]
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L. W. Kraiss, A. S. Weyrich, N. M. Alto, D. A. Dixon, T. M. Ennis, V. Modur, T. M. McIntyre, S. M. Prescott, and G. A. Zimmerman Fluid flow activates a regulator of translation, p70/p85 S6 kinase, in human endothelial cells Am J Physiol Heart Circ Physiol, May 1, 2000; 278(5): H1537 - H1544. [Abstract] [Full Text] [PDF]
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X. Bao, C. B. Clark, and J. A. Frangos Temporal gradient in shear-induced signaling pathway: involvement of MAP kinase, c-fos, and connexin43 Am J Physiol Heart Circ Physiol, May 1, 2000; 278(5): H1598 - H1605. [Abstract] [Full Text] [PDF]
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H. Park, Y.-M. Go, R. Darji, J.-W. Choi, M. P. Lisanti, M. C. Maland, and H. Jo Caveolin-1 regulates shear stress-dependent activation of extracellular signal-regulated kinase Am J Physiol Heart Circ Physiol, April 1, 2000; 278(4): H1285 - H1293. [Abstract] [Full Text] [PDF]
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M. A. Haidekker, N. L'Heureux, and J. A. Frangos Fluid shear stress increases membrane fluidity in endothelial cells: a study with DCVJ fluorescence Am J Physiol Heart Circ Physiol, April 1, 2000; 278(4): H1401 - H1406. [Abstract] [Full Text] [PDF]
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Y. Kano, K. Katoh, and K. Fujiwara Lateral Zone of Cell-Cell Adhesion as the Major Fluid Shear Stress-Related Signal Transduction Site Circ. Res., March 3, 2000; 86(4): 425 - 433. [Abstract] [Full Text] [PDF]
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J.-i. Abe, M. Okuda, Q. Huang, M. Yoshizumi, and B. C. Berk Reactive Oxygen Species Activate p90 Ribosomal S6 Kinase via Fyn and Ras J. Biol. Chem., January 21, 2000; 275(3): 1739 - 1748. [Abstract] [Full Text] [PDF]
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S. Q. Liu Focal Expression of Angiotensin II Type 1 Receptor and Smooth Muscle Cell Proliferation in the Neointima of Experimental Vein Grafts : Relation to Eddy Blood Flow Arterioscler Thromb Vasc Biol, November 1, 1999; 19(11): 2630 - 2639. [Abstract] [Full Text] [PDF]
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A. I. Barakat, E. V. Leaver, P. A. Pappone, and P. F. Davies A Flow-Activated Chloride-Selective Membrane Current in Vascular Endothelial Cells Circ. Res., October 29, 1999; 85(9): 820 - 828. [Abstract] [Full Text] [PDF]
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Y.-M. Go, R. P. Patel, M. C. Maland, H. Park, J. S. Beckman, V. M. Darley-Usmar, and H. Jo Evidence for peroxynitrite as a signaling molecule in flow-dependent activation of c-Jun NH2-terminal kinase Am J Physiol Heart Circ Physiol, October 1, 1999; 277(4): H1647 - H1653. [Abstract] [Full Text] [PDF]
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M. Okuda, M. Takahashi, J. Suero, C. E. Murry, O. Traub, H. Kawakatsu, and B. C. Berk Shear Stress Stimulation of p130cas Tyrosine Phosphorylation Requires Calcium-dependent c-Src Activation J. Biol. Chem., September 17, 1999; 274(38): 26803 - 26809. [Abstract] [Full Text] [PDF]
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J. J. Chiu, B. S. Wung, H. J. Hsieh, L. W. Lo, and D. L. Wang Nitric Oxide Regulates Shear Stress–Induced Early Growth Response-1 : Expression via the Extracellular Signal–Regulated Kinase Pathway in Endothelial Cells Circ. Res., August 6, 1999; 85(3): 238 - 246. [Abstract] [Full Text] [PDF]
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O. Traub, T. Ishida, M. Ishida, J. C. Tupper, and B. C. Berk Shear Stress-mediated Extracellular Signal-regulated Kinase Activation Is Regulated by Sodium in Endothelial Cells. POTENTIAL ROLE FOR A VOLTAGE-DEPENDENT SODIUM CHANNEL J. Biol. Chem., July 16, 1999; 274(29): 20144 - 20150. [Abstract] [Full Text] [PDF]
