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Second Department of Internal Medicine, Hirosaki University School of Medicine, Hirosaki, 036-8562 Japan
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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We tested the hypothesis
that at sites of vascular damage, vessel homeostasis is maintained
through the cross talk of shear-induced production of prostacyclin and
nitric oxide (NO) in vascular smooth muscle cells (VSMC). Confluent
A7r5 cells derived from rat aortic VSMC and mesenteric VSMC were
exposed to shear stress at 15 dyn/cm2 for 90 min with the
use of a cone-plate device, and productions of prostacyclin and NO were
examined. Shear stress increased cumulative production of prostacyclin
by 3- to 3.5-fold and that of NO by 6- to 7.5-fold. Western blot
analysis showed that inducible NO synthase protein was expressed after
shear stress in both types of VSMC. Inhibition of NO synthase enhanced
the shear-induced production of prostacyclin from 40 to 60%.
Shear-induced production of NO was suppressed by 70% after treatment
with 10
4 M of indomethacin. A7r5 cells adhesiveness for
monocytes was suppressed by 50% after shear stress. This suppression
was abolished by pretreatment with 104 M of indomethacin,
whereas inhibition of NO synthase only minimally inhibited it. We
conclude that there is a cross talk of shear-induced production of
prostacyclin and NO in VSMC. At sites of vascular damage, prostacyclin
synthesis may prevent monocyte adhesiveness for VSMC through the
concomitant enhancement of NO production.
shear stress; vascular smooth muscle cells
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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THE PASSAGE OF BLOOD THROUGH the cardiovascular system generates hemodynamic forces. Shear stress, a type of flow force across the cell surface (32), influences the function of endothelial cells and induces initiation of intracellular signaling (23, 25, 26, 33), stimulation of the synthesis and secretion of various bioactive molecules (9, 10, 22), inhibition of cell proliferation (2), and elongation of the cells in the direction of flow through cytoskeletal rearrangement (8, 43). Although vascular smooth muscle cells (VSMC), another type of vessel-composed cells, are separated from the laminal blood flow by endothelial cells that line the intimal surface of the vasculature, they could be directly exposed to shear stress at sites of vascular damage caused by the rupture of atherosclerotic lesions or invasive techniques, such as angioplasty.
Vessels are composed of cells that secrete prostacyclin and nitric oxide (NO), and these compounds synergistically contribute to vessel homeostasis by reducing VSMC tone (30, 36) and growth (11, 18), platelet aggregation (14), leukocyte adhesion to endothelium (35, 36, 40), and susceptibility to thrombosis (24). We (28) recently demonstrated that vessel homeostasis might be maintained through an increase in prostacyclin production in vascular endothelial cells when NO synthesis is impaired. However, the cross talk of prostacyclin and NO at sites of severe vascular damage, where VSMC can be directly exposed to blood flow, still remains to be elucidated. Furthermore, in these regions, the adhesive interaction of monocytes and VSMC is an initial process of the cellular activation, which is closely related to the pathogenesis of atherosclerosis (15). Accordingly, this study was designed to investigate the interaction between prostacyclin and NO production in VSMC under shear stress and to examine whether shear stress inhibits monocyte adhesiveness by triggering the release of prostacyclin and NO.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Materials.
The A7r5 cells, derived from rat aortic VSMC, and THP-1 monocytes were
purchased from American Type Culture Collection. Wistar-Kyoto rats were
obtained from Charles River. Dulbecco's modified Eagle's medium
(DMEM), phenol red-free MEM (nitrate free), Hanks' balanced salt
solution (HBSS), fetal bovine serum (FBS), phosphate-buffered saline,
trypsin (0.05%)-EDTA (0.02%), and petri dishes were from GIBCO-BRL
(Grand Island, NY). A tritiated 6- keto-prostaglandin (PG)
F1
tracer and L-arginine were purchased from
DuPont-New England Nuclear (Boston, MA). Ono Pharmaceutical provided
the antibody 6-keto-PGF1 and standard. Inducible NO
synthase (iNOS) antibody was purchased from Santa Cruz Biotechnology
(Santa Cruz, CA). The nitrate and nitrite assay kit and
PGH2 were purchased from Cayman Chemical (Ann Arbor, MI).
The toxicolor test was purchased from Seikagaku Kogyo (Tokyo, Japan).
The amplified alkaline phosphatase immunoblot kit and other reagents
for Western blot analysis were purchased from Bio-Rad (Hercules, CA).
Indomethacin, NG-nitro-L-arginine
methyl ester (L-NAME), HEPES, and all of the other reagents
were purchased from Sigma (St. Louis, MO).
