© 1999 American Heart Association, Inc.
Original Contributions |
Temporal Gradient in Shear But Not Steady Shear Stress Induces PDGF-A and MCP-1 Expression in Endothelial Cells
Role of NO, NF
B, and egr-1
From the Department of Bioengineering, University of California, San Diego, La Jolla
Correspondence to Dr John A. Frangos, Department of Bioengineering, University of California, San Diego, 6407 Engineering Bldg Unit 1, 9500 Gilman Dr, La Jolla, CA 92093-0412. E-mail frangos{at}ucsd.edu
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Abstract |
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Abstract—Three well-defined laminar flow profiles were created to distinguish the influence of a gradient in shear and steady shear on platelet-derived growth factor A (PDGF-A) and monocyte chemoattractant protein-1 (MCP-1) expression in human endothelial cells. The flow profiles (16 dyne/cm2 maximum shear stress) were ramp flow (shear stress smoothly transited at flow onset), step flow (shear stress abruptly applied at flow onset), and impulse flow (shear stress abruptly applied for 3 s only). Ramp flow induced only minor expression of PDGF-A and did not increase MCP-1 expression. Step flow increased PDGF-A and MCP-1 mRNA levels 3- and 2-fold at 1.5 hours, respectively, relative to ramp flow. In contrast, impulse flow increased PDGF-A and MCP-1 expression 6- and 7-fold at 1.5 hours, and these high levels were sustained for at least 4 hours. These results indicate that a temporal gradient in shear (impulse flow and the onset of step flow) and steady shear (ramp flow and the steady component of step flow) stimulates and diminishes the expression of PDGF-A and MCP-1, respectively. NO synthase inhibitor NG-amino-L-arginine (L-NAA) was found to markedly enhance MCP-1 and PDGF-A expression induced by step flow, but decrease their expression induced by impulse flow, in a dose-dependent manner. NO donor spermine-NONOate (SPR/NO) dose-dependently reduced the MCP-1 and PDGF-A expression induced by impulse flow. Moreover, impulse flow was found to stimulate sustained (4 hours) I
B-
degradation and
egr-1 mRNA induction. L-NAA prevented IB-
degradation, whereas SPR/NO increased IB- resynthesis 2 hours
after impulse flow. Both L-NAA and SPR/NO inhibited the impulse flow
inducibility of egr-1 4 hours after the flow
stimulation. The results show that both NO induced by steady shear and
NO donor inhibit temporal gradient in shear-induced MCP-1 and PDGF-A
expression by downregulation of their respective transcription factors
NFB and egr-1, whereas NO induced by impulse flow
stimulates MCP-1 and PDGF-A expression by upregulation of the
transcription factors. The above findings suggest distinct roles of
temporal gradient in shear and steady shear in atherogenesis in
vivo.
Key Words: shear stress • gene expression • nitric oxide • transcription factor • atherosclerosis
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Introduction |
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Because of the focal distribution of atherosclerotic plaques, hemodynamic forces including fluid shear stress have long been suggested to potentiate atherogenesis.1 2 The exact nature and influence of local shear involved in endothelial dysfunction leading to susceptibility for atherogenesis, however, remains unclear. Endothelial cells (EC) throughout the vasculature experience a variety of flow environments both with spatial variances and with temporal gradients in wall shear stress. In the venous system wall shear stress is lower and minimal gradients in shear stress exist because of the nonpulsatile nature of the blood flow. In the arterial system the flow conditions are generally assumed to be laminar and to present the endothelium with a high mean wall shear stress in addition to large temporal gradients in shear stress. At arterial bifurcations and curvatures, locations known to be highly prone to atherogenesis, disturbed flow patterns may develop that result in low mean wall shear stress, but still present the EC with large temporal gradients in shear stress.3 These observations, combined with other in vitro evidence,4 5 6 suggest that gradients in shear and steady shear represent different biomechanical stimuli that differentially regulate local endothelial function by distinct signaling pathway, and thus contribute to the characteristic distribution pattern of atherosclerosis.
A host of endothelial genes exhibit differential responses to shear stress stimuli that may be involved in the focal localization of atherogenic plaques.7 The application of step shear on EC in vitro has been shown to induce an initial transient expression of monocyte chemoattractant protein-1 (MCP-1), a potent chemotactic agent for monocytes, and platelet-derived growth factor A (PDGF-A), a potent mitogen and chemotactic agent for smooth muscle cells, giving a peak response at 1.5 hours followed by sustained downregulation.8 9 MCP-1 and PDGF-A are believed to participate in the early events of atherosclerosis.10 11 12 In contrast, three potential antiatherogenic genes—manganese superoxide dismutase (Mn SOD), cyclooxygenase-2 (COX-2), and endothelial nitric oxide synthase (eNOS)—were demonstrated to be continuously upregulated by steady laminar shear but not turbulent shear.13 Although the pathways and biochemical factors that control the dynamic balance of expression levels of these various genes have not been completely characterized, it is clear that imbalanced regulation of these effector molecules occurs at lesion-prone sites. Given that the onset of fluid shear in vitro can be decomposed into two separate physical stimuli, an initial step-change in shear (or a large temporal gradient in shear) followed by steady shear, we hypothesized that the induction of those atherogenesis-related genes (eg, PDGF-A and MCP-1) is associated with the temporal gradient in shear but not steady shear.