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K.-D. Chen, Y.-S. Li, M. Kim, S. Li, S. Yuan, S. Chien, and J. Y-J. Shyy Mechanotransduction in Response to Shear Stress. ROLES OF RECEPTOR TYROSINE KINASES, INTEGRINS, AND Shc J. Biol. Chem., June 25, 1999; 274(26): 18393 - 18400. [Abstract] [Full Text] [PDF]
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J. M. Muller, M. J. Davis, L. Kuo, and W. M. Chilian Changes in coronary endothelial cell Ca2+ concentration during shear stress- and agonist-induced vasodilation Am J Physiol Heart Circ Physiol, May 1, 1999; 276(5): H1706 - H1714. [Abstract] [Full Text] [PDF]
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L.-H. Yeh, Y. J. Park, R. J. Hansalia, I. S. Ahmed, S. S. Deshpande, P. J. Goldschmidt-Clermont, K. Irani, and B. R. Alevriadou Shear-induced tyrosine phosphorylation in endothelial cells requires Rac1-dependent production of ROS Am J Physiol Cell Physiol, April 1, 1999; 276(4): C838 - C847. [Abstract] [Full Text] [PDF]
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J. Fan and K. B. Walsh Mechanical Stimulation Regulates Voltage-Gated Potassium Currents in Cardiac Microvascular Endothelial Cells Circ. Res., March 5, 1999; 84(4): 451 - 457. [Abstract] [Full Text] [PDF]
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M. Ikeda, T. Takei, I. Mills, H. Kito, and B. E. Sumpio Extracellular signal-regulated kinases 1 and 2 activation in endothelial cells exposed to cyclic strain Am J Physiol Heart Circ Physiol, February 1, 1999; 276(2): H614 - H622. [Abstract] [Full Text] [PDF]
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C. Yan, M. Takahashi, M. Okuda, J.-D. Lee, and B. C. Berk Fluid Shear Stress Stimulates Big Mitogen-activated Protein Kinase 1 (BMK1) Activity in Endothelial Cells. DEPENDENCE ON TYROSINE KINASES AND INTRACELLULAR CALCIUM J. Biol. Chem., January 1, 1999; 274(1): 143 - 150. [Abstract] [Full Text] [PDF]
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H. Hagiwara, M. Mitsumata, T. Yamane, X. Jin, and Y. Yoshida Laminar Shear Stress–Induced GRO mRNA and Protein Expression in Endothelial Cells Circulation, December 8, 1998; 98(23): 2584 - 2590. [Abstract] [Full Text] [PDF]
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H. Park, Y.-M. Go, P. L. St. John, M. C. Maland, M. P. Lisanti, D. R. Abrahamson, and H. Jo Plasma Membrane Cholesterol Is a Key Molecule in Shear Stress-dependent Activation of Extracellular Signal-regulated Kinase J. Biol. Chem., November 27, 1998; 273(48): 32304 - 32311. [Abstract] [Full Text] [PDF]
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Y.-M. Go, H. Park, M. C. Maland, V. M. Darley-Usmar, B. Stoyanov, R. Wetzker, and H. Jo Phosphatidylinositol 3-kinase gamma mediates shear stress-dependent activation of JNK in endothelial cells Am J Physiol Heart Circ Physiol, November 1, 1998; 275(5): H1898 - H1904. [Abstract] [Full Text] [PDF]
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M. F Bellamy, J. Goodfellow, A. C Tweddel, F. D.J Dunstan, M. J Lewis, and A. H Henderson Syndrome X and endothelial dysfunction Cardiovasc Res, November 1, 1998; 40(2): 410 - 417. [Abstract] [Full Text] [PDF]
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V. Rizzo, A. Sung, P. Oh, and J. E. Schnitzer Rapid Mechanotransduction in Situ at the Luminal Cell Surface of Vascular Endothelium and Its Caveolae J. Biol. Chem., October 9, 1998; 273(41): 26323 - 26329. [Abstract] [Full Text] [PDF]
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T. Murase, N. Kume, R. Korenaga, J. Ando, T. Sawamura, T. Masaki, and T. Kita Fluid Shear Stress Transcriptionally Induces Lectin-like Oxidized LDL Receptor-1 in Vascular Endothelial Cells Circ. Res., August 10, 1998; 83(3): 328 - 333. [Abstract] [Full Text] [PDF]
