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Circulation. 1996;94:1682-1689

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(Circulation. 1996;94:1682-1689.)
© 1996 American Heart Association, Inc.

Articles

Fluid Flow Inhibits Endothelial Adhesiveness

Nitric Oxide and Transcriptional Regulation of VCAM-1

Philip S. Tsao, PhD; Ricardo Buitrago, MD; Jason R. Chan, BS; John P. Cooke, MD, PhD

the Falk Cardiovascular Research Center, Stanford, Calif.

Correspondence to John P. Cooke, MD, PhD, Falk Cardiovascular Research Center, 300 Pasteur Dr, Stanford, CA 94305-5246.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Background In the arterial tree, regions exposed to reduced shear stress (low and/or disturbed flow) are predisposed to atherogenesis. Fluid flow is a potent stimulus for the release of endothelium-derived nitric oxide (NO). Because NO inhibits monocyte–endothelial cell interaction, we speculated that the effects of flow in inhibiting atherogenesis might be mediated in part by NO.

Methods and Results Confluent monolayers of human aortic endothelial cells were exposed to static or fluid flow conditions for 4 hours. The medium was replaced, and cells were then incubated with native LDL (50 µg/mL), oxidized LDL (30 µg/mL), or lipopolysaccharide (LPS) (10 ng/mL) + tumor necrosis factor-{alpha} (TNF-{alpha}) (10 U/mL) for an additional 4 hours. Functional binding assays using THP-1 monocytes were then performed. Superoxide production by human aortic endothelial cells was monitored by lucigenin chemiluminescence, and expression of the adhesion molecules vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 were quantified by flow cytometry. Whereas native LDL had little effect, incubation with either oxidized LDL or LPS/TNF-{alpha} significantly increased superoxide production, nuclear factor-{kappa}B activity, VCAM-1 expression, and endothelial adhesiveness for monocytes. Previous exposure to fluid flow inhibited these sequelae of exposure to cytokines or oxidized lipoprotein. The effect of fluid flow appears to be due in part to shear-induced release of NO, because coincubation with nitro-L-arginine completely abolished these effects of flow. Furthermore, the NO donor PAPA-NONOate and 8-Br-cGMP (but not 8-Br-cAMP) mimicked the effects of flow.

Conclusions Previous exposure to fluid flow decreased cytokine- or lipoprotein-stimulated endothelial cell superoxide production, VCAM-1 expression, and monocyte binding; the effects of flow appear to be due to NO. Flow-mediated NO-dependent regulation of oxidant-responsive transcription may influence the site of a lesion.


Key Words: blood flow • endothelium-derived factors • adhesion molecules • atherosclerosis • free radicals


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
The distribution of atherosclerotic lesions throughout the vascular tree is nonuniform. As early as the 19th century, the great pathologists Rokitansky1 and Virchow2 independently speculated that this nonuniformity was due to local alterations in hemodynamic forces impinging on the vascular tree. Indeed, at sites vulnerable to plaque formation—bends, branches, and bifurcations—unidirectional laminar flow is disturbed, with areas of recirculation characterized by low and fluctuating shear stress.3 4 5 6 7 In these regions of low shear stress, the vulnerability to plaque formation may be a result of enhanced monocyte binding. Adhesive interactions between leukocytes in the vessel wall are less likely to be disrupted under conditions of low shear stress. Furthermore, experimental reductions of blood flow enhance endothelial adhesiveness for monocytes.8 9 This may be in part because low-shear regions exhibit enhanced permeability to macromolecules, including LDL10 11 ; accumulation and modification of LDL in the vessel wall could trigger a cascade of events leading to changes in the chemotactic and adhesive properties of the endothelium.10 11

Tractive forces of fluid flow also modulate the gene expression of endothelial adhesion molecules and cytokines that participate in monocyte binding. In the New Zealand White rabbit, an adhesion molecule homologous to the human VCAM-1 is upregulated by hypercholesterolemia and is expressed at sites of early lesion formation.12 13 The expression of VCAM-1 in a murine endothelial cell line is reduced by 75% after 24 hours of exposure to laminar fluid flow.14 The expression of VCAM-1 is regulated in part by oxidant-responsive transcriptional activation.15 Oxidized LDL and cytokines induce the expression of VCAM-1 via a transcriptional pathway modulated by NF-{kappa}B; this NF-{kappa}B–mediated gene expression can be abrogated by antioxidants.15