In response to fluid shear stress, EC elaborate various bioactive
substances that may be involved in the regulation of these
atherogenesis-related genes, such as NO.14 15 The loss of
endothelium-derived NO control of vasomotion precedes
the onset of atherogenesis.16 In addition to being a
potent vasodilator, NO is also an inhibitor of platelet
aggregation and smooth muscle cell proliferation, properties that have
been shown to be antiatherogenic.17 Exposure of EC to
fluid shear also stimulates the activation of transcription factors,
such as egr-1,18 AP-1,19
and NFB.19 20 Activated egr-1 binds
to the proximal PDGF-A promoter by displacing sp-1 from
their overlapping recognition element, and the egr-1 binding
site has been identified as a cis-element for the induction of PDGF-A
by fluid shear.18 The promoter of MCP-1 gene contains
both NFB and AP-1 binding sites, which may coordinately modulate
shear inducibility of MCP-1.20 21 22 NO may affect
expression of these genes by activation or inactivation of their
transcription factors.
The present study focuses on the differential responses of two
atherogenesis-related genes, PDGF-A and MCP-1, to steady
shear and temporal gradient in shear in human umbilical vein EC
(HUVEC). To separate these two distinct mechanical stimuli, various
flow regimens were used that included impulse, step, and ramp flow
profiles. By creating these simplified flow profiles, we can better
characterize the responses of EC to physiological
flows by deconstructing the shear stress profile into two components:
steady (or slowly transiting) shear stress and impulse (or temporal
gradient) in shear stress, as exists in rapidly fluctuating flow. The
latter profile was chosen not to simulate
physiological flow, but rather to subject the cells
to one component of physiological flow. We
demonstrate, for the first time, that the rate of change in shear
stress rather than steady shear stress is a potent stimulus for the
expression of such atherogenesis-related genes as PDGF-A and MCP-1. By
using NO synthase inhibitors and NO donors, and by
measuring expression of the transcription factors NFB and
egr-1, a dual role for NO in regulating the expression of
MCP-1 and PDGF-A is also demonstrated in this study.
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Methods |
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Cell Culture and Experimental Preparation
Primary HUVEC isolation was performed as previously described.23 Briefly, cells were harvested from human umbilical veins by collagenase (Boehringer Mannheim) treatment and were then seeded onto glass microscope slides (cell area, 16 cm2). The cells were grown to confluence within 3 to 4 days in ATP-free M199 medium supplemented with 20% FBS (Hyclone), and with 2 mmol/L L-glutamine, 0.5 U/mL penicillin, and 0.05 mg/mL streptomycin (Sigma). Before shear exposure, the confluent cells were serum-starved for 48 hours in 0.5% FBS-supplemented M199 to establish quiescence in the monolayers. For NO synthase inhibitor and NO donor studies, cell monolayers were preincubated with NG-amino-L-arginine (L-NAA; Alexis), spermine-NONOate (SPR/NO; Alexis), or spermine (SPR; Alexis) at different concentrations for 1 hour before being subjected to shear. All cell cultures were maintained in a humidified 5% CO2/95% air incubator at 37°C.
Flow Experiments
Cell monolayers on glass slides were subjected to well-defined
laminar flow for various times in a parallel-plate flow chamber, in
which perfusing medium was driven by a syringe pump (pump 44, Harvard
Apparatus) or a constant head flow loop as described
previously.23 The computer-controlled syringe pump was
programmed to generate different flow profiles. The flow rate (and
hence shear stress) was changed in 1-s microsteps with volume/flow
increments determined from the slope of the overall increase in shear
stress. Four well-defined laminar flow profiles were generated to which
EC were exposed (Figure 1
). These
profiles were designed and used to separate the effect of different
dynamic flow stimuli presented to EC. The following profiles
were applied: (1) step flow (instantaneous shear stress increase from 0
to 16 dyne/cm2, followed by steady shear for a
sustained period); (2) ramp flow (shear stress smoothly transited from
0 to 16 dyne/cm2 during 10 minutes, and then
sustained for a desired period); (3) impulse flow (a 3-s impulse of 16
dyne/cm2); and (4) reverse impulse flow (a step
increase to 16 dyne/cm2, sustained for 3 s,
followed by a ramped decrease to 0 dyne/cm2
during 10 minutes).
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The entire flow device was placed in an air curtain incubator and maintained at 37°C throughout the experiment. The fluid used to shear the cells was the same as the medium used to preincubate the cells before the shear exposure, and was placed in the incubator for 2 to 3 hours before the flow experiments. Dextran (1.25%, 2 000 000 MW; Sigma) was added to the medium. This addition increased the media viscosity from 1 to 3 cP. The shear stress was calculated as previously described.24 After impulse flow exposure, slides were removed from the flow chamber, placed back into Petri dishes, and kept in the incubator for the appropriate matched period. Time-matched stationary (no-flow) controls were performed in Petri dishes.