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S. Dimmeler, B. Assmus, C. Hermann, J. Haendeler, and A. M. Zeiher Fluid Shear Stress Stimulates Phosphorylation of Akt in Human Endothelial Cells : Involvement in Suppression of Apoptosis Circ. Res., August 10, 1998; 83(3): 334 - 341. [Abstract] [Full Text] [PDF]
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S. Kim-Schulze, W. L. Lowe Jr, and H. W. Schnaper Estrogen Stimulates Delayed Mitogen-Activated Protein Kinase Activity in Human Endothelial Cells via an Autocrine Loop That Involves Basic Fibroblast Growth Factor Circulation, August 4, 1998; 98(5): 413 - 421. [Abstract] [Full Text] [PDF]
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S. Lehoux and A. Tedgui Signal Transduction of Mechanical Stresses in the Vascular Wall Hypertension, August 1, 1998; 32(2): 338 - 345. [Abstract] [Full Text] [PDF]
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O. Traub and B. C. Berk Laminar Shear Stress : Mechanisms by Which Endothelial Cells Transduce an Atheroprotective Force Arterioscler Thromb Vasc Biol, May 1, 1998; 18(5): 677 - 685. [Abstract] [Full Text] [PDF]
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S. Jalali, Y.-S. Li, M. Sotoudeh, S. Yuan, S. Li, S. Chien, and J. Y-J. Shyy Shear Stress Activates p60src-Ras-MAPK Signaling Pathways in Vascular Endothelial Cells Arterioscler Thromb Vasc Biol, February 1, 1998; 18(2): 227 - 234. [Abstract] [Full Text] [PDF]
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E. M. Redmond, P. A. Cahill, and J. V. Sitzmann Flow-Mediated Regulation of G-Protein Expression in Cocultured Vascular Smooth Muscle and Endothelial Cells Arterioscler Thromb Vasc Biol, January 1, 1998; 18(1): 75 - 83. [Abstract] [Full Text] [PDF]
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S. Chien, S. Li, and J. Y-J. Shyy Effects of Mechanical Forces on Signal Transduction and Gene Expression in Endothelial Cells Hypertension, January 1, 1998; 31(1): 162 - 169. [Abstract] [Full Text] [PDF]
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K. G. Birukov, S. Lehoux, A. A. Birukova, R. Merval, V. A. Tkachuk, and A. Tedgui Increased Pressure Induces Sustained Protein Kinase C–Independent Herbimycin A–Sensitive Activation of Extracellular Signal–Related Kinase 1/2 in the Rabbit Aorta in Organ Culture Circ. Res., December 19, 1997; 81(6): 895 - 903. [Abstract] [Full Text]
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J. M. Pyles, K. L. March, M. Franklin, K. Mehdi, R. L. Wilensky, and L. P. Adam Activation of MAP Kinase In Vivo Follows Balloon Overstretch Injury of Porcine Coronary and Carotid Arteries Circ. Res., December 19, 1997; 81(6): 904 - 910. [Abstract] [Full Text]
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O. Traub, B. P. Monia, N. M. Dean, and B. C. Berk PKC-epsilon Is Required for Mechano-sensitive Activation of ERK1/2 in Endothelial Cells J. Biol. Chem., December 12, 1997; 272(50): 31251 - 31257. [Abstract] [Full Text] [PDF]
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J.J. Chiu, B.S. Wung, J. Y.J. 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, December 1, 1997; 17(12): 3570 - 3577. [Abstract] [Full Text]
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M. T. Franklin, C. L.-A. Wang, and L. P. Adam Stretch-dependent activation and desensitization of mitogen-activated protein kinase in carotid arteries Am J Physiol Cell Physiol, December 1, 1997; 273(6): C1819 - C1827. [Abstract] [Full Text] [PDF]
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S. Li, M. Kim, Y.-L. Hu, S. Jalali, D. D. Schlaepfer, T. Hunter, S. Chien, and J. Y-J. Shyy Fluid Shear Stress Activation of Focal Adhesion Kinase. LINKING TO MITOGEN-ACTIVATED PROTEIN KINASES J. Biol. Chem., November 28, 1997; 272(48): 30455 - 30462. [Abstract] [Full Text] [PDF]