Cell culture. Mesenteric VSMC were isolated from ether-anesthetized Wistar-Kyoto rats, as described previously (27). Mesenteric VSMC and A7r5 cells were maintained in DMEM supplemented with 10% FBS, 100 µg/ml streptomycin, and 100 U/ml penicillin (complete DMEM) at 37°C under 5% CO2 and 95% air. The VSMC were subcultured every 5-7 days with trypsin-EDTA, and media in all cultures were renewed every 2-3 days. The culture supplemented with various chemicals was incubated at 37°C for 30 min before exposure to shear stress. THP-1 monocytes were maintained in RPMI 1640 supplemented with 10% FBS, 100 µg/ml of streptomycin, and 100 U/ml of penicillin. The viability of the cells, determined by trypan blue excretion, was generally >95%.
Flow method.
Confluent VSMC, last fed with the complete growth media, were exposed
to a fluid shear stress with the use of a cone-plate viscometer
specifically designed to accept standard tissue culture plates
(21). Shear stress magnitude (Tss = µw/
) was computed by using relations derived by Sdougos
et al. (37), where µ is fluid viscosity, w is
rotational velocity, and is the cone angle. In the steady laminar
mode, a cone of 1° angle spinning at 4 and 6.7 rps was used to
achieve an average shear stress magnitude of 15 and 25 dyn/cm2. The cells inside the viscometer were kept at a
temperature of 37°C and a humidified atmosphere with 5%
CO2. The viability of the cells exposed to shear stress was
assessed by the measurement of protein content of the dishes and the
staining with trypan blue.
Protocol. The study was performed primarily on A7r5 cells. To confirm that the findings obtained from A7r5 cells can be observed in another type of VSMC, the experiments on the productions of NO and prostacyclin and the protein of iNOS in response to shear stress were performed in mesenteric VSMC.
VSMC in 75-cm2 flasks were subcultured with trypsin-EDTA into four 100-mm petri dishes and were maintained in the complete media. Confluent monolayers in petri dishes were gently washed three times with phosphate-buffered saline and replaced with either 5 ml of serum-free DMEM for the assessment of 6-keto-PGF1 or 5 ml of serum-free and phenol red-free MEM for that of nitrite. One
monolayer of the petri dish was kept under a static condition (control), and the others were exposed to shear stress (15 and 25 dyn/cm2) for 90 min at 37°C under 5% CO2. To
examine the reproducibility of shear-induced production of prostacyclin
and NO, A7r5 cells already exposed to shear stress at 15 dyn/cm2 were further subjected to the same shear stress for
another 90-min period after the 30-min static interval. The medium (100 µl) was taken from the cultures with and without shear stress
conditions for the measurements of 6-keto-PGF1 and
nitrite at 20, 40, 60, and 90 min after the initiation of the study. To
determine the enzymatic source of NO, Western blot analysis for iNOS
protein was performed with or without shear stress conditions in both types of VSMC. In another set of experiments, the confluent monolayer was preincubated with 104 M of
L-NAME or 105 M of indomethacin for 30 min,
and replaced with 5 ml of fresh medium in both types of VSMC. These
cells were subjected to shear stress at 15 dyn/cm2 for 90 min, and the medium (100 µl) was taken for the measurements of
6-keto-PGF1 and nitrite.
The direct effect of 104 M of L-NAME on
cyclooxygenase activity was assessed by the conversion of exogenous
arachidonic acid to prostacyclin and exogenous PGH2 to
prostacyclin in A7r5 cells. The cells on 24-well plates were incubated
in serum-free DMEM containing 10 ng/ml of arachidonic acid or 10 ng/ml
of PGH2 in both the presence and absence of
L-NAME for 30 min at 37°C, and the medium was taken for
the measurement of 6-keto-PGF1. The direct effect of
105 M of indomethacin on NOS activity was
assessed by the conversion of L-arginine to
L-citrulline in the A7r5 cell lysate after shear stress
according to the method of Bredt and Snyder (7).
Assay for PG.
The concentration of 6-keto-PGF1 was measured by
radioimmunoassay according to the method of Inagawa et al.
(17), with the use of a specific antibody to
6-keto-PGF1. The cross-reaction rate of the
6-keto-PGF1 antiserum was 9.1% for PGE2,
5.0% for PGF2, and undetectable for thromboxane
B2. Cellular protein was determined by Bradford's method
(6). The amount of 6-keto-PGF1 secreted
from the cells was expressed as picograms per 1 µg protein.