RNA Isolation and Northern Blotting
Slides were removed from the flow chamber or Petri dishes, and
cell monolayers were washed twice with PBS and then lysed. Total RNA
was then isolated using an RNAeasy Total RNA Kit (Qiagen). After
quantification by measuring absorbance at 260 nm, equal amounts of
total RNA were loaded and electrophoresed on 1% formaldehyde-agarose
gels, transferred to nylon membranes (Micron Separation), and then
fixed by ultraviolet irradiation. The following cDNA fragments were
labeled with [32P]dCTP (ICN Radiochemical) and
used as hybridization probes: for PDGF-A, a 1.3-kb EcoRI
fragment of PDGF-A-13.125 ; for MCP-1, a 0.7-kb
EcoRI/BamHI fragment of MCP-1 (ATCC); for
glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an
0.8-kb XbaI/PstI fragment of pHcGAP
(ATCC); and for egr-1, a 3.1-kb
EcoRI/BamHI fragment of
egr-1.26 Nylon filters with uniformly
transferred RNA were prehybridized for 2 to 4 hours and then hybridized
at 42°C in hybridization buffer overnight with the appropriate
32P-labeled DNA probes. The membranes were then
washed and exposed to Kodak X-omat film at -70°C for
autoradiography. For some membranes the bound probes
were stripped off by washing the membranes in stripping buffer (1
mmol/L TrisCl, pH 8; 1 mmol/L EDTA, pH 8; and 0.1x Denhardts) at
75°C for 2 hours. The same membranes were subsequently rehybridized
with other probes. Densitometry was performed using a model IS-10000
image analyzer (Alpha Innotech).
Protein Preparation and Western Blotting
Cells were immediately washed with ice-cold PBS and lysed at
4°C in lysis buffer (20 mmol/L sodium HEPES, pH 7.4; 1
mmol/L EDTA; 2 mmol/L MgCl2; 10 µg/mL
PMSF; 2 µg/mL leupeptin; 2 µg/mL aprotinin), followed by scraping,
sonication, centrifugation (15 minutes at
14 000g), and boiling (5 minutes at 100°C). Total cell
lysates (50 µg/protein) were separated by SDS-polyacrylamide
gel electrophoresis (10% running, 4% stacking). The protein was then
transferred to nitrocellulose filters (Millipore). Blocking was
performed overnight at 4°C in Tris-buffered saline (TBS) plus 5%
nonfat dry milk. The blots were incubated with affinity-purified rabbit
antibody to IB- (1:1000; Santa Cruz) overnight in buffer
containing TBS with 0.05% Tween-20 and 5% nonfat dry milk (Blotto),
followed by incubation with the horseradish
peroxidase–conjugated goat anti-rabbit antibody (1:2000; New
England) in Blotto for 45 minutes. Immunodetection was accomplished
using chemiluminescence (Super Signal CL-HRP, Pierce Chemical Corp).
NO Determination
Confluent EC were incubated in modified Eagle's minimum
essential medium (MEM, Sigma) containing 2% FBS overnight, and then
incubated for 4 hours in serum-deprived modified MEM before exposure to
shear. Two aliquots of 0.5 mL of perfusing media were obtained
immediately before and 4 hours after flow shear treatment. NO levels
(NO2- +
NO3-) in the perfusing media
were measured by an NO chemiluminescence analyzer (Sievers
270B).
Statistics
The results are presented as mean±SE compared with
controls and among separate experiments. Significance was determined by
unpaired Student's t test (P<0.05).
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Results |
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Effects of Temporal Gradient in Shear and Steady Shear Stress on Gene Expression of MCP-1 and PDGF-A
To differentiate the effects of temporal gradient in shear stress versus steady shear stress on endothelial MCP-1 and PDGF-A expression, confluent HUVEC were subjected to ramp, step, or impulse laminar flow at a maximal shear stress of 16 dyne/cm2 for 1.5 hours. The level and duration of shear stress was selected because an average shear stress of 16 dyne/cm2 is within the physiological range in arterial vasculature and has been demonstrated to induce significant increases in MCP-18 and PDGF-A9 mRNA levels in HUVEC, with the maximal response at 1.5 hours. Ramp flow consistently elicited only minor increases in MCP-1 and PDGF-A expression relative to the static levels. Step flow produced a 2-fold increase in MCP-1 mRNA levels greater than that induced by ramp flow. Impulse flow resulted in a 7-fold increase in MCP-1 expression relative to ramp flow. For PDGF-A expression, step flow elicited a 4-fold increase in mRNA level, whereas impulse flow produced a 6-fold increase in mRNA level relative to ramp flow (Figure 2
).
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To account for the possible effect of small differences in the duration
of steady flow caused by linear ramp on MCP-1 and PDGF-A mRNA levels,
HUVEC were subjected to two different ramp flows with the shear stress
smoothly transited from 0 to 16 dyne/cm2 during
10 minutes, and then continuing for 80 minutes (Figure 2
) or 90
minutes (data not shown) at 16 dyne/cm2. No
significant differences in gene expression were observed between these
two ramps.
To account for possible stimulation of cells by the manipulation during assembly of the cell-covered slide on the chamber, the slide was placed gently on the chamber without flow for 10 minutes, and then placed back in the Petri dish for another 80 minutes. This procedure of manipulating slides did not induce significant gene expression (data not shown).
Because in every case ramp flow elicited minor increases in gene
expression relative to the static condition (Figure 2), the
effect of the small increments in flow rate during the ramp period was
investigated. Cells were subjected to ramp shear stress, which
increased from 0 to 0.4 dyne/cm2 at the same
increments (0.03 dyne/cm2 per increment) and time
steps (1 increment/s) as the ramp flow, and then maintained at 0.4
dyne/cm2 for 90 minutes. This low level of shear
stress induced MCP-1 gene expression at a level equivalent to that
produced by ramp flow (data not shown). The result indicates that the
small steps of 0.03 dyne/cm2 used to ramp the
flow are themselves sufficient to stimulate MCP-1 expression, albeit
not at the level of impulse flow, suggesting that further refinement in
smoothly applying ramp flow should lead to a reduction of this ramp
flow-induced gene expression.