Flow is also a potent stimulus for endothelial elaboration of NO.16 17 18 Recent evidence indicates that in addition to its role as a potent vasodilator, NO downregulates oxidative enzyme activity and reduces intracellular oxidative stress.19 20 We therefore propose that flow inhibits VCAM-1 expression by triggering the release of endothelium-derived NO and thereby inhibiting oxidant-responsive transcriptional activation. Accordingly, in this investigation we tested the hypothesis that flow inhibits, in an NO-dependent manner, the effects of oxidized LDL and cytokines in inducing endothelial oxidative stress, to activate NF-{kappa}B–mediated expression of VCAM-1, and to increase endothelial adhesiveness for monocytes.


*    Methods
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 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Endothelial Cell Culture
HAECs (Clonetics), passages 3 through 6, were grown to confluence in endothelial growth medium (Clonetics) on 60-mm culture dishes in a 5% CO2 atmosphere at 37°C. Endothelial growth medium contains epidermal growth factor (10 ng/mL), hydrocortisone (1 µg/mL), bovine brain extract (10 µg/mL), FCS (2%), gentamicin (50 µg/mL), and amphotericin-B (50 ng/mL). These cells have a normal cobblestone phenotype and test positive for factor VIII antigen. THP-1 (ATCC) was cultured in RPMI medium (Life Technologies) containing 10% FCS. Viability of THP-1 cells, as determined by trypan blue exclusion, was >95%.

Fluid Flow
Confluent monolayers were placed in serum-free medium for 1 hour and then exposed to static conditions or flow. Flow was induced by placement of confluent 60-mm culture dishes on a mixing table (Thermylene) rotating at 120 rpm for 4 hours. Compared with the well-defined cone-plate viscometer,21 22 this technique induces qualitatively similar changes in cell alignment, NOx production, and NOS mRNA transcription.23 Fluid flow with the mixing table caused endothelial cells to produce levels of NOx comparable to those attained by the cone-plate viscometer generating a shear stress of 12 dynes/cm2. After 4 hours of flow, NOx levels in the conditioned medium increased by 105% compared with static controls.23

In some experiments, cells were treated with the NOS inhibitors L-NA (100 µmol/L), 8-Br-cGMP (100 µmol/L), or 8-Br-cAMP (100 µmol/L) or the NO donor PAPA-NONOate (100 µmol/L) (Caymen Chemical) in static conditions. PAPA-NONOate is a stable NO donor that dissociates to the free amine and NO according to first-order kinetics at physiological pH (pH 7.4) and has a half-life of 72 minutes. All drugs were incubated with the endothelial cells for 4 hours before addition of lipoproteins or cytokines; then the drugs were removed with fresh HBSS medium. After 4 hours of either flow or static conditions, cells were placed in static conditions and exposed to native LDL (50 µg/mL), oxLDL (30 µg/mL), or LPS (10 ng/mL)+TNF-{alpha} (10 U/mL) (Sigma) for 4 hours.

Lipoprotein Preparation
LDL was isolated by density gradient ultracentrifugation of normal human plasma collected in EDTA (1 mg/mL). The protein fraction was quantified by Lowry assay with BSA as standard. oxLDL was prepared by incubation of LDL (100 µg/mL) in 2 mL F-10 medium containing CuSO4 (10 µmol/L) in a 37°C incubator for 24 hours. BHT was then added to halt the oxidation process. The extent of oxidation was monitored by measurement of TBARS at 550 Å as previously described. Copper oxidation of LDL routinely produced 40 to 60 nmol TBARS/mg LDL.

Adhesion Assay
Monocyte adhesion assays were performed as previously described.23 Briefly, HAECs exposed to the above conditions were washed with HBSS (Irvine Scientific) containing (in mmol/L) CaCl2 2, MgCl2 2, and HEPES 20. Culture dishes were then placed on a rocking platform, and THP-1 cells were incubated with HAECs for 30 minutes, with dishes rotated 120° clockwise every 10 minutes to ensure even distribution of cells. Medium was aspirated and replaced with fresh HBSS to remove nonadherent cells. After a second washing, dishes were returned to the rocker platform for an additional 5 minutes. Medium was again aspirated and replaced with HBSS containing 2% glutaraldehyde. After overnight fixation, adherent cells were quantified by light microscopy.