Assay for NO metabolites. The nitrate and nitrite anion content in the conditioned media was measured to determine the enzymatic production of NO from L-arginine by cultured cells (12). After conversion of nitrate to nitrite in the presence of nitrate reductase and cofactor, aliquots of conditioned media were mixed with an equal volume of Greiss reagent (a 1:1 mixture of 1% sulfanilamide and 0.1% naphthylethylenediamine in 2% phosphoric acid). After the color was developed, the optical density was determined at 540 nm. The concentration of unknowns was determined by comparison with values from a standard curve obtained with sodium nitrite.
Western blot analysis. After cells were exposed to shear stress at 15 dyn/cm2 or kept under a static condition for 90 min, they were solubilized in sodium dodecyl sulfate (SDS) sample buffer (125 mM of Tris buffer, pH 6.8, 2% SDS, 5% glycerol, and 1% mercaptoethanol), heated at 95°C for 1 min, and subjected to SDS-polyacrylamide gel electrophoresis using a 4-20% separating gel. The proteins were transferred electrophoretically to a nitrocellulose membrane for 1 h at 100 V. The membranes were soaked overnight in 5% skim milk and then treated with anti-iNOS antibody (1:300 dilution) for 2 h at room temperature. After the membranes were washed, they were treated with alkaline phosphatase-conjugated anti-rabbit antibody and stained by amplified alkaline phosphatase immunoblot kits.
Monocyte adhesion assay.
After A7r5 cells on culture dishes were exposed to shear or static
condition for 90 min, they were washed with HBSS containing (in mmol/l)
2 Ca2+, 2 Mg2+, and 20 HEPES, and then
incubated with THP-1 cells in HBSS at a concentration of 1.5 × 106 cells/ml on a rocking platform for 30 min. The medium
was aspirated and replaced with fresh binding medium twice to remove
nonadherent cells, and the dishes were returned to the rocker platform
for an additional 5-min incubation. Medium was removed and replaced with binding medium containing 2% gluteraldehyde. After overnight fixing, adherent cells were quantified by microscopy. To determine whether shear-induced alteration in adhesiveness was due to either NO
or prostacyclin, in some experiments, cells were incubated with
L-NAME (104 M) or indomethacin
(105 M) for 30 min before the application of shear or
static condition and throughout it.
Measurement of endotoxin. The conditioned media before and after exposure of the cells to 90-min shear stress were added to an aliquot of toxicolor test (a lyophilized mixture of amebocyte lysate from Trachypleus tridentatus and a chromogenic substrate, Boc-Leu-Gly-Arg-pNA) dissolved in Tris · HCl buffer, pH 8.0, and the mixture was incubated at 37°C for 30 min. Absorbance was measured at 545 nm.
Statistics. Results are presented as means ± SE. Differences in the time-dependent production of prostacyclin and NO metabolites between conditions with and without shear and with and without L-NAME and indomethacin were analyzed by two-way analysis of variance (ANOVA). Differences in the monocyte adhesiveness among conditions with and without shear and with and without L-NAME and indomethacin were analyzed by one-way ANOVA. Comparison of two variables was performed by paired or unpaired Student's t-test. Difference was considered significant at an error probability of P < 0.05.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Viability of the cells. In A7r5 cells, the protein contents were 236 ± 21 µg/dish in a static condition and 222 ± 15 and 204 ± 43 µg/dish in a shear stress condition at 15 and 25 dyn/cm2, respectively, after the period of 90 min (P = ns). The successive exposures to shear stress at 15 dyn/cm2 also did not affect the protein contents. Treatment with indomethacin or L-NAME had no influence to the protein content after 90-min exposure to shear stress. The number of trypan blue-positive cells was undetectable in both static control and shear conditions.
Prostacyclin production in A7r5 cells and mesenteric VSMC.
In A7r5 cells, 6-keto-PGF1 in the conditioned medium
accumulated in a time-dependent manner in a static condition (Fig. 1, open circles), and the level at 90 min
was 4.4 ± 2.3 pg/µg of protein (see Fig. 1). Sudden onset of
laminar shear stress at 15 dyn/cm2 after culture in a
static condition caused a rapid and marked increase in prostacyclin
production (Fig. 1, closed circles). Shear-induced increase in
prostacyclin production was time dependent, and the level after 90 min
of exposure was threefold greater than that in a static control
condition. Further exposure of the cells for another 90-min period to
the same shear at 15 dyn/cm2 after the 30-min static
interval caused the similar increase in prostacyclin production
(12.4 ± 2.9 pg/µg of protein after the initial shear and
11.9 ± 2.4 pg/µg of protein after the second shear,
n = 3, P = not significant, ns). When
prostacyclin production under two levels of shear stress was compared,
it was 12.0 ± 3.5 pg/µg of protein under shear at 15 dyn/cm2 and 18.9 ± 4.5 pg/µg of protein under shear
at 25 dyn/cm2 (P < 0.05 by paired
t-test).