To further evaluate the effects of gradient in shear stress, cells
exposed to impulse flow were placed under static incubation for a
longer time period. The levels of MCP-1 and PDGF-A mRNA induced by
impulse flow at 4 hours were significantly higher than the mRNA levels
at 1.5 hours, indicating sustained and increasing expression (Figure 2). The high levels of expression for PDGF-A and MCP-1 induced
by impulse flow were sustained even at 8 hours (data not shown). It
appears, therefore, that the induction of gene expression by impulse
flow is not as transient as that by step flow, which has been shown to
return to or even fall below basal levels after 4
hours.8 9
Impulse flow can be further decomposed to two stimuli: step increase
and step decrease in shear. To elucidate the possible effect of a step
decrease on gene expression, the responses to reverse impulse flow (see
Figure 1) were analyzed. The levels of MCP-1 and PDGF-A
mRNA induced by reverse impulse flow were equivalent to that induced by
impulse flow at 1.5 hours (Figure 2A). The result suggests that
either the step decrease in flow has no effect or the stimulus of the
step increase in flow saturated the response.
Effect of NO on the Temporal Gradient in Shear-Induced
Endothelial MCP-1 and PDGF-A Expression
The potential role of steady flow-associated production of
NO14 in MCP-1 and PDGF-A expression induced by the
temporal gradient in shear was investigated by using L-NAA, a NOS
inhibitor, in cells subjected to step flow for 4 hours.
L-NAA (100 µmol/L) significantly increased step flow-induced
MCP-1 and PDGF-A mRNA expression (0.6- and 3.2-fold, respectively)
(Figure 3A and 3B), and correspondingly
decreased step flow-induced NO production by 82% (inset,
Figure 3B) compared with that induced by step flow without
treatment with L-NAA.
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To determine the effect of exogenous NO, cells subjected to impulse
flow were treated with SPR/NO or SPR at various concentrations for 4
hours. The NO donor downregulated impulse flow-induced MCP-1 and PDGF-A
expression in a dose-dependent manner (Figure 3A and 3C). SPR/NO
(1, 10, and 100 µmol/L) decreased impulse flow-induced MCP-1
mRNA by 11%, 36%, and 80%, and PDGF-A mRNA by 23%, 50%, and 57%,
respectively, compared with impulse flow alone. No significant effect
on mRNA levels was observed with SPR alone.
Because impulse flow stimulates a burst in production of
NO,4 14 we further investigated whether this burst in NO
is involved in impulse-flow-induced gene expression. L-NAA (50, 100,
and 300 µmol/L) dose-dependently decreased the impulse
flow-induced MCP-1 mRNA by 18%, 22%, and 41%, and PDGF-A mRNA by
34%, 74%, and 76%, respectively, compared with expression induced by
impulse flow alone (Figure 4). Similar
results were observed with another NOS inhibitor,
NG-nitro-L-arginine
(data not shown). Thus it appears that impulse flow-induced gene
expression is mediated by NO production.
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Effect of NO on IB- Expression Induced by Temporal Gradients
in Shear
The promoter of the human MCP-1 gene contains functionally active
NFB binding sites.27 The activation of NFB by tumor
necrosis factor- has been shown to occur within 15 minutes and
coincides with phosphorylation and degradation of
IB-, which is inhibited by NO after 2 hours.28 To
determine how exogenous NO regulates the activation of NFB induced
by impulse flow, we investigated the fate of impulse flow-stimulated
IB- expression in the presence and absence of SPR/NO (100
µmol/L). Exposure to impulse flow resulted in sustained (from 15
minutes to 4 hours) 6- to 7-fold IB- degradation. SPR/NO
initially (15 minutes) reduced impulse flow-induced IB-
expression, but increased its expression by 4- to 5-fold at 2 and 4
hours (Figure 5A). This indicated that NO
inhibits NFB activation not by stabilization of IB- but rather
by increasing IB- resynthesis.
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To determine whether endogenous NO is involved in impulse
flow-induced IB- degradation, EC were treated with L-NAA
(300 µmol/L) 1 hour before subjecting to impulse flow. L-NAA
inhibited impulse flow-induced IB- degradation by 3-fold at 15
minutes (Figure 5B), but had no significant
inhibitory effect on the expression of IB- in static
cells. The inhibitory effect of L-NAA on impulse
flow-induced IB- degradation was also observed at 4 hours (data
not shown). All data presented were normalized by nonspecific
bands, which correspond to the amount of the protein loading.
Effect of NO on egr-1 mRNA Expression Induced by
Temporal Gradient in Shear
Because the egr-1 binding site in the proximal PDGF-A
promoter is responsible for its shear stress
inducibility,18 we investigated the effect of NO on
egr-1 mRNA expression. EC were exposed to impulse flow in
the presence or absence of L-NAA and SPR/NO for 4 hours. L-NAA
(100 µmol/L) almost completely abolished impulse flow-induced
egr-1 mRNA expression. On the other hand, addition of SPR/NO
(10 µmol/L) also significantly decreased the impulse-flow
induced egr-1 mRNA expression. SPR alone did not cause a
significant change in egr-1 mRNA levels (Figure 6).