Flow Cytometry
To detect changes in the expression of endothelial glycoprotein adhesion molecules, we performed flow cytometry using specific monoclonal antibodies to VCAM-1 and ICAM-1. Confluent HAECs were exposed to shear stress or static conditions for 4 hours and then one of the described conditions for an additional 4 hours. Cells were gently detached with 5 mmol/L EDTA. Subsequently, 10% FCS was added to the cell suspension, and the cells were processed for fluorescence analysis by a highly modified dual-laser FACS IV (Becton Dickinson). Nonviable cells were detected by the technique of propidium iodide (1 µg/mL) incorporation. Propidium iodide–positive cells were excluded by electronic gating. In general, viability was judged to be >95%. To assess the expression of endothelial adhesion molecules, cell suspensions (2x106/mL) were incubated for 25 minutes on ice with anti-human VCAM-1 monoclonal antibody (1:100) (Endogen, Inc), anti–human ICAM-1 monoclonal antibody (Genzyme), or an isotype-matched control antibody. Nonspecific binding was blocked by incubation of the cells with human serum. Subsequently, the cells were stained with goat anti-mouse monoclonal antibody conjugated with Texas Red (0.5 mg/mL) (Molecular Probes).

Superoxide Production
Superoxide anion production by HAECs was monitored by modification of the method previously reported by Pagano et al.24 After undergoing the described protocols, HAECs were detached from culture dishes with EDTA, washed with PBS, and resuspended in HBSS containing lucigenin (250 µmol/L). In some experiments, the copper-zinc SOD inhibitor diethyldithiocarbamate (10 mmol/L) was added to enhance superoxide production. Superoxide was monitored in a Turner Designs luminometer for 1 minute with 30-second delay. The relative specificity of lucigenin-induced chemiluminescence by superoxide anion is demonstrated by the lack of effect of scavengers of hydrogen peroxide and by the potent effect of 4,5-dihydroxy-1,3-benzene disulfonic acid (100 µmol/L), an intracellular scavenger of superoxide, in blocking the signal.

Electrophoretic Mobility Shift Assay
Nuclear extracts were prepared as described by Dignam et al.25 Cells from the appropriate conditions were harvested, centrifuged to pellet cells, and washed in ice-cold PBS. The remaining steps were performed on ice or at 4°C. Cells were resuspended in buffer A (in mmol/L: PMSF 0.5, HEPES 10 [pH 7.8], MgCl2 1.5, KCl 10, and DTT 0.5) containing 0.1% Nonidet P-40 and disrupted in a tight-fitting Dounce homogenizer. Nuclei were then pelleted by centrifugation (25 000g, 20 minutes, 4°C). Crude nuclei were resuspended in buffer C (in mmol/L: HEPES 20 [pH 7.8], NaCl2 0.42, MgCl2 1.5, EDTA 0.2, DTT 0.5, and PMSF 0.5; 25 vol% glycerol) and incubated on ice for 30 minutes. The mixture was then spun at 25 000g for 20 minutes at 4°C, the supernatant was collected, and protein was quantified. Nuclear proteins were stored at -85°C until gel shift assay. Binding reactions were carried out by mixing nuclear proteins with a double-stranded oligonucleotide corresponding to the published NF-{kappa}B–binding domain (5'-AGT TGA GGG GAC TTT CCC AGG C). Reactions were performed with 32P-labeled DNA oligonucleotide in the presence of (in mmol/L) MgCl2 1, EDTA 0.5, DTT 0.5, NaCl 50, and Tris-HCl 10 (pH 7.5) and 0.05 µg/mL polydeoxyinosinic-deoxycytidylic acid in 20 vol% glycerol. Samples were separated on a 4% nondenaturing polyacrylamide gel and exposed to x-ray film overnight.

Data Analysis
Data are expressed as mean±SEM. Comparisons of multiple means were made by ANOVA followed by a Fisher's protected least significant difference test. A value of P<.05 was accepted as statistically significant.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Effect of Flow on Endothelial Adhesiveness
Incubation of HAECs with native LDL for 4 hours had little effect on endothelial adhesiveness for THP-1 monocytes compared with control cells not exposed to lipoproteins (Fig 1Down). In contrast, exposure of HAECs to oxLDL (50 µg/mL) increased THP-1 binding to HAECs by 58±14% compared with control cells (P<.05) (Fig 2Down). In either case, previous exposure to either flow or the NO donor PAPA-NONOate markedly reduced monocyte binding. The cGMP analogue 8-Br-cGMP had no significant effect on endothelial adhesiveness in native LDL–treated cells, whereas it had a modest effect on monocyte–endothelial cell interactions induced by oxLDL.