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T. Caulin-Glaser, G. Garcia-Cardena, P. Sarrel, W. C. Sessa, and J. R. Bender 17ß-Estradiol Regulation of Human Endothelial Cell Basal Nitric Oxide Release, Independent of Cytosolic Ca2+ Mobilization Circ. Res., November 19, 1997; 81(5): 885 - 892. [Abstract] [Full Text]
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Y. Hu, L. Cheng, B.-W. Hochleitner, and Q. Xu Activation of Mitogen-Activated Protein Kinases (ERK/JNK) and AP-1 Transcription Factor in Rat Carotid Arteries After Balloon Injury Arterioscler Thromb Vasc Biol, November 1, 1997; 17(11): 2808 - 2816. [Abstract] [Full Text]
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L. M. Khachigian and T. Collins Inducible Expression of Egr-1–Dependent Genes : A Paradigm of Transcriptional Activation in Vascular Endothelium Circ. Res., October 19, 1997; 81(4): 457 - 461. [Full Text]
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J. K. Miyashiro, V. Poppa, and B. C. Berk Flow-Induced Vascular Remodeling in the Rat Carotid Artery Diminishes With Age Circ. Res., September 19, 1997; 81(3): 311 - 319. [Abstract] [Full Text]
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E. O. Harrington, J. Loffler, P. R. Nelson, K. C. Kent, M. Simons, and J. A. Ware Enhancement of Migration by Protein Kinase Calpha and Inhibition of Proliferation and Cell Cycle Progression by Protein Kinase Cdelta in Capillary Endothelial Cells J. Biol. Chem., March 14, 1997; 272(11): 7390 - 7397. [Abstract] [Full Text] [PDF]
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J. M. Muller, W. M. Chilian, and M. J. Davis Integrin Signaling Transduces Shear Stress–Dependent Vasodilation of Coronary Arterioles Circ. Res., March 1, 1997; 80(3): 320 - 326. [Abstract] [Full Text]
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H. Jo, K. Sipos, Y.-M. Go, R. Law, J. Rong, and J. M. McDonald Differential Effect of Shear Stress on Extracellular Signal-regulated Kinase and N-terminal Jun Kinase in Endothelial Cells. Gi2- AND Gbeta /gamma -DEPENDENT SIGNALING PATHWAYS J. Biol. Chem., January 10, 1997; 272(2): 1395 - 1401. [Abstract] [Full Text] [PDF]
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M. Kusuhara, A. Chait, A. Cader, and B. C. Berk Oxidized LDL Stimulates Mitogen-Activated Protein Kinases in Smooth Muscle Cells and Macrophages Arterioscler Thromb Vasc Biol, January 1, 1997; 17(1): 141 - 148. [Abstract] [Full Text]
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M. A. Corson, 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., November 1, 1996; 79(5): 984 - 991. [Abstract] [Full Text]
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S. R.P. Gudi, C. B. Clark, and J. A. Frangos Fluid Flow Rapidly Activates G Proteins in Human Endothelial Cells: Involvement of G Proteins in Mechanochemical Signal Transduction Circ. Res., October 1, 1996; 79(4): 834 - 839. [Abstract] [Full Text]
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T. Ishida, T. E. Peterson, N. L. Kovach, and B. C. Berk MAP Kinase Activation by Flow in Endothelial Cells: Role of ß1 Integrins and Tyrosine Kinases Circ. Res., August 1, 1996; 79(2): 310 - 316. [Abstract] [Full Text]
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J.-i. Abe, M. Kusuhara, R. J. Ulevitch, B. C. Berk, and J.-D. Lee Big Mitogen-activated Protein Kinase 1(BMK1) Is a Redox-sensitive Kinase J. Biol. Chem., July 12, 1996; 271(28): 16586 - 16590. [Abstract] [Full Text] [PDF]
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K. Ayajiki, 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., May 1, 1996; 78(5): 750 - 758. [Abstract] [Full Text]
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P. F. Davies NO Flow Helps Clear Murky Waters? Circ. Res., May 1, 1996; 78(5): 945 - 946. [Full Text]
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