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Discussion |
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Hemodynamic shear stress influences the physiology of EC and participates in the development and progression of arterial diseases.29 30 31 In addition to steady shear stress, recent attention has also focused on gradients in shear stress as a contributing factor that may link disturbed fluid dynamics to atherosclerosis.4 5 6 Exposure of endothelial cells to fluid flow in vitro has been used as a simplified means of understanding the precise role of fluid shear in endothelial function. The concept that fluid flow provides two distinct stimuli—temporal gradient in shear and steady shear—to the endothelium has been previously suggested in prostacyclin production,23 and demonstrated in a number of studies, including NO and cGMP production4 14 and pinocytosis.32 This study, for the first time, directly compared the induction of certain atherogenesis-related genes (PDGF-A and MCP-1) in EC exposed to temporal gradient in shear and steady shear, using three well-defined laminar flow profiles: impulse, step, and ramp flow.
The present experiments demonstrate the enhanced expression of
MCP-1 and PDGF-A mRNA at 1.5 hours in EC exposed to impulse flow
relative to step flow and even more pronounced elevations with respect
to ramp flow (Figure 2). In addition, impulse flow was shown to
further increase expression of these two genes after 1.5 hours as
observed at 4 hours after shear stimulation (Figure 2). This is
in sharp contrast to the effect of step flow at 4 hours, when gene
expression returns to basal or sub-basal levels.8 9 Both
step and impulse profiles contain a sharp gradient in shear stress.
However, a component present in both step and ramp profiles—steady
shear stress—is absent in impulse flow. The contrasting abilities of
step and impulse flow, and the inability of ramp flow to elicit
significant gene expression, suggest that temporal gradient in shear
but not steady shear stimulates the expression of these
atherogenesis-related genes.
Temporal gradients in shear in the absence of steady shear stress induced a larger and sustained expression. Thus, one can hypothesize that the continued exposure of EC to steady shear promotes a downregulation of these atherogenesis-related genes. This hypothesis is supported by the observation that steady shear downregulates the expression of vascular cell adhesion molecule-1 (VCAM-1), another potential proatherogenic leukocyte adhesion molecule.33 Together with the observation of the selective and sustained upregulation of three endothelial antiatherogenic genes (eNOS, COX-2, and MnSOD) by steady shear,13 our findings suggest that steady shear may exert a coordinated influence on endothelial function, the net effect of which is antiatherogenic. In contrast, temporal gradients in shear, such as may be found in areas with disturbed hemodynamic flow, induce endothelial atherogenesis-related gene expression, and thus serve as a biomechanical risk factor.
The present study focused on a physiological
level of steady shear of 16 dyne/cm2 that in vivo
approximates the average wall shear typically encountered in
lesion-protected areas of the vasculature. We and others have shown,
however, that exposure of EC to prolonged steady low shear (2
dyne/cm2) induces sustained increases in
endothelin-1 (a mitogen for vascular smooth muscle cells) release, as
well as NFB, MCP-1, and VCAM-1 expression.20 34 35
These observations suggest that steady low shear may be another
biomechanical risk factor. Taken together, the coordinated induction of
these atherogenic factors may occur at the lesion-prone area, where the
disturbed hemodynamic flow patterns—low mean shear
levels associated with temporal gradients in shear—prevail, and thus
may be one of the mechanisms contributing to the focal distribution of
early atherosclerotic lesions.
Until now the role of NO in regulation of gene expression has been
controversial because both stimulatory and inhibitory
effects have been observed. For example, NO has been shown to inhibit
cytokine-induced expression of VCAM-1, ICAM-1, and
MCP-136 37 38 39 through cGMP-independent pathways, whereas NO
can stimulate the expression of certain immediate early genes through
cGMP-dependent pathways.40 41 42 43 Here we observed the
effects of NO induced by different flow profiles, as well as those of
exogenous NO, on gene expression. We found that NO induced by impulse
flow stimulated MCP-1 and PDGF-A expression (Figure 4), whereas
sustained NO release, generated by steady shear (or NO donors),
inhibited the expression of these atherogenesis-related genes (Figure 3). This dual action of NO is further supported by the
activation of NFB (Figure 5) and the induction of
egr-1 mRNA (Figure 6) by impulse flow-induced NO
production, and downregulation of these transcription factors
by sustained NO release (Figures 5 and 6). The results we
provided demonstrate for the first time that NO has two opposing
(stimulatory and inhibitory) actions on the same genes, and
NO exerts its dual actions on gene expression by coordinately
regulating its transcription factor. This apparently conflicting action
of NO may be related to the kinetics and signaling of NO
production induced by different flow profiles. Previous work
has demonstrated that temporal gradient in shear (impulse flow)
produces a transient burst of NO, whereas steady shear (ramp flow)
induces a sustained low rate of NO in EC; the former is dependent on
and the latter is independent of calcium and
G-protein.4 14 It needs to be further elucidated,
however, how this bimodal NO production differently regulates
gene expression.