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4 M of
L-NAME, which was confirmed by the undetectable
concentration of NO metabolites in the media, enhanced the
shear-induced production of prostacyclin (P < 0.05 by
two-way ANOVA) by 40%. The levels after 90 min in cultures exposed to
shear in the absence (closed circles in Fig. 1) and presence of
L-NAME (closed squares in Fig. 1) were 12.4 ± 2.9 and
17.1 ± 2.4 pg/µg of protein, respectively. Prostacyclin production in a static condition was unaffected by the treatment of
L-NAME. Neither the conversion of arachidonic acid to
prostacyclin (789 ± 54 in the absence and 810 ± 48 pg/well
in the presence of L-NAME, n = 4, P = ns) nor that of PGH2 to prostacyclin
(570 ± 56 in the absence and 584 ± 65 pg/well in the
presence of L-NAME, n = 4, P = ns) was affected by 104M
L-NAME.
In mesenteric VSMC, the level of 6-keto-PGF1 was
2.7 ± 0.4 pg/µg of protein at 90 min in a static condition
(n = 4), and was significantly increased to 9.4 ± 1.1 pg/µg of protein after 90-min shear stress at 15 dyn/cm2 (n = 4, P 
0.05). Treatment with 104 M of L-NAME
significantly enhanced this shear-induced production of
6-keto-PGF1 to 15.1 ± 0.3 pg/µg of protein
(n = 4, P 0.05).
NO production in A7r5 cells and mesenteric VSMC.
In A7r5 cells, NO metabolites (nitrate and nitrite) in the conditioned
media accumulated in a static condition (Fig. 2, open circles), and the level at 90 min was 23 ± 6 pmol/µg of
protein. Exposure of the cells to 15 dyn/cm2 of shear stress significantly enhanced the
time-dependent increase in NO metabolites concentration in the media
(Fig. 2, closed circles), and the level after 90 min (174 ± 33 pmol/µg of protein) was 7.5-fold greater than that in a static
baseline production (P < 0.01 by two-way ANOVA).
Further exposure of the cells for another 90-min period to the same
shear at 15 dyn/cm2 induced the similar production of NO
metabolites (158 ± 19 pmol/µg of protein, n = 3). Shear stress at 25 dyn/cm2 enhanced NO metabolites
production (301 ± 54 pmol/µg of protein) by twofold compared
with that at 15 dyn/cm2. NO metabolites concentration in
the media before shearing the cells was undetectable. The concentration
of endotoxin in the conditioned media before and after exposure of the
cells to 90-min shear stress was 1 pg/ml.
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5 M totally inhibited prostacyclin
production in a shear condition (0.1 ± 0.1 pg/µg of protein at
90 min). The production of NO metabolites under shear stress was
significantly suppressed (P < 0.05 by two-way ANOVA)
by 70% after indomethacin in A7r5 cells (Fig. 2). The levels after 90 min were 52 ± 11 in the presence and 174 ± 33 pmol/µg of
protein in the absence of indomethacin. NO metabolites production in a
static condition was unaffected even after treatment with indomethacin
(17 ± 9 pmol/µg of protein at 90 min). NOS activity was
13.5 ± 2.1 in the absence and 13.9 ± 1.5 pmol/mg of protein
per 30 min in the presence of indomethacin (n = 4, P = ns).
In mesenteric VSMC, the level of NO metabolites was 6.0 ± 3.5 pmol/µg of protein at 90 min in a static condition (n = 4), and was significantly increased to 37.2 ± 8.6 pmol/µg of
protein after 90-min shear stress at 15 dyn/cm2
(n = 4, P < 0.05). Treatment with
105 M indomethacin significantly suppressed this
shear-induced production of NO metabolites to 15.1 ± 8.7 pmol/µg of protein (n = 4, P < 0.05).
Western blot analysis in A7r5 cells and mesenteric VSMC.
Representative blots of A7r5 cells and mesenteric VSMC are shown in
Fig. 3. In A7r5 cells, the protein of
iNOS was undetectable under a static condition, whereas it was clearly
detectable after 90-min shear stress at 15 dyn/cm2. In
mesenteric VSMC, the protein of iNOS was slightly detectable in a
static condition, and it was significantly enhanced after 90-min shear
stress at 15 dyn/cm2. The same results were obtained from
the other three separated experiments, done in both types of VSMC.