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  		Figure 1. Histogram illustrating the inhibitory effects of flow and PAPA-NONOate (PAPA-NO) on monocyte binding to HAECs exposed to native LDL (50 µg/mL). Incubation of HAECs with native LDL did not increase adhesiveness compared with control cells. The inhibitory effect on monocyte binding by flow and NO donor was not mimicked by the stable analogue 8-Br-cGMP. Bars represent mean±SEM. *P<.05 vs static.



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  		Figure 2. Flow decreases endothelial adhesiveness induced by oxLDL (30 µg/mL). Exposure of HAECs to oxLDL increased THP-1 binding compared with control cells. Previous exposure to flow or, in static conditions, NO donor or 8-Br-cGMP inhibited the monocyte adhesion induced by oxLDL. Bars represent mean±SEM. *P<.05; **P<.01 vs static.

Fig 3Down illustrates the more potent effect of LPS and the cytokine TNF-{alpha} to increase monocyte adhesion (210±18%) compared with cells treated with vehicle (P<.01). Prior exposure to flow protected the endothelial cells from this agonist-induced adhesion (94±12% of control; P<.01 from static+LPS/TNF-{alpha}). The NO donor PAPA-NONOate mimicked the effect of flow; in contrast, 8-Br-cGMP had very little effect. Incubation of HAECs with the cAMP analogue 8-Br-cAMP (100 µmol/L) for 4 hours augmented monocyte binding induced by oxLDL or cytokines (data not shown).



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  		Figure 3. Flow decreases endothelial adhesiveness induced by LPS/TNF. Exposure of HAECs to LPS 10 ng/mL+TNF-{alpha} 10 U/mL significantly increased THP-1 adhesion to HAECs compared with control cells. The inhibitory effect of flow was mimicked by NO donor but not the stable cGMP analogue 8-Br-cGMP. Bars represent mean±SEM. *P<.05; **P<.01 vs static.

Effect of Flow on Endothelial Adhesion Molecules
To determine whether flow exerted its effect on monocyte binding by altering the expression of endothelial adhesion molecules, we performed flow cytometric analysis of HAECs for ICAM-1 and VCAM-1 that had been stimulated with oxLDL or LPS+TNF-{alpha}. As illustrated in the representative tracing shown in Fig 4Down, oxLDL (30 µg/mL) increased expression of ICAM-1 in HAECs after 4 hours, as did the combination of LPS+TNF-{alpha} over basal expression. Previous exposure to flow had no effect on the surface expression of ICAM-1 to either agonist. Likewise, the NO donor PAPA-NONOate in static conditions did not affect ICAM-1 levels.



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  		Figure 4. Flow cytometric analysis of HAECs using antibody to ICAM-1. Left, Representative plots of Static (no oxLDL, dotted line), Static+oxLDL (30 µg/mL; solid line), and Flow+oxLDL (broken line). Right, Representative plots of Static (no LPS/TNF; dotted line), Static+LPS/TNF (LPS 10 ng/mL, TNF-{alpha} 10 U/mL; solid line), and Flow+LPS/TNF (broken line). A total of 10 000 cells were analyzed for each condition. Relative cell numbers (vertical) and fluorescence intensity on log10 scale (horizontal) are indicated.

Flow cytometric analysis revealed that HAECs express lower levels of VCAM-1 under basal conditions than of ICAM-1 (Fig 5Down). Exposure of endothelial cells to oxLDL (P<.05) or LPS/TNF-{alpha} (P<.01) markedly elevated VCAM-1 expression compared with cells exposed to vehicle. Induced expression of VCAM-1 by both oxLDL and cytokines was significantly suppressed by previous exposure to flow. The effect of flow is largely due to shear stress–induced NO production, since exposure of HAECs to L-NA (1x10-4) abrogated the effect of flow on VCAM-1 (Fig 6Down). Furthermore, the effect of flow was mimicked by the NO donor PAPA-NONOate in the absence of shear stress (Fig 6Down). Data from five separate experiments are summarized in Table 1Down.



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  		Figure 5. Flow cytometric analysis of HAECs using antibody to VCAM-1. Conditions are the same as in Fig 4Up. A total of 10 000 cells were analyzed for each condition. Relative cell numbers (vertical) and fluorescence intensity on log10 scale (horizontal) are indicated.