Evidence provided here shows that NO plays a crucial role in temporal
gradients in shear-induced stimulatory effects as well as steady
shear-mediated inhibitory effects. It is not clear,
however, why NOS inhibition enhanced expression of MCP-1 and PDGF-A by
step flow (Figure 3). One speculative possibility regards the
involvement of superoxide
(O2-). Fluid shear can induce
sustained production of reactive oxygen species,44
which induces the expression of ICAM-1 and
c-fos.45 46 The breakdown of
O2- occurs mainly by reaction
with NO to form peroxynitrite
(ONOO-),47 which has cytoprotective
effects similar to those of NO at low concentration.48
Although eNOS can produce NO as well as
O2-, NOS inhibitors
may only block NO production,49 50 leading to
excess O2-. Superoxide, in
turn, can activate the transcription factors
AP-1,51 NFB,52 and
egr-1,53 and therefore induce
expression of MCP-1 and PDGF-A. Another source of
O2- is activated
Ras.54 Step flow has been shown to induce a transient and
rapid activation of Ras in EC, leading to the induction of MCP-1
expression.55 NO has also been shown to inhibit Ras
activation.56 With the inhibition of NO production
by L-NAA, activation of Ras may be more sustained. Therefore, the
Ras-O2- pathway may be another
source for O2- involved in step
flow-induced MCP-1 and PDGF-A expression when sustained NO
production is inhibited.
In summary, we have shown that temporal gradients in shear stress lead
to enhanced and sustained expression of MCP-1 and PDGF-A in HUVEC,
whereas the presence of steady laminar shear stress reduces these
levels. Both the stimulatory effects of temporal gradients in shear and
the inhibitory effects of steady shear on gene expression
have been found to be mediated by NO, probably by activation and
inactivation of their transcription factors, NFB and
egr-1. Although additional investigation is necessary to
further elucidate and refine the concepts presented in this
paper, understanding the distinct contribution of each component in the
complicated flow regimen may provide an important clue to the focal
development of atherosclerosis and vascular disease in
general.
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Acknowledgments |
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This work was supported by National Heart, Lung, and Blood Institute grant HL-40696. X. Bao is the recipient of a National Research Service Award from the National Institute of Health. We thank T. McAllister for his excellent technical assistance, and C. Shilling and C.B. Clark for their helpful discussions. We also express our sincere appreciation to the nurses at the maternity wards of Sharp Memorial Hospital and Mercy Hospital of San Diego for supplying umbilical cords.
Received June 4, 1998; accepted September 30, 1998.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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1. Asakura T, Karino T. Flow patterns and spatial distribution of atherosclerotic lesions in human coronary arteries. Circ Res. 1990;66:1045–1066.
2. Cornhill JF, Akins D, Hutson M, Chandler AB. Localization of atherosclerotic lesions in the human basilar artery. Atherosclerosis. 1980;35:77–86.[Medline] [Order article via Infotrieve]
3. Glagov S, Zarins C, Giddens DP, Ku DN. Hemodynamics and atherosclerosis: insights and perspectives gained from studies of human arteries. Arch Pathol Lab Med. 1988;112:1018–1031.[Medline] [Order article via Infotrieve]
4. Frangos JA, Huang TY, Clark CB. Steady shear and step changes in shear stimulate endothelium via independent mechanisms: superposition of transient and sustained nitric oxide production. Biochem Biophys Res Commun. 1996;224:660–665.[Medline] [Order article via Infotrieve]
5.
Ojha M. Wall shear stress temporal gradient and
anastomotic intimal hyperplasia. Circ Res. 1994;74:1227–1231.
6. Kleinstreuer C, Lei M, Archie JP Jr. Flow input waveform effects on the temporal and spatial wall shear stress gradients in a femoral graft-artery connector. ASME J Biomech Eng. 1996;118:506–510.[Medline] [Order article via Infotrieve]
7. Resnick N, Gimbrone MA. Hemodynamic forces are complex regulators of endothelial gene expression. FASEB J. 1995;9:874–882.[Abstract]
8.
Shyy YJ, Hsieh HJ, Usami S, Chien S. Fluid shear
stress induces a biphasic response of human monocyte chemotactic
protein 1 gene expression in vascular endothelium.
Proc Natl Acad Sci U S A. 1994;91:4678–4682.
9.
Hsieh HJ, Li NQ, Frangos JA. Shear stress increases
endothelial platelet derived growth factor mRNA
levels. Am J Physiol. 1991;260:H642–H646.
10. Cushing SD, Berliner JA, Valente AJ, Territo MC, Navab M, Parhami F, Gerrity R, Schwartz CJ, Fogelman AM. Minimally modified low density lipoprotein induces monocyte chemotactic protein 1 in human endothelial cells and smooth muscle cells. Proc Natl Acad Sci U S A. 1990;87:134–138.
11. Wilcox JN, Smith KM, Williams LT, Schwartz SM, Gordon D. Platelet-derived growth factor mRNA detection in human atherosclerotic plaques by in situ hybridization. J Clin Invest. 1988;82:1134–1143.
12.
Rekhter MD, Gordon D. Does platelet-derived growth
factor-A chain stimulate proliferation of arterial
mesenchymal cells in human atherosclerotic plaques? Circ
Res. 1994;75:410–417.
13.
Topper JN, Cai J, Falb D, Gimbrone MA. 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 U S A. 1996;93:10417–10422.
14.
Kuchan MJ, Frangos JA. Role of calcium and
calmodulin in flow-induced nitric oxide production
in endothelial cells. Am J Physiol. 1994;266:C628–C636.
15.
Rubanyi GM, Romero JC, Vanhoutte PM. Flow-induced
release of endothelium derived relaxing factor.
Am J Physiol. 1986;250:H1145–H1149.
16.
Cohen RA, Zitnay KM, Haudenschild CC, Cunningham LD.
Loss of selective endothelial cell vasoactive functions
caused by hypercholesterolemia in pig
coronary arteries. Circ Res. 1988;63:903–910.