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Effect of shear stress on A7r5 cells adhesiveness.
THP-1 monocytes adhered to A7r5 cells in a static condition. Exposure
of the cells to shear stress induced a significant reduction (53 ± 5% of the static condition) in the binding of THP-1 cells (Fig.
4). Treatment with L-NAME at
104 M unaffected the binding of THP-1 cells in a
static condition, whereas it significantly attenuated shear-induced
suppression of THP-1 adhesion (72 ± 2% of the static condition)
(P < 0.05 vs. shear-induced suppression without
L-NAME). Incubation of A7r5 cells with indomethacin at
105 M slightly increased the THP-binding in a static
condition (114 ± 6% of the static condition) (P = ns vs. the static control without the inhibitors), and completely
reversed the suppressant effect of shear stress on A7r5 cells
adhesiveness (104 ± 19% of the static condition).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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With the use of cultured VSMC (A7r5 cells and mesenteric VSMC), the present study showed that shear stress at 15 dyn/cm2 accomplished the following: 1) it increased the cumulative production of prostacyclin and NO in VSMC; 2) shear stress potentiated the expression of iNOS protein; 3) shear-induced production of prostacyclin was significantly enhanced by L-NAME, whereas shear-induced production of NO was remarkably attenuated by indomethacin; and 4) shear stress suppresses VSMC adhesiveness for monocytes and this effect was abolished by pretreatment with indomethacin.
Shear-induced production of prostacyclin and NO from VSMC. The VSMC can be exposed to shear stress levels of at least 15 dyn/cm2 when directly exposed to blood flow after deep vessel-wall injury. Even VSMC within the intact vessel wall are exposed directly to flow driven by the transmural pressure gradient (42). Fluid movement through the interstitial spaces of the intact vessel wall, which continually provides nutrients to the surrounding tissue (19), generates shear stress in a manner similar to blood flow through vessels. Calculations indicate that VSMC are likely to be exposed to shear stress levels as high as 2-3 dyn/cm2 in intact blood vessels (19). However, changes in hydraulic conductivity across the endothelial cell lining and pathological conditions due to elevations of blood pressure would dramatically increase the shear stress on the VSMC. We demonstrated that shear stress levels approaching these magnitudes are capable of a rapid increase in prostacyclin production and a slower but marked increase in NO. Because the NO metabolite concentration in the media before the cells were sheared was undetectable, the increase in NO metabolites in the media of sheared cells reflects de novo NO synthesis. The present shear-induced responses are consistent with those of the previous reports on VSMC (29, 38). We demonstrated in this study that shear-induced production of prostacyclin in VSMC is magnitude dependent, whereas Alshihabi et al. (1) showed that the peak rate of prostacyclin production is independent of the magnitude of shear stress. This discrepancy may be due to the difference of maximal magnitude of shear stress used.
The mechanism by which shear stress augments the production of these vasodilator compounds has been well known in vascular endothelial cells: shear-induced prostacyclin production is associated with the activation of phospholipase A2, which is mediated by pertussis toxin-sensitive GTP-binding protein Gi3, and the subsequent release of arachidonic acid from membrane phospholipids (3, 5, 10, 13). In contrast, NO production is mediated by the activation of phospholipase C linked to pertussis toxin-insensitive GTP-binding proteins, which rapidly stimulates the hydrolysis of phosphatidylinositol 4,5-bisphosphate into the second messengers inositol 1,4,5-trisphosphate and diacylglycerol (4, 24, 34). It seems reasonable to presume that the enzymatic source of NO by shear stress is iNOS, because the expression of its enzyme protein was significantly enhanced after shear stress in both types of VSMC. Papadaki et al. (31) showed that fluid flow rapidly stimulates NO production in cultured human aortic VSMC in a Ca2+ calmodulin-dependent manner. They also demonstrated that a calmodulin antagonist had little effect at later time and fluid flow elicited no overall cytosolic Ca2+ changes in human aortic VSMC. Thus the burstlike flow-induced NO production in human cells may depend on Ca2+ calmodulin signaling pathways, whereas the slower but marked increase in NO production observed in the present rat-derived VSMC appears to depend on the mechanism via iNOS. Endotoxin is a representative agent for iNOS induction. However, its induction by endotoxin seems negligible, because the concentration of endotoxin in the media was1 pg/ml. Blockade of prostacyclin
production partially suppressed shear-induced increase in NO
metabolites, indicating that at least the prostacyclin-dependent
mechanism contributes to the shear-induced iNOS induction.