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  		Figure 6. Flow cytometric analysis of HAECs demonstrating the effect of NO in the flow-mediated inhibition of VCAM-1 expression. Left, Representative plots of Static (no oxLDL, dotted line), Static+oxLDL (30 µg/mL; solid line), Flow+oxLDL+L-NA (broken line), and Static+oxLDL+PAPA-NONOate (PAPA-NO, broken gray line). Right, Representative plots of Static (no LPS/TNF, dotted line), Static+LPS/TNF (30 µg/mL; solid line), Flow+LPS/TNF+L-NA (broken line), and Static+LPS/TNF+PAPA-NONOate (broken gray line). A total of 10 000 cells were analyzed for each condition. Relative cell numbers (vertical) and fluorescence intensity on log10 scale (horizontal) are indicated.


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  		Table 1. Results of Flow Cytometry: Percent of Cells Expressing Adhesion Molecules

Effect of Flow on Oxidant-Sensitive Transcription
VCAM-1 has been shown to be an oxidant-responsive gene in endothelial cells. Therefore, to characterize the oxidative stress of endothelial cells exposed to oxidized LDL or cytokines, we monitored endothelial superoxide anion production using lucigenin chemiluminescence. Whereas native LDL had minimal effect, oxLDL significantly increased endothelial superoxide production. LPS+TNF caused a further increase in the generation of superoxide anion (Table 2Down). Previous exposure to flow opposed the effect of oxidized LDL or cytokines in increasing endothelial superoxide anion generation. The reduction in superoxide anion appears to be a sustained effect of NO, since media containing PAPA-NONOate or conditioned media after shear stress were removed before superoxide measurements. The addition of the NOS inhibitor L-NA 15 minutes before superoxide measurements had no effect on the shear stress– or NO donor–mediated reduction in superoxide production. Therefore, it is unlikely that the effect of NO in this system is simply due to a scavenging of superoxide anions but rather to a more chronic effect on the endothelial generation of superoxide anion.


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  		Table 2. Superoxide Production by HAECs

Molecular cloning of VCAM-1 has provided evidence for NF-{kappa}B–binding domains in the promoter region of this gene. Since NF-{kappa}B has been reported to respond to oxidative stress, we examined whether NO may have an effect on NF-{kappa}B activity. Nuclear extracts from HAECs were isolated, and gel-shift analysis was performed with an oligonucleotide containing the putative NF-{kappa}B binding site. As shown in Fig 7Down, both oxLDL (lane 2) and LPS+TNF-{alpha} (lane 5) induced activation of NF-{kappa}B. This effect was greatly reduced in cells previously exposed to flow (lanes 3 and 6). Exposure of HAECs to PAPA-NONOate in static conditions had a similar effect of inhibiting NF-{kappa}B activity. Furthermore, the effect of flow in inhibiting NF-{kappa}B activation was mimicked by PAPA-NONOate in the absence of shear stress.



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  		Figure 7. Electrophoretic mobility shift assay showing the effect of flow on the activation of NF-{kappa}B. Nuclear extracts were isolated from HAECs that underwent static or flow conditions for 4 hours and then were stimulated with either oxLDL (30 µg/mL) or LPS/TNF (LPS 10 ng/mL, TNF 10 U/mL) under static conditions. Four separate experiments produced qualitatively similar results.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
The salient findings of this study are that (1) oxidized LDL or cytokines induce endothelial elaboration of superoxide anion, elevate NF-{kappa}B binding activity, augment VCAM-1 expression, and increase endothelial adhesiveness for monocytes; (2) prior exposure of the endothelial cells to flow abrogates these lipid- or cytokine-induced alterations; (3) the effects of flow appear to be partially mediated by NO, since they are blocked by NOS antagonists and mimicked by NO donors; and (4) the effects of NO are likely to be mediated in part by non-cGMP pathways, because 8-Br-cGMP only partially mimicked the effects of flow on NO donors.

Oxidant stress may play a central role in precipitating endothelial cell–monocyte interaction in atherogenesis. Recent evidence indicates the existence of oxidant-responsive genes encoding proteins that modulate endothelial adhesiveness.15 26 We hypothesized that flow-mediated NO-dependent regulation of intracellular oxidative enzyme activity would chronically inhibit oxidant-responsive transcriptional pathways that regulate endothelial adhesiveness. The present investigation confirms this hypothesis and suggests a mechanism by which hemodynