© 1998 American Heart Association, Inc.
Original Contributions |
Influence of Oscillatory and Unidirectional Flow Environments on the Expression of Endothelin and Nitric Oxide Synthase in Cultured Endothelial Cells
From the Division of Hypertension and Vascular Medicine, Lausanne University Hospital, Switzerland.
Correspondence to Thierry Ziegler, Division d'Hypertension, CHUV, 1011 Lausanne, Switzerland. E-mail thierry.ziegler{at}chuv.hospvd.ch
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Abstract—In vivo, endothelial cells (ECs) are subjected to a complex mechanical environment composed of shear stress, pressure, and circumferential stretch. The aim of this study was to subject bovine aortic ECs to a pulsatile pressure oscillating from 70 to 130 mm Hg (mean of 100 mm Hg) in combination with pulsatile shear stresses from 0.1 to 6 dyne/cm2 (1 dyne/cm2=0.1 N/m2) with or without a cyclic circumferential stretch of 4% for 1, 4, and 24 hours. The effect of highly reversing oscillatory shear stress (range -3 to +3 dyne/cm2, mean of 0.3 dyne/cm2) typical of regions prone to the development of atherosclerotic plaques was also studied at 4 and 24 hours. Endothelin-1 (ET-1) and endothelial constitutive nitric oxide synthase (ecNOS) mRNA expression was time and mechanical force dependent. ET-1 mRNA was maximal at 4 hours and decreased to less than static culture expression at 24 hours, whereas ecNOS mRNA increased over time. Pressure combined with low shear stress upregulated ET-1 and ecNOS mRNA compared with static control. Additional increase in expression for both genes was observed under a combination of higher shear stress and pressure. A cyclic circumferential stretch of 4% did not induce a further increase in ET-1 and ecNOS mRNA at either low or high shear stress. Oscillatory shear stress with pressure induced a higher expression of ET-1 mRNA but lower expression of ecNOS mRNA compared with unidirectional shear stress and pressure. We have shown that the combination of pressure and oscillatory shear stress can downregulate ecNOS levels, as well as upregulate transient expression of ET-1, compared with unidirectional shear stress. These results provide a new insight into the exact role of mechanical forces in endothelial dysfunction in regions prone to the development of atherosclerosis.
Key Words: mechanical stress • hemodynamics • vascular endothelium • nitric oxide synthase • endothelin
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The fluid mechanics of blood are known to transiently regulate vascular tone. In addition to short-term action, fluid mechanics have also been shown to regulate vascular remodeling in the case of long-term changes in pressure (hypertension) and flow rate (pregnancy, arteriovenous shunts, and exercise).1 More importantly, it has been demonstrated that the localization of the atherosclerotic plaque is correlated with regions characterized by oscillatory shear stress environment.2 This regulation has been demonstrated in part to occur via two molecules secreted by the ECs and which have been identified as ET-1 and NO.
ET-1 is a 21–amino acid peptide acting as a powerful vasoconstrictor and an SMC mitogen.3 ET-1 is thought to play an active role in SMC proliferation during remodeling of arteries. ET-1 mRNA expression has been shown to be transiently stimulated at 1 to 4 hours in cultured porcine aortic ECs exposed to a shear stress of 5 dyne/cm2 (1 dyne/cm2=0.1 N/m2) and 30 minutes in BAECs exposed to 15 dyne/cm2.4 5 Longer time exposure to 15 dyne/cm2 resulted in a decrease in ET-1 mRNA in BAECs.5 This decrease was not observed when ECs were exposed to an oscillatory shear stress ranging from -21 to 21 dyne/cm2.5 Another study using venous ECs suggested that there exists a threshold value of steady shear stress around 5 to 6 dyne/cm2 above which ET-1 mRNA is downregulated and below which it is upregulated.6 7 It should be noted, however, that the perfusion system used in the latter study subjects ECs to both shear stress and unspecified pressure. A recent study has demonstrated an increased ET-1 release by hydrostatic pressure as low as 40 mm Hg.8 No change in ET-1 mRNA levels was shown after the cells were exposed to a 20% stretch for 2 to 10 hours.5 Other studies, however, found an upregulation of ET-1 mRNA, as well as an increased ET-1 secretion into the medium, by cyclic stretch of 5% to 20% by a transcriptional regulation mechanism.9
NO is the product of conversion of L-arginine into L-citrulline by NOS and was found to play an important role in the regulation of vascular tone.10 NO was shown to inhibit SMC growth through a cyclic GMP–dependent mechanism.11 NO is released in bigger amounts by ECs subjected to both steady and pulsatile shear stresses.12 13 Both mRNA and protein expressions of ecNOS and NO release are enhanced as early as 3 hours after exposure to 15 or 25 dyne/cm2.12 13 Although a lower shear stress of 4 dyne/cm2 has been shown to induce mRNA expression, this level was not sufficient to induce higher protein content than in static culture.13 Using a vacuum-powered stretching device, it was demonstrated that ecNOS mRNA and protein, as well as the release of NO, were increased in BAECs exposed to 1 Hz cyclic stretch of 6% to 10%.14 15
Many different systems have been designed to subject ECs to mechanical stresses (for review see Reference 1616 ). In all of these systems, one or a maximum of two mechanical forces can be applied at the same time. However, it is known that the physiological mechanical environment generated during the cardiac cycle has a more complex nature, which includes different combinations of shear stress, pressure, and cyclic circumferential stretch. Using a perfusion system designed in our laboratory that subjects ECs cultured on the inside of elastic tubes to combined mechanical forces, we have shown that BAECs elongate and align in a direction parallel to the major axis of the tube when subjected to shear stresses oscillating from 3 to 6 dyne/cm2 and cyclic circumferential stretch values from 2% to 10% after 24 hours.17 Furthermore, the combination of shear stress and cyclic stretch synergistically enhanced cell elongation and alignment.17
In the present study, we subjected BAECs to two different combinations of mechanical stresses. The first one, corresponding to that found in areas free of atherosclerotic plaques in vivo is characterized by a nonreversing pulsatile shear stress.2 The second is composed of forces corresponding to those found in lesion-prone areas and is characterized by a highly reversing shear stress with a low mean value of 0.3 dyne/cm2.2 The novelty in this study is that these two shear stress patterns were always combined to a pulsatile pressure with a mean value of 100 mm Hg and in some cases to a cyclic circumferential stretch of 4%. We assessed the effect of different time exposures to such mechanical environments on the expression of ET-1 and ecNOS mRNA, as well as on the level of ecNOS protein.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Cell Culture
BAECs were isolated from aortae obtained from the local slaughterhouse. The aorta was cannulated using plastic syringes and all collaterals were clamped. The aorta was then filled with a solution containing 1% collagenase CLS4 (Worthington) in modified EMEM (GIBCO) and incubated 15 minutes at 37°C. After this time period, the collagenase solution was centrifuged 5 minutes at 1000 rpm and the supernatant discarded. The pellet was resuspended in culture medium composed of EMEM supplemented with 10% FBS (Seromed), 100 U/mL penicillin-streptomycin (GIBCO), 2 mmol/L glutamine (GIBCO), and nonessential amino acids (Seromed) and seeded into several T-75 (Falcon, Inotech). All experiments were carried out using cells at passage 4 from the same aorta.