17. Moncada S, Higgs EA. Endogenous nitric oxide: physiology, pathology and clinical relevance. Eur J Clin Invest. 1991;21:361–374.[Medline] [Order article via Infotrieve]
18.
Khachigian LM, Anderson KR, Halnon NJ, Gimbrone MAJ,
Resnick N, Collins T. Egr-1 is activated in
endothelial cells exposed to fluid shear stress and
interacts with a novel shear-stress response element in the PDGF
A-chain promoter. Arterioscler Thromb Vasc Biol. 1997;17:2280–2286.
19.
Lan Q, Mercurius KO, Davies PF. Stimulation of
transcription factors NFB and AP-1 in
endothelial cells subjected to shear stress.
Biochem Biophys Res Commun. 1994;201:950–956.[Medline]
[Order article via Infotrieve]
20.
Mohan S, Mohan N, Sprague EA. Differential activation
of NFB in human aortic endothelial cells
conditioned to specific flow environments. Am J
Physiol. 1997;273:C572–C578.
21.
Shyy JY, Lin MC, Han J, Lu Y, Petrime M, Chien S. The
cis-acting phorbol ester
"12-O-tetradecanoylphorbol
13-acetate"-responsive element is involved in shear stress-induced
monocyte chemotactic protein-1 gene expression. Proc Natl Acad
Sci U S A. 1995;92:8069–8073.
22.
Khachigian LM, Resnick N, Gimbrone MAJ, Collins T.
Nuclear factor-B interacts functionally with the
platelet-derived growth factor B-chain shear stress response
element in vascular endothelial cells exposed to fluid
shear stress. J Clin Invest. 1995;96:1169–1175.
23.
Frangos JA, Eskin SG, McIntire LV, Ives CL. Flow
effects on prostacyclin production by cultured human
endothelial cells. Science. 1985;227:1477–1479.
24. Frangos JA. Physical Forces and the Mammalian Cell. San Diego, CA: Academic Press; 1993.
25. Betsholtz C, Johnsson A, Heldin C-H, Westermark B, Lind P, Urdea MS, Eddy R, Shows TB, Philpott K, Mellor AL, Knott TJ, Scott J. cDNA sequence and chromosomal localization of human platelet-derived growth factor A-chain and its expression in tumor cell lines. Nature. 1986;320:695–699.[Medline] [Order article via Infotrieve]
26. Sukhatme VP, Cao XM, Chang LC, Tsai-Morris CH, Stamenkovich D, Ferreira PC, Cohen DR, Edwards SA, Shows TB, Curran T, Le Beau MM, Adamson ED. A zinc finger-encoding gene coregulated with c-fos during growth and differentiation, and after cellular depolarization. Cell. 1988;53:37–43.[Medline] [Order article via Infotrieve]
27.
Uead A, Okuda K, Ohno S, Shirai A, Igareshi T,
Matsunaga K, Fukushina J, Kawamoto S, Ishigatsubo J, Okubo T.
NF-B, and Sp-1 regulate transcription of the human monocyte
chemoattractant protein-1 gene. J Immunol. 1994;153:2052–2063.[Abstract]
28.
Spiecker M, Peng HB, Liao JK. Inhibition of
endothelial vascular cell adhesion molecule-1
expression by nitric oxide involves the induction and nuclear
translocation of IB. J Biol Chem. 1997;272:30969–30974.
29. Nerem RM. Hemodynamics and the vascular endothelium. J Biomech Eng. 1993;115:510–514.[Medline] [Order article via Infotrieve]
30.
Langille BL, O'Donnell F. Reductions in
arterial diameter produced by chronic decreases in blood
flow are endothelium-dependent. Science. 1986;231:405–407.
31.
Davies PF. Flow-mediated endothelial
mechanotransduction. Physiol Rev. 1995;75:519–560.
32. Davies PF, Dewey CFJ, Bussolari SR, Gordon EJ, Gimbrone MA Jr. Influence of hemodynamic forces on vascular endothelial function in vitro studies of shear stress and pinocytosis in bovine aortic cells. J Clin Invest. 1984;73:1121–1129.
33.
Ando J, Tsuboi H, Korenaga R, Takada Y,
Toyama-Sorimachi N, Miyasaka M, Kamiya A. Shear stress inhibits
adhesion of cultured mouse endothelial cells to
lymphocytes by downregulating VCAM-1 expression. Am J
Physiol. 1994;267:C679–C687.
34.
Kuchan MJ, Frangos JA. Shear stress regulates
endothelin-1 release via protein kinase C and cGMP in cultured
endothelial cells. Am J Physiol. 1993;264:H150–H156.
35. Sprague EA, Luo J. Prolonged low shear stress flow conditioning of cultured human aortic endothelial cell enhances monocyte adhesion, MCP-1 and VCAM-1 expression. Circulation. 1994;90:I-84. Abstract.
36. Biffl WL, Moore EE, Moore FA, Barnett CC. Nitric oxide reduces endothelial expression of intercellular adhesion molecule (ICAM-1). J Surg Res. 1996;63:328–332.[Medline] [Order article via Infotrieve]
37.
Khan BV, Harrison DG, Olbrych MT, Alexander RW, Medford
RM. Nitric oxide regulates vascular cell adhesion molecule 1 gene
expression and redox-sensitive transcriptional events in human vascular
endothelial cells. Proc Natl Acad Sci
U S A. 1996;93:9114–9119.
38. Caterina RD, Libby P, Peng HB, Thannickal VJ, Rajavashisth TB, Gimbrone MA Jr, Shin WS, Liao JK. Nitric oxide decreases cytokine-induced endothelial activation. J Clin Invest. 1995;96:60–68.
39.
Zeiher AM, Fisslthaler B, Schray-Utz B, Busse R. Nitric
oxide modulates the expression of monocyte chemoattractant protein-1 in
cultured human endothelial cells. Circ Res. 1995;76:980–986.
40. Haby C, Lisovoski F, Aunis D, Zwiller J. Stimulation of the cyclic GMP pathway by NO induces expression of the immediate early genes c-fos and junB in PC12 cells. J Neurochem. 1991;62:496–501.[Medline] [Order article via Infotrieve]
41. Peunova N, Enikolopov G. Amplification of calcium-induced gene transcription by nitric oxide in neuronal cells. Nature. 1993;364:450–453.[Medline] [Order article via Infotrieve]
42. Pilz RB, Suhasini M, Idriss S, Meinkoth JL, Boss GR. Nitric oxide and cGMP analogs activate transcription from AP-1-responsive promoters in mammalian cells. FASEB J. 1995;9:552–558.[Abstract]
43.
Gudi T, Huvar I, Meinecke M, Lohmann SM, Boss GR, Pilz
RB. Regulation of gene expression by cGMP-dependent protein kinase.
J Biol Chem. 1996;271:4597–4600.
44.
Laurindo FRM, Pedro MA, Barbeiro HV, Pileggi F,
Carvalho MHC, Augusto O, Luz PL. Vascular free radical release: ex vivo
and in vivo evidence for a flow-dependent endothelial
mechanism. Circ Res. 1994;74:700–709.
45.
Chiu JJ, Wung BS, Shyy JYJ, Hsieh HJ, Wang DL. Reactive
oxygen species are involved in shear stress-induced intercellular
adhesion molecule-1 expression in endothelial cells.
Arterioscler Thromb Vasc Biol. 1997;17:3570–3577.
46. Hsieh HJ, Cheng CC, Wu ST, Chiu JJ, Wung BS, Wang DL. 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. 1998;175:156–162.[Medline] [Order article via Infotrieve]
47.
Beckman JS, Koppenol WH. Nitric oxide, superoxide
and peroxynitrite: the good, the bad and the ugly. Am J
Physiol. 1996;271:C1424–C1437.
48. Lefer DJ, Scalia R, Campbell B, Nossuli T, Hayward R, Salamon M, Grayson J, Lefer AM. Peroxynitrite inhibits leukocyte-endothelial cell interactions and protects against ischemia-reperfusion injury in rats. J Clin Invest. 1997;99:684–691.[Medline] [Order article via Infotrieve]
49. List BM, Klosch B, Volker C, Gorren ACF, Sessa WC, Werner ER, Kukovetz WR, Schmidt K, Mayer B. Characterization of bovine endothelial nitric oxide synthase as a homodimer with down-regulated uncoupled NADPH oxidase activity: tetrahydrobiopterin binding kinetics and role of haem in dimerization. Biochem J. 1997;323:159–165.
50.
Chen PF, Tsai AL, Berka V, Wu KK. Mutation of Glu-361
in human endothelial nitric-oxide synthase selectively
abolishes L-arginine binding without perturbing the
behavior of heme and other redox centers. J Biol Chem. 1997;272:6114–6118.
51.
Wung BS, Cheng JJ, Hsieh HJ, Shyy YJ, Wang DL. Cyclic
strain-induced monocyte chemotactic protein-1 gene expression in
endothelial cells involves reactive oxygen species
activation of activator protein-1. Circ Res. 1997;81:1–7.
52.
Marumo T, Schini-Kerth VB, Fisslthaler B, Busse R.
Platelet-derived growth factor-stimulated superoxide anion
production modulates activation of transcription factor
NF-kappaB and expression of monocyte chemoattractant protein-1 in human
aortic smooth muscle cells. Circulation. 1997;96:2361–2367.
53. Puri PL, Avantaggiati ML, Burgio VL, Chirillo P, Collepardo D, Natoli G, Balsano C, Levrero M. Reactive oxygen intermediates (ROIs) are involved in the intracellular transduction of angiotensin II signal in C2C12 cells. Ann N Y Acad Sci. 1995;752:394–405.[Medline] [Order article via Infotrieve]
54.
Irani K, Xia Y, Zweier JL, Sollott SJ, Der CJ, Fearon
ER, Sundaresan M, Finkel T, Goldschmidt-Clermont PJ.
Mitogenic signaling mediated by oxidants in Ras-transformed
fibroblasts [see comments]. Science. 1997;275:1649–1652.
55. Li YS, Shyy JY, Li S, Lee J, Su B, Karin M, Chien S. The Ras-JNK pathway is involved in shear-induced gene expression. Mol Cell Biol. 1996;16:5947–5954.[Abstract]
56. Clancy RM, Rediske J, Tang X, Nijher N, Frenkel S, Philips M, Abramson SB. Outside-in signaling in the chondrocyte: nitric oxide disrupts fibronectin-induced assembly of a subplasmalemmal actin/rho A/focal adhesion kinase signaling complex. J Clin Invest. 1997;100:1789–1796.[Medline] [Order article via Infotrieve]
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