Cross talk of prostacyclin and NO production. Inhibition of NO synthase with L-NAME enhanced the shear-induced production of prostacyclin, indicating that endogenous NO functions as an inhibitor of prostacyclin production. This effect of NO may be associated with cell death due to its cytotoxic action. However, the result showed no significant reduction in protein content of the dishes. In addition, trypan blue studies demonstrated no loss of viability in VSMC, suggesting that NO shows its effects by mechanisms other than cytotoxicity. In contrast, the cumulative production of NO under shear stress was remarkably attenuated after the complete inhibition of cyclooxygenase, indicating that endogenous prostacyclin functions as a powerful stimulator of NO production, presumably via the expression of iNOS gene, under shear in an autocrine or paracrine fashion. Indeed, it was reported that cAMP as the second messenger of prostacyclin stimulates the expression of iNOS mRNA (16). We previously demonstrated that this positive effect of prostacyclin was not observed in vascular endothelial cells (28). Thus, in sheared VSMC, the impaired production of prostacyclin would attenuate NO production.
In vivo role of the cross talk between prostacyclin and NO.
In vascular endothelial cells, adhesive interaction with leukocytes was
demonstrated to induce the expression of adhesion molecules
(38), which was attenuated under shear stress largely due
to flow-stimulated release of prostacyclin and NO (39,
40). In contrast, the interaction between VSMC and leukocytes
was investigated only in a static condition, and the production of
tumor necrosis factor- (15) and the secretion of matrix
metalloproteinase through an interleukin-1-dependent mechanism
(20) were demonstrated. In the present study, we clearly
showed that shear stress suppressed monocyte adhesiveness to A7r5
cells, and it was largely attenuated after treatment with indomethacin
and, to a lesser extent, with L-NAME. One interpretation of
the data is that prostacyclin synthesis may play a crucial role in the
prevention of monocyte adhesiveness for VSMC through the synergistic
effect of NO production under moderate shear stress. Another
interpretation suggests that the binding of THP-1 may be inhibited
primarily by prostacyclin, rather than by NO, and that the role of NO
may be to stabilize prostacyclin. It is noted that basal release of NO
and prostacyclin was different between types of VSMC. Thus the in vivo
role of these vasodilators under shear stress would be different at the
site of vessels.
Limitations. A mechanism for the cross talk between NO and prostacyclin production under shear stress was investigated with the use of their inhibitors. However, because the shear-induced intracellular signaling pathway is unknown in VSMC, we could not conclude the molecular mechanism for the cross talk of prostacyclin and NO. Further investigation will be required to elucidate its mechanism in a shear condition and the significance of prostacyclin at sites of vascular damage in vivo.
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FOOTNOTES |
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Address for reprint requests and other correspondence: T. Osanai, Second Dept. of Internal Medicine, Hirosaki Univ. School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan (E-mail: osanait{at}cc.hirosaki-u.ac.jp).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 7 January 2000; accepted in final form 23 February 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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|---|
1.
Alshihabi, SN,
Chang YS,
Frangos JA,
and
Tarbell JM.
Shear stress-induced release of PGE2 and PGI2 by vascular smooth muscle cells.
Biochem Biophys Res Commun
224:
808-814,
1996[ISI][Medline].
2.
Ando, J,
Nomura H,
and
Kamiya A.
The effects of fluid shear stress on the migration and proliferation of cultured endothelial cells.
Microvasc Res
33:
62-70,
1987[ISI][Medline].
3.
Berthiaume, F,
and
Frangos JA.
Flow-induced prostacyclin production is mediated by a pertussis toxin-sensitive G protein.
FEBS Lett
308:
277-279,
1992[ISI][Medline].
4.
Bhagyalakshmi, A,
Berthiaume F,
Reich KM,
and
Frangos JA.
Fluid shear stress stimulates membrane phospholipid metabolism in cultured human endothelial cells.
J Vasc Res
29:
443-449,
1992[ISI][Medline].
5.
Bhagyalakshmi, A,
and
Frangos JA.
Mechanism of shear-induced prostacyclin production in endothelial cells.
Biochem Biophys Res Commun
158:
31-37,
1989[ISI][Medline].
6.
Bradford, MM.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal Biochem
284:
248-254,
1976.
7.
Bredt, DS,
and
Snyder SH.
Isolation of nitric oxide synthase, a calmodulin-requiring enzyme.
Proc Natl Acad Sci USA
87:
682-685,
1990
8.
Dewey, CF,
Bussolari SR,
Gimbrone MA,
and
Davies PF.
The dynamic response of vascular endothelial cells to fluid shear stress.
J Biomech Eng
103:
177-185,
1981[ISI][Medline].
9.
Diamond, SL,
Eskin SG,
and
McIntire LV.
Fluid flow stimulates tissue plasminogen activator secretion by cultured endothelial cells.
Science
243:
1483-1485,
1989
10.
Frangos, JA,
McIntire LV,
Eskin SG,
and
Ives CL.
Flow effects on prostacyclin production by cultured human endothelial cells.
Science
227:
1477-1479,
1985
11.
Garg, UC,
and
Hassid A.
Nitric oxide-generating vasodilators and 8-bromo-cyclic guanosine monophosphate inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells.
J Clin Invest
83:
1774-1777,
1989.
12.
Green, LC,
Wanger DA,
Glogowski J,
Skipper PL,
Wishnok JS,
and
Tannenbaum SR.
Analysis of nitrate, nitrite and [15N] nitrate in biological fluids.
Anal Biochem
126:
131-138,
1982[ISI][Medline].
13.
Gudi, SRP,
Clark CB,
and
Frangos JA.
Fluid flow rapidly activates G proteins in human endothelial cells. Involvement of G proteins in mechanochemical signal transduction.
Circ Res
79:
834-839,
1996
14.
Ignarro, LJ.
Biological actions and properties of endothelium-derived nitric oxide formed and released from artery and vein.
Circ Res
65:
1-21,
1989
15.
Ikeda, U,
Maeda Y,
Funayama H,
Hojo Y,
Ikeda M,
Minota S,
Kano S,
and
Shimada K.
Monocyte-vascular smooth muscle cell interaction enahnces nitric oxide production.
Cardiovasc Res
37:
820-825,
1998
16.
Imai, T,
Hirata Y,
Kanno K,
and
Marumo F.
Induction of nitric oxide synthase by cyclic AMP in rat vascular smooth muscle cells.
J Clin Invest
93:
543-549,
1994.
17.
Inagawa, T,
Ohki S,
Sawada M,
and
Hirata F.
Studies on extraction, separation and estimation of prostaglandins by radioimmunoassay.
Yakugaku Zashi
92:
1187-1194,
1972[ISI][Medline].
18.
Kato, H,
How J,
Chobanian AV,
and
Brecher P.
Effects of angiotensin II infusion and inhibition of nitric oxide synthase on the rat aorta.
Hypertension
28:
153-158,
1996
19.
Kim, WS,
and
Tarbell JM.
Macromolecular transport through the deformable porous media of an artery wall.
J Biomech Eng
116:
156-163,
1994[ISI][Medline].
20.
Lee, E,
Grodzinsky AJ,
Libby P,
Clinton SK,
Lark MW,
and
Lee RT.
Human vascular smooth muscle cell-monocyte interactions and metalloproteinase secretion in culture.
Arterioscler Thromb Vasc Biol
15:
2284-2289,
1995
21.
Malek, A,
and
Izumo S.
Physiological fluid shear stress causes downregulation of endothelin-1 mRNA in bovine aortic endothelium.
Am J Physiol Cell Physiol
263:
C389-C396,
1992
22.
Mitsumata, M,
Fishel RS,
Nerem RM,
Alexander RW,
and
Berk BC.
Fluid shear stress stimulates platelet-derived growth factor expression in endothelial cells.
Am J Physiol Heart Circ Physiol
265:
H3-H8,
1993
23.
Mo, M,
Eskin SG,
and
Schilling WP.
Flow-induced changes in Ca2+ signaling of vascular endothelial cells: effect of shear stress and ADP.
Am J Physiol Heart Circ Physiol
260:
H1698-H1707,
1991
24.
Murata, T,
Ushikubi F,
Matsuoka T,
Hirata M,
Yamasaki A,
Sugomoto Y,
Ichikawa A,
Aze Y,
Tanaka T,
Yoshida N,
Ueno A,
Ohishi S,
and
Narumiya S.
Altered pain perception and inflammatory response in mice lacking prostacyclin receptor.
Nature
388:
678-682,
1997[Medline].
25.
Nollert, MU,
Eskin SG,
and
McIntire LV.
Shear stress increases inositol trisphosphate levels in human endothelial cells.
Biochem Biophys Res Commun
10:
281-287,
1990.
26.
Olesen, SP,
Clapham DE,
and
Davies PF.
Hemodynamic shear stress activates a K+ current in vascular endothelial cells.
Nature
331:
168-170,
1988[Medline].
27.
Osanai, T,
and
Dunn MJ.
Phospholipase C responses in cells from spontaneously hypertensive rats.
Hypertension
19:
446-455,
1992
28.
Osanai, T,
Fujita N,
Fujiwara N,
Nakano T,
Takahashi K,
Guan W,
and
Okumura K.
Cross talk of shear-induced production of prostacyclin and nitric oxide in endothelial cells.
Am J Physiol Heart Circ Physiol
278:
H233-H238,
2000
29.
Osanai, T,
Kanazawa T,
Kamada T,
Okuguchi T,
Kosugi T,
Mio Y,
Imaoka Y,
Metoki H,
Oike Y,
and
Onodera K.
Interaction between flow-induced prostacyclin production and nitric oxide synthesis in vascular smooth muscle cells.
Hypertens Res
17:
227-232,
1994.
30.
Palmer, RMJ,
Ferrige AG,
and
Moncada S.
Nitric oxide accounts for the biological activity of endothelium-derived relaxing factor.
Nature
327:
524-526,
1987[Medline].
31.
Papadaki, M,
Tilton RG,
Eskin SG,
and
McIntire LV.
Nitric oxide production by cultured human aortic smooth muscle cells: stimulation by fluid flow.
Am J Physiol Heart Circ Physiol
274:
H616-H626,
1998
32.
Patrick, CW,
and
McIntire LV.
Shear stress and cyclic strain modulation of gene expression in vascular endothelial cells.
Blood Purif
13:
112-124,
1995[ISI][Medline].
33.
Patrick, CW,
and
McIntire LV.
Fluid shear stress effects on endothelial cell cytosolic pH.
Tissue Eng
1:
53-70,
1995.
34.
Prasad, ARS,
Logan SA,
Nerem RM,
Schwartz CJ,
and
Sprague EA.
Flow-related responses of intracellular inositol phosphate levels in cultured aortic endothelial cells.
Circ Res
72:
827-836,
1993
35.
Radomski, M,
Vallance P,
Whitley G,
Foxwell N,
and
Moncada S.
Platelet adhesion to human vascular endothelium is modulated by constitutive and cytokine-induced nitric oxide.
Cardiovasc Res
27:
1380-1382,
1993
36.
Ribeiro, MO,
Antunes E,
de Nucci G,
Lovisolo SM,
and
Zatz R.
Chronic inhibition of nitric oxide synthesis. A new model of arterial hypertension.
Hypertension
20:
298-303,
1992
37.
Sdougos, HP,
Bussolari SR,
and
Dewey CF, Jr.
Secondary flow and turbulence in a cone-plate device.
J Fluid Mech
138:
379-404,
1984.
38.
Takahashi, M,
Ikeda U,
Masuyama J,
Kitagawa S,
Kasahara T,
Shimpo M,
Kano S,
and
Shimada K.
Monocyte-endothelial cell intreraction induces expression of adhesion molecules on human umbilical cord endothelial cells.
Cardiovasc Res
32:
422-429,
1996
39.
Tsao, PS,
Buitrago R,
Chan JR,
and
Cooke JP.
Fluid flow inhibits endothelial adhesiveness. Nitric oxide and transcriptional regulation of VCAM-1.
Circulation
94:
1682-1689,
1996
40.
Tsao, PS,
Lewis NP,
Alpert S,
and
Cooke JP.
Exposure to shear stress alters endothelial adhesiveness. Role of nitric oxide.
Circulation
92:
3513-3519,
1995
41.
Wagner, CT,
Durante W,
Christodoulides N,
Hellums JD,
and
Schafer AI.
Hemodynamic forces induce the expression of heme oxygenase in cultured vascular smooth muscle cells.
J Clin Invest
100:
589-596,
1997[ISI][Medline].
42.
Wang, DM,
and
Tarbell JM.
Modeling interstitial flow in an artery wall allows estimation of wall shear stress on smooth muscle cells.
J Biomech Eng
117:
358-363,
1995[ISI][Medline].
43.
Wechezak, AR,
Vigger RF,
and
Sauvage LR.
Fibronectin and F-actin redistribution in cultured endothelial cells exposed to shear stress.
Lab Invest
53:
639-647,
1985[ISI][Medline].
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), cells were kept under a
static condition for 90 min at 37°C under 5% CO2. Data
are means ± SE for 7 experiments in duplicate. *P < 0.05 by two-way analysis of variance (ANOVA).