After extensive cleaning, five silicone tubes (see following section) were treated with 70% sulfuric acid (Merck) for 2 minutes. After several rinses with deionized water, each tube was mounted onto an individual fitting and longitudinally stretched by 10%. This amount of stretching in the longitudinal direction prevented buckling of the tube under pressure. All fittings were sterilized by autoclaving. After cooling, the tubes were incubated overnight with a solution of PBS containing 10 µg/mL bovine fibronectin (Sigma). The following day, the tubes were seeded with a suspension of BAECs at a density of 150 000/cm2. The cells were allowed to attach under rotation for 4 hours in a tissue-culture incubator, at which time confluence was checked under the microscope.
Experimental System
The system has been described in more detail elsewhere
(Reference 1717 and Fig 1A
). Briefly, it is
composed of compliant tubes made of Sylgard 184 (Dow Corning Europe) of
6 mm diameter and 76 mm length. The tubes are mounted onto
specially designed fittings, which in turn are inserted in parallel
into a perfusion loop composed of a reservoir and a gear pump
(Ismatec). The pump is controlled by a function generator (Hewlett
Packard) to produce a 1-Hz sinusoidal flow rate. Medium in the
reservoir is composed of culture medium with 2% dextran,
Mr=70 000 (Sigma), to increase the
viscosity to 1.07x10-3 N ·
m-2 · s-1. The
reservoir is constantly gassed by a 5% CO2/95%
air mixture to keep a constant pH of 7.2. Both reservoir and fittings
were kept at a constant temperature of 37°C in a water bath.
|
Flow rate was measured with ultrasonic flowmeters (Transonic System, Inc) with a precision of ±2%. Tube diameter and diameter fluctuation over time were measured with a specially designed ultrasonic echotracking device (NIUS, Asulab) with a precision of 3 µm.18 Pressure was measured using pressure catheters (Millar Instruments) inserted at the level of each tube. Shear stress was computed from flow rate and diameter measurements using a Womersley approximation.19 The percentage of circumferential stretch is calculated as the ratio of the diameter amplitude to the mean diameter.
Each tube was exposed to a different mechanical environment (Fig 1A
).
Tubes 1 and 4 were exposed to the combination of shear stress, cyclic
stretch, and pressure. Tubes 2 and 3 were exposed to shear stress and
pressure, while cyclic stretching was prevented by adding a rigid shell
around each tube. Tubes 3 and 4 were exposed to a lower shear stress
(0.1 to 0.3 dyne/cm2) than tubes 1 and 2. A fifth
tube was kept in a tissue-culture incubator to expose cells to a purely
static environment.
In this system, tubes 1 through 4 were all exposed to a pulsatile pressure with a mean of 100 mm Hg and an amplitude of 60 mm Hg. The different levels of shear stress assessed can be classified into three categories: low shear stress of 0.08 and 0.3 dyne/cm2 with little pulsatility, oscillatory shear stress of 0.3 dyne/cm2 with a high pulsatility, and unidirectional shear stress of 6 dyne/cm2 with a high pulsatility. In addition, we evaluated the effect of a 4% cyclic circumferential stretch in combination with the stresses described above. Flow experiments were run for 1, 4, and 24 hours.
Cell Harvesting and Northern Blot Analysis
At the end of the experiment, the fittings were quickly
dismounted. The tubes were then rinsed once with PBS and filled with 3
mL of a trypsin-EDTA solution (GIBCO). After 3 minutes, the cells were
completely detached by gentle tapping of the tubes and the cell
suspension obtained was centrifuged for 10 minutes at
800g and 4°C. The cell pellet was lysed in a buffer
containing guanidinium isothiocyanate and total RNA was isolated using
a kit (RNeasy, Quiagen). Total RNA (5 µg) was loaded into wells and
separated according to its size in a 1% agarose/6% formaldehyde gel.
RNA was transferred overnight by capillarity to a nylon membrane
(Hybond-N, Amersham). The membranes were prehybridized at 42°C for a
minimum of 1 hour in 50% formamide, 0.2% polyvinylpyrrolidone, 0.2%
BSA, 0.2% Ficoll, 0.05 mol/L Tris (pH 7.5), 1.0 mol/L NaCl, 0.1%
sodium pyrophosphate, 1% SDS, 10% dextran sulfate, and 100 µg/mL
denatured salmon sperm DNA (Boehringer Mannheim). Hybridization
was carried out in the same solution containing
32P-random-primed–labeled cDNA
(Boehringer Mannheim) specific for bovine preproendothelin-1
(gift of Dr Quetermous), bovine ecNOS (gift of Dr D. Harrison, Atlanta,
Ga), rat ß-actin or human GAPDH. After overnight incubation, the
membranes were washed in 2x SSC/0.1% SDS at room temperature for 30
minutes and in 0.1x SSC/0.1% SDS (for ET-1 and ecNOS) or 0.5%
SSC/0.1% SDS (ß-actin and GAPDH) at 60°C for 1 hour and then
exposed to X-ray film (Kodak X-Omat) at -80°C. Transcript levels
were quantified using an electronic autoradiography
apparatus (Instant Imager, Packard) or by scanning the
x-ray film (Apple OneScanner) followed by densitometric
analysis (NIH Image).
Protein Harvesting and Western Blot Analysis
Total cellular protein was solubilized in lysis buffer (50
mmol/L Tris, pH 6.8, 2% SDS) containing 1 mmol/L PMSF (Sigma) and
10 µmol/L leupeptin (Sigma). Samples were boiled for 5 minutes
and protein concentration was measured using a protein assay (BCA,
Pierce). Samples of protein (10 µg) were separated on a 7%
SDS-polyacrylamide gel under reducing conditions and
transferred to nitrocellulose membranes (Hybond-ECL, Amersham) using a
semidry transfer cell (Bio-Rad). The membrane was blocked in PBS
containing 3% nonfat milk and incubated overnight at 4°C with a
monoclonal antibody directed against ecNOS (Transduction Laboratories).
The following day, the membrane was washed in PBS with Tween 20 (Sigma)
and incubated 1 hour at room temperature with a secondary horseradish
peroxidase conjugate (Amersham). After washing, the proteins were
visualized by chemiluminescence using the ECL revealing kit (Amersham).
Transcript levels were quantified as for Northern blot
analysis.
Cell Staining and Measurement of Cell Elongation
The borders of the cells were silver stained as previously
described.17 The perimeter, area, and major axis
of single cells were measured from images taken with a confocal
microscope (MRC 500, Bio-Rad) using a dedicated software (SOM,
Bio-Rad). The SI was then computed as 4x
xcell
area/perimeter2. The SI defines the degree of
cell elongation and is equal to 1 for a circle and 0 for a straight
line. The angle of orientation was also calculated as the angle between
the cell major axis and the longitudinal axis of the tube. SIs and
angles of orientation of 80 randomly chosen cells (40 per experiment)
were averaged for each tube and expressed as mean±SEM. Statistical
analysis was performed by nonparametric tests using
Instat 2 software (GraphPad).
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Effect of Mechanical Forces on ET-1 mRNA Expression
Fig 2A
and 2B show a time course of
ET-1 mRNA induction by mechanical forces. After 1 hour of exposure of
BAECs to a unidirectional flow environment composed of a shear stress
of 6±3 dyne/cm2, 4% stretch, and pressure, we
observed a slight induction of ET-1 mRNA by mechanical forces (tubes 1
through 4) compared with static control (tube 5). At 4 hours, ET-1 mRNA
induction was maximally induced in all tubes compared with static
control values. The effect of pressure, shear stress, and cyclic
stretch on ET-1 mRNA expression in BAECs seen at the 1-hour and 4-hour
time points was no longer observed at 24 hours. Furthermore, the
expression levels showed a return to those of the static culture.
|
A dose response of the effect of shear stress in the presence of
pressure on BAEC ET-1 mRNA at 4 hours is shown in Fig 3A. Very low steady shear stresses of
0.08 to 0.3 dyne/cm2 were sufficient to induce a
2.5-fold to 3-fold increase in ET-1 mRNA. A pulsatile shear stress of 6
(range 3 to 9) dyne/cm2 further upregulated ET-1
mRNA expression (3.7-fold). Interestingly, oscillatory shear stress
(mean of 0.3, range from -3 to +3 dyne/cm2)
maximally induced ET-1 mRNA, with an approximate 6-fold increase
compared with static control. The presence of a 4% cyclic stretch did
not significantly affect ET-1 mRNA expression at both low and high
shear stress values and in the presence of oscillatory shear stresses
(Fig 3A).
|
Effect of Mechanical Forces on ecNOS mRNA Expression
The pattern of ecNOS mRNA expression in BAECs exposed to the same
unidirectional environment described in the previous paragraphs for 1,
4, and 24 hours is shown in Fig 4A and 4B. At 1 hour and 4 hours, there was no effect of any combination of
mechanical environment on ecNOS mRNA expression. However, by 24 hours,
there was a dramatic increase in ecNOS mRNA expression in all tubes
exposed to mechanical forces compared with static control.
|
Very low shear stress values of 0.08 dyne/cm2 in
combination with pressure did not induce ecNOS mRNA at all time points
studied (Fig 3B). A 6-fold induction of ecNOS mRNA was observed in ECs
cultured in the presence of a steady shear stress of 0.3
dyne/cm2. Increasing both the mean and the
pulsatility of shear stress (mean of 6 dyne/cm2,
range from 3 to 9) resulted in a further induction of ecNOS mRNA
expression (13-fold compared with static). At 24 hours and in the
presence of pressure, oscillatory shear stress induced a lower
normalized level of ecNOS mRNA compared with unidirectional shear
stresses of 0.3 and 6 dyne/cm2 (Fig 3B).
Similarly to ET-1, cyclic stretch had no additional effect on the
expression of ecNOS mRNA in the presence of low, high, and oscillatory
shear stresses (Fig 3B).
These results were confirmed at the protein level by Western blot
analysis of total cell lysate of BAECs exposed to
unidirectional flow environment for 24 hours, as depicted in Fig 5. In comparison to increasing mRNA
expression, cyclic stretch did not increase ecNOS protein level in the
presence of either low or high shear stress. Oscillatory shear stress
in the presence or in the absence of a 4% circumferential stretch only
slightly induced ecNOS protein expression in BAECs compared with static
culture (Fig 5).
|
In the light of the different results obtained with oscillatory shear
stress, we exposed BAECs to oscillatory shear stress to assess their
shape change and compare it with that seen under unidirectional shear
stress. Elongation and alignment of BAECs has been well documented in
the presence of shear stress only20 21 but less
when shear stress is combined with pressure and/or cyclic
circumferential stretch.17 In this study, we
found again that BAECs exposed to a high unidirectional shear stress of
6 (range 3 to 9) dyne/cm2 for 24 hours were
elongated and aligned with the direction of flow (Fig 6). In the presence of oscillatory shear
stress of 0.3 dyne/cm2 (range -3 to +3), the SI
of BAECs did not change compared with cells exposed to a low shear
stress of 0.3 dyne/cm2 (range 0.2 to 0.4),
implying that the cells did not elongate. Furthermore, no particular
alignment of the cells was observed in both low and oscillatory shear
stresses. When a cyclic stretch of 4% was combined with the
oscillatory shear stress, the SI decreased, demonstrating cell
elongation. However, these morphological changes were less pronounced
than those with the combination of high unidirectional shear stress and
stretch.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Previous studies of how mechanical forces affect EC gene expression have used a variety of different apparatuses that look at individual force effects (for review see Reference 1616 ). Our study relies on a perfusion system in which ECs are exposed to a more complex mechanical environment. In addition, we can clarify more fully the contribution of an individual force to EC morphology and function in a complex environment. Using this system, we have previously shown a synergistic effect of pressure, shear stress, and cyclic stretch on EC elongation and alignment.17
The main findings of this study are that (1) pressure in combination with a shear stress lower than 0.1 dyne/cm2 induced ET-1 mRNA expression at 4 hours but failed to change ecNOS mRNA expression at 4 to 24 hours; (2) higher shear stress magnitudes increased ecNOS mRNA and protein at 24 hours but not at 4 hours; (3) the presence of a 4% cyclic circumferential stretch did not alter ET-1 and ecNOS mRNA expression induced by shear stresses ranging from 0.08 to 6 dyne/cm2; and (4) oscillatory shear stress upregulated ET-1 mRNA at 4 hours and downregulated ecNOS mRNA at 24 hours compared with unidirectional shear stress.
The effect of shear stress on ET-1 mRNA expression in cultured ECs is controversial, with some studies showing a downregulation of ET-1 at levels similar to the ones used in our study.6 However, it has been demonstrated that a nonpulsatile shear stress of 5 dyne/cm2 at atmospheric pressure increased ET-1 mRNA expression by ECs in a time-dependent manner.4 22 Our study has shown that very low unidirectional shear stresses in combination with a pulsatile pressure ranging from 70 to 130 mm Hg induced ET-1 mRNA in BAECs in a similar manner. As this level of expression was further upregulated by a higher pulsatile shear stress, we have demonstrated that a pulsatile shear stress can individually affect EC expression in a pressurized system.
In the presence of a pulsatile pressure, lowering shear stress to less than 0.1 dyne/cm2 had no effect on expression levels of ET-1 mRNA. This observation could suggest that the effect observed in tube 3 may be mainly due to pressure and not shear stress. However, because we are unable to show the effect of shear stress alone in our current system, we have recently designed a new perfusion apparatus that will allow us to further investigate the effect of pressure on ET-1 expression. This new generation of flow device will also allow for measurement of secreted ET-1.
Although small increases in mRNA expression were observed at 1 hour, ecNOS mRNA was not significantly upregulated by mechanical forces at 4 hours. A more dramatic change was observed at 24 hours, with a 7-fold to 13-fold increase in mRNA expression compared with the static control. The combination of pressure and a shear stress of 0.1 dyne/cm2, a level previously shown not to induce any increase in ecNOS content and NO release,23 slightly upregulated ecNOS mRNA (1.5-fold) at 24 hours. However, our results show that a slightly higher shear stress magnitude to 0.3 dyne/cm2 resulted in a 7-fold increase in ecNOS mRNA at 24 hours. This observation suggests that the ecNOS expression level is not affected by the presence of a pulsatile pressure but may be mostly due to the small magnitude of shear stress.
At all levels of shear stress studied here, the presence of a 4% cyclic circumferential stretch did not significantly alter either ET-1 or the ecNOS mRNA response. Other studies have shown that ECs cultured on flexible membranes and stretched up to 20% release ET-1 after 4 hours due to induced gene expression.9 24 25 Similarly, NO release and ecNOS mRNA expression were increased after exposure of BAECs to 20% stretch.14 15 The main differences between these stretch studies and our experiments are the presence of pressure and shear stress and the lower level of stretch used here. Further experiments to show whether stretch levels greater than 4% can affect expression of ET-1 mRNA in the presence of pressure and low and high shear stresses are being carried out at present in our laboratory. It may be that the stretch effect on ecNOS and ET-1 mRNA and protein levels is blunted by the shear stress effect. This result shows that the only way to evaluate the relative importance of pressure, shear stress, and cyclic stretch is by combining mechanical forces in a single experiment.
Oscillatory flow, characterized by a low mean component and an important amount of flow in the reverse direction, has been widely studied because it was demonstrated to be correlated with the localization of the atherosclerotic plaque.2 In these regions, the phenotype of the endothelium is characterized by a rounded shape with an increased proliferation rate and increased permeability.20 In this study, we found that a 24-hour exposure of BAECs to an oscillatory shear stress ranging from -3 to +3 dyne/cm2 did not induce elongation and alignment of the cells with the direction of flow. This result is consistent with in vivo data and in vitro works which show that an environment composed of a highly reversing shear stress with a low mean component did not affect cell shape and extracellular matrix composition.21 26 Moreover, pressure more than anything else was the factor inducing changes in the quantity and organization of fibronectin.26 Unlike unidirectional shear stress, which acted synergistically to enhance elongation and alignment in response to cyclic stretch,17 the presence of an oscillatory shear stress did not change the morphological response of BAECs to cyclic stretch.
We have found that BAECs exposed to a combination of pressure and oscillatory shear stress produced more ET-1 mRNA at 4 hours and less ecNOS mRNA at 24 hours than BAECs exposed to pressure and unidirectional shear stress. This enhanced ET-1 mRNA production and lower ecNOS expression in cells exposed to oscillatory shear stress is consistent with data showing increased release of ET-1 at the level of the atherosclerotic plaque,27 as well as higher levels of free radicals. It should be emphasized that these in vitro results cannot be directly extrapolated to in vivo conditions because both the biochemical environment and time of exposure to mechanical forces are different. However, these results imply that oscillatory shear stress may contribute to the endothelial dysfunction by inducing increased ET-1 and decreased NO production, thus leading to cellular toxicity, SMC proliferation, and atherogenesis, as hypothesized by Loscalzo and Welch.10 This difference in cell behavior due to the highly reversing nature of flow (which is the only difference between tube 1 and tube 3, in which ECs were exposed to a low amplitude of shear stress) is worth studying in greater details.
Using a novel perfusion apparatus, we have shown that both ET-1 and ecNOS mRNA were upregulated by the combination of pressure and shear stress but at different time points. Oscillatory shear stress, characteristic of regions prone to the development of atherosclerotic plaque and characterized by an important amount of stress in the reverse direction, was shown to induce more ET-1 mRNA at 4 hours and less ecNOS mRNA and protein at 24 hours than unidirectional shear stress. Overall, these results emphasize the importance of using a quasiphysiological mechanical environment combining all stresses to assess endothelial function in vitro.
|
Selected Abbreviations and Acronyms |
|---|
|
|
Acknowledgments |
|---|
This work was supported by a grant to D.H. from the Swiss National Foundation (No. 32–42515-94). The authors wish to thank Dr David Harrison for the ecNOS cDNA and Dr Quetermous for ET-1 cDNA. V.J.H. is funded by a Wellcome European Travelling Fellowship.
Received May 2, 1997; accepted November 3, 1997.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Rubanyi GM, Freay AD, Kauser K, Johns A, Harder DR. Mechanoreception by the endothelium: mediators and mechanisms of pressure- and flow-induced vascular responses. Blood Vessels. 1990;27:246–257.[Medline] [Order article via Infotrieve]
2.
Ku DN, Giddens DP, Zarins CK, Glagov S. Pulsatile flow
and atherosclerosis in the human carotid bifurcation:
positive correlation between plaque location and low and oscillating
shear stress. Arteriosclerosis. 1985;5:293–302.
3. Dubin D, Pratt RE, Cooke JP, Dzau VJ. Endothelin, a potent vasoconstrictor, is a vascular smooth muscle mitogen. J Vasc Med Biol. 1989;1:13–17.
4. Yoshizumi M, Kurihara H, Sugiyama T, Takaku F, Yanagisawa M, Masaki T, Yazaki Y. Hemodynamic shear stress stimulates endothelin production by cultured endothelial cells. Biochem Biophys Res Commun. 1989;161:859–864.[Medline] [Order article via Infotrieve]
5. Sharefkin JB, Diamond SL, Eskin SG, McIntire LV, Dieffenbach CW. Fluid flow decreases preproendothelin mRNA levels and suppresses endothelin-1 peptide release in cultured human endothelial cells. J Vasc Surg. 1991;14:1–9.[Medline] [Order article via Infotrieve]
6.
Malek AM, Izumo S. Physiological
fluid shear stress causes downregulation of endothelin-1 mRNA in bovine
aortic endothelium. Am J Physiol. 1992;263:C389–C396.
7.
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.
8.
Hishikawa K, Nakaki T, Marumo T, Suzuki H, Kato R,
Saruta T. Pressure enhances endothelin-1 release from cultured human
endothelial cells. Hypertension. 1995;25:449–452.
9. Macarthur H, Warner TD, Wood EG, Corder R, Vane JR. Endothelin-1 release from endothelial cells in culture is elevated both acutely and chronically by short periods of mechanical stretch. Biochem Biophys Res Commun. 1994;200:395–400.[Medline] [Order article via Infotrieve]
10. Loscalzo J, Welch G. Nitric oxide and its role in the cardiovascular system. Prog Cardiovasc Dis. 1995;38:87–104.[Medline] [Order article via Infotrieve]
11. Garg UC, Hassid A. Nitric oxide (NO) and 8-bromo-cyclic GMP inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells. J Clin Invest. 1989;83:1974–1977.
12.
Uematsu M, Ohara Y, Navas JP, Nishida K, Murphy TJ,
Alexander RW, Nerem RM, Harrison DG. Regulation of
endothelial cell nitric oxide synthase mRNA expression
by shear stress. Am J Physiol. 1995;269:C1371–C1378.
13.
Ranjan V, Xiao Z, Diamond SL. Constitutive NOS
expression in cultured endothelial cells is elevated by
fluid shear stress. Am J Physiol. 1995;269:H550–H555.
14. Awolesi MA, Widmann MD, Sessa WC, Sumpio BE. Cyclic strain increases endothelial nitric oxide synthase activity. Surgery. 1994;116:439–444.[Medline] [Order article via Infotrieve]
15. Awolesi MA, Sessa WC, Sumpio BE. Cyclic strain upregulates nitric oxide synthase in cultured bovine aortic endothelial cells. J Clin Invest. 1995;96:1449–1454.
16. Resnick N, Gimbrone MA Jr. Hemodynamic forces are complex regulators of endothelial gene expression. FASEB J. 1995;9:874–882.[Abstract]
17.
Zhao S, Suciu A, Ziegler T, Moore JE Jr, Burki E,
Meister JJ, Brunner HR. Synergistic effects of fluid shear stress and
cyclic circumferential stretch on vascular endothelial
cell morphology and cytoskeleton. Arterioscler Thromb Vasc
Biol. 1995;15:1781–1786.
18.
Hayoz D, Tardy Y, Rutschmann B, Mignot JP, Achakri H,
Feihl F, Meister JJ, Waeber B, Brunner HR. Spontaneous diameter
oscillations of the radial artery in humans. Am
J Physiol. 1993;264:H2080–H2084.
19. He X, Ku DN, Moore JE Jr. Simple calculation of the velocity profiles for pulsatile flow in a blood vessel using Mathematica. Ann Biomed Eng. 1993;21:45–49.[Medline] [Order article via Infotrieve]
20. Schwartz CJ, Valente AJ, Sprague EA, Kelley JL, Nerem RM. The pathogenesis of atherosclerosis: an overview. Clin Cardiol. 1991;14(suppl):I1–16.
21. Helmlinger G, Geiger RV, Schreck S, Nerem RM. Effects of pulsatile flow on cultured vascular endothelial cell morphology. J Biomech Eng. 1991;113:123–131.[Medline] [Order article via Infotrieve]
22. Morita T, Kurihara H, Maemura K, Yoshizumi M, Yazaki Y. Disruption of cytoskeletal structures mediates shear stress–induced endothelin-1 gene expression in cultured porcine aortic endothelial cells. J Clin Invest. 1993;92:1706–1712.
23.
Kanai AJ, Strauss HC, Truskey GA, Crews AL, Grunfeld S,
Malinski T. Shear stress induces ATP-independent transient nitric oxide
release from vascular endothelial cells, measured
directly with a porphyrinic microsensor. Circ Res. 1995;77:284–293.
24. Wang DL, Wung BS, Peng YC, Wang JJ. Mechanical strain increases endothelin-1 gene expression via protein kinase C pathway in human endothelial cells. J Cell Physiol. 1995;163:400–406.[Medline] [Order article via Infotrieve]
25. Stula M, von Harsdorf R, Menzel S, Yu JCM, Orzechowski HD, Paul M, Dietz R. Mechanical stretch induces the endothelin system in endothelial cells. In: Program and abstracts of the European Research Conference on Blood Pressure and Cardiovascular Disease, October 19–21, 1996; Noordwijkerhout, the Netherlands. Abstract.
26. Thoumine O, Nerem RM, Girard PR. Oscillatory shear stress and hydrostatic pressure modulate cell-matrix attachment proteins in cultured endothelial cells. In Vitro Cell Dev Biol. 1995; 31:45–54.
27.
Zeiher AM, Goebel H, Schachinger V, Ihling C. Tissue
endothelin-1 immunoreactivity in the active coronary
atherosclerotic plaque: a clue to the mechanism of increased
vasoreactivity of the culprit region in unstable angina.
Circulation. 1995;91:941–947.
This article has been cited by other articles:
 |
|
 |
|
  D. H.J. Thijssen, E. A. Dawson, T. M. Tinken, N. T. Cable, and D. J. Green Retrograde Flow and Shear Rate Acutely Impair Endothelial Function in Humans Hypertension, June 1, 2009; 53(6): 986 - 992. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Ai, M. Rouhanizadeh, J. C. Wu, W. Takabe, H. Yu, M. Alavi, R. Li, Y. Chu, J. Miller, D. D. Heistad, et al. Shear stress influences spatial variations in vascular Mn-SOD expression: implication for LDL nitration Am J Physiol Cell Physiol, June 1, 2008; 294(6): C1576 - C1585. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. E. Pyke, V. Poitras, and M. E. Tschakovsky Brachial artery flow-mediated dilation during handgrip exercise: evidence for endothelial transduction of the mean shear stimulus Am J Physiol Heart Circ Physiol, June 1, 2008; 294(6): H2669 - H2679. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. C. Newcomer, C. L. Sauder, N. T. Kuipers, M. H. Laughlin, and C. A. Ray Effects of posture on shear rates in human brachial and superficial femoral arteries Am J Physiol Heart Circ Physiol, April 1, 2008; 294(4): H1833 - H1839. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. H. Laughlin, S. C. Newcomer, and S. B. Bender Importance of hemodynamic forces as signals for exercise-induced changes in endothelial cell phenotype J Appl Physiol, March 1, 2008; 104(3): 588 - 600. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. C. W. Groenendijk, K. Van der Heiden, B. P. Hierck, and R. E. Poelmann The Role of Shear Stress on ET-1, KLF2, and NOS-3 Expression in the Developing Cardiovascular System of Chicken Embryos in a Venous Ligation Model Physiology, December 1, 2007; 22(6): 380 - 389. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Won, S.-N. Zhu, M. Chen, A.-M. Teichert, J. E. Fish, C. C. Matouk, M. Bonert, M. Ojha, P. A. Marsden, and M. I. Cybulsky Relative Reduction of Endothelial Nitric-Oxide Synthase Expression and Transcription in Atherosclerosis-Prone Regions of the Mouse Aorta and in an in Vitro Model of Disturbed Flow Am. J. Pathol., November 1, 2007; 171(5): 1691 - 1704. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. F. Kelly and H. M. Snow Characteristics of the response of the iliac artery to wall shear stress in the anaesthetized pig J. Physiol., July 15, 2007; 582(2): 731 - 743. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. A. Himburg, S. E. Dowd, and M. H. Friedman Frequency-dependent response of the vascular endothelium to pulsatile shear stress Am J Physiol Heart Circ Physiol, July 1, 2007; 293(1): H645 - H653. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. S. Chatzizisis, A. U. Coskun, M. Jonas, E. R. Edelman, C. L. Feldman, and P. H. Stone Role of Endothelial Shear Stress in the Natural History of Coronary Atherosclerosis and Vascular Remodeling: Molecular, Cellular, and Vascular Behavior J. Am. Coll. Cardiol., June 26, 2007; 49(25): 2379 - 2393. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Gambillara, C. Chambaz, G. Montorzi, S. Roy, N. Stergiopulos, and P. Silacci Plaque-prone hemodynamics impair endothelial function in pig carotid arteries Am J Physiol Heart Circ Physiol, June 1, 2006; 290(6): H2320 - H2328. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. C. Isenberg, C. Williams, and R. T. Tranquillo Small-Diameter Artificial Arteries Engineered In Vitro Circ. Res., January 6, 2006; 98(1): 25 - 35. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-L. E. Helm, M. E. Fleury, A. H. Zisch, F. Boschetti, and M. A. Swartz Synergy between interstitial flow and VEGF directs capillary morphogenesis in vitro through a gradient amplification mechanism PNAS, November 1, 2005; 102(44): 15779 - 15784. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. R. Alegria, J. Herrmann, D. R. Holmes Jr, A. Lerman, and C. S. Rihal Myocardial bridging Eur. Heart J., June 2, 2005; 26(12): 1159 - 1168. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Ganguli, L. Persson, I. R. Palmer, I. Evans, L. Yang, R. Smallwood, R. Black, and E. E. Qwarnstrom Distinct NF-{kappa}B Regulation by Shear Stress Through Ras-Dependent I{kappa}B{alpha} Oscillations: Real-Time Analysis of Flow-Mediated Activation in Live Cells Circ. Res., April 1, 2005; 96(6): 626 - 634. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Herrmann, S. T. Higano, R. J. Lenon, C. S. Rihal, and A. Lerman Myocardial bridging is associated with alteration in coronary vasoreactivity Eur. Heart J., December 1, 2004; 25(23): 2134 - 2142. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Sun, A. Huang, and G. Kaley Mechanical compression elicits NO-dependent increases in coronary flow Am J Physiol Heart Circ Physiol, December 1, 2004; 287(6): H2454 - H2460. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. B. Dancu, D. E. Berardi, J. P. Vanden Heuvel, and J. M. Tarbell Asynchronous Shear Stress and Circumferential Strain Reduces Endothelial NO Synthase and Cyclooxygenase-2 but Induces Endothelin-1 Gene Expression in Endothelial Cells Arterioscler. Thromb. Vasc. Biol., November 1, 2004; 24(11): 2088 - 2094. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.-H. Juan, J.-J. Chen, C.-H. Chen, H. Lin, C.-F. Cheng, J.-C. Liu, M.-H. Hsieh, Y.-L. Chen, H.-H. Chao, T.-H. Chen, et al. 17{beta}-Estradiol inhibits cyclic strain-induced endothelin-1 gene expression within vascular endothelial cells Am J Physiol Heart Circ Physiol, September 1, 2004; 287(3): H1254 - H1261. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. A. Miyakawa, M. de Lourdes Junqueira, and J. E. Krieger Identification of two novel shear stress responsive elements in rat angiotensin I converting enzyme promoter Physiol Genomics, April 13, 2004; 17(2): 107 - 113. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. R. White, M. A. Haidekker, H. Y. Stevens, and J. A. Frangos Extracellular signal-regulated kinase activation and endothelin-1 production in human endothelial cells exposed to vibration J. Physiol., March 1, 2004; 555(2): 565 - 572. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. A. VanderLaan, C. A. Reardon, and G. S. Getz Site Specificity of Atherosclerosis: Site-Selective Responses to Atherosclerotic Modulators Arterioscler. Thromb. Vasc. Biol., January 1, 2004; 24(1): 12 - 22. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. Vita and G. F. Mitchell Effects of shear stress and flow pulsatility on endothelial function: Insights gleaned from external counterpulsation therapy J. Am. Coll. Cardiol., December 17, 2003; 42(12): 2096 - 2098. [Full Text] [PDF]
|
|
|
|
|
|
J. S. McNally, M. E. Davis, D. P. Giddens, A. Saha, J. Hwang, S. Dikalov, H. Jo, and D. G. Harrison Role of xanthine oxidoreductase and NAD(P)H oxidase in endothelial superoxide production in response to oscillatory shear stress Am J Physiol Heart Circ Physiol, December 1, 2003; 285(6): H2290 - H2297. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-C. Liu, J.-J. Chen, P. Chan, C.-F. Cheng, and T.-H. Cheng Inhibition of Cyclic Strain-Induced Endothelin-1 Gene Expression by Resveratrol Hypertension, December 1, 2003; 42(6): 1198 - 1205. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. Florian, J. R. Kosky, K. Ainslie, Z. Pang, R. O. Dull, and J. M. Tarbell Heparan Sulfate Proteoglycan Is a Mechanosensor on Endothelial Cells Circ. Res., November 14, 2003; 93 (10): e136 - e142. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. K. HSIAI, S. K. CHO, P. K. WONG, M. ING, A. SALAZAR, A. SEVANIAN, M. NAVAB, L. L. DEMER, and C.-M. HO Monocyte recruitment to endothelial cells in response to oscillatory shear stress FASEB J, September 1, 2003; 17(12): 1648 - 1657. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Pellieux, A. Desgeorges, C. H. Pigeon, C. Chambaz, H. Yin, D. Hayoz, and P. Silacci Cap G, a Gelsolin Family Protein Modulating Protective Effects of Unidirectional Shear Stress J. Biol. Chem., August 1, 2003; 278(31): 29136 - 29144. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Reckova, C. Rosengarten, A. deAlmeida, C. P. Stanley, A. Wessels, R. G. Gourdie, R. P. Thompson, and D. Sedmera Hemodynamics Is a Key Epigenetic Factor in Development of the Cardiac Conduction System Circ. Res., July 11, 2003; 93(1): 77 - 85. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. G. Carlier, L. C.A. van Damme, C. P. Blommerde, J. J. Wentzel, G. van Langehove, S. Verheye, M. M. Kockx, M. W.M. Knaapen, C. Cheng, F. Gijsen, et al. Augmentation of Wall Shear Stress Inhibits Neointimal Hyperplasia After Stent Implantation: Inhibition Through Reduction of Inflammation? Circulation, June 3, 2003; 107(21): 2741 - 2746. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Ceravolo, R. Maio, A. Pujia, A. Sciacqua, G. Ventura, M. C. Costa, G. Sesti, and F. Perticone Pulse pressure and endothelial dysfunction in never-treated hypertensive patients J. Am. Coll. Cardiol., May 21, 2003; 41(10): 1753 - 1758. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. M. Reber, B. Y. Su, K. R. Clark, D. L. Pohlman, C. E. Miller, and P. T. Nowicki Developmental expression of eNOS in postnatal swine mesenteric artery Am J Physiol Gastrointest Liver Physiol, December 1, 2002; 283(6): G1328 - G1335. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F Markos, B A Hennessy, M Fitzpatrick, J O'Sullivan, and H M Snow The effect of tezosentan, a non-selective endothelin receptor antagonist, on shear stress-induced changes in arterial diameter of the anaesthetized dog J. Physiol., November 1, 2002; 544(3): 913 - 918. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. K. Hsiai, S. K. Cho, S. Reddy, S. Hama, M. Navab, L. L. Demer, H. M. Honda, and C. M. Ho Pulsatile Flow Regulates Monocyte Adhesion to Oxidized Lipid-Induced Endothelial Cells Arterioscler. Thromb. Vasc. Biol., November 1, 2001; 21(11): 1770 - 1776. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Silacci, A. Desgeorges, L. Mazzolai, C. Chambaz, and D. Hayoz Flow Pulsatility Is a Critical Determinant of Oxidative Stress in Endothelial Cells Hypertension, November 1, 2001; 38(5): 1162 - 1166. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H M Snow, F Markos, D O'Regan, and K Pollock Characteristics of arterial wall shear stress which cause endothelium-dependent vasodilatation in the anaesthetized dog J. Physiol., March 15, 2001; 531(3): 843 - 848. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Suvatne, A. I. Barakat, and M. E. O'Donnell Flow-induced expression of endothelial Na-K-Cl cotransport: dependence on K+ and Cl{-} channels Am J Physiol Cell Physiol, January 1, 2001; 280(1): C216 - C227. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R Schulz, D Schmidt, A Blum, X Lopes-Ribeiro, C Lücke, K Mayer, H Olschewski, W Seeger, and F Grimminger Decreased plasma levels of nitric oxide derivatives in obstructive sleep apnoea: response to CPAP therapy Thorax, December 1, 2000; 55(12): 1046 - 1051. [Abstract] [Full Text]
|
|
|
|
 |
|
 L.-m. Gan, L. Selin-Sjogren, R. Doroudi, and S. Jern Temporal regulation of endothelial ET-1 and eNOS expression in intact human conduit vessels exposed to different intraluminal pressure levels at physiological shear stress Cardiovasc Res, October 1, 2000; 48(1): 168 - 177. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Peng, F. A. Recchia, B. J. Byrne, I. S. Wittstein, R. C. Ziegelstein, and D. A. Kass In vitro system to study realistic pulsatile flow and stretch signaling in cultured vascular cells Am J Physiol Cell Physiol, September 1, 2000; 279(3): C797 - C805. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Ueno, P. Kanellakis, A. Agrotis, and A. Bobik Blood Flow Regulates the Development of Vascular Hypertrophy, Smooth Muscle Cell Proliferation, and Endothelial Cell Nitric Oxide Synthase in Hypertension Hypertension, July 1, 2000; 36(1): 89 - 96. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
 |
|
 T. V. Bilfinger and G. B. Stefano Human aortocoronary grafts and nitric oxide release: relationship to pulsatile pressure Ann. Thorac. Surg., February 1, 2000; 69(2): 480 - 485. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Parent and M. Lavallee Endothelin-dependent effects limit flow-induced dilation of conductance coronary vessels after blockade of nitric oxide formation in conscious dogs Cardiovasc Res, January 14, 2000; 45(2): 470 - 477. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. M. Malek, S. L. Alper, and S. Izumo Hemodynamic Shear Stress and Its Role in Atherosclerosis JAMA, December 1, 1999; 282(21): 2035 - 2042. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Ziegler, P. Silacci, V. J. Harrison, and D. Hayoz Nitric Oxide Synthase Expression in Endothelial Cells Exposed to Mechanical Forces Hypertension, August 1, 1998; 32(2): 351 - 355. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Dai, O. Tsukurov, M. Chen, J. P. Gertler, and R. D. Kamm Endothelial nitric oxide production during in vitro simulation of external limb compression Am J Physiol Heart Circ Physiol, June 1, 2002; 282(6): H2066 - H2075. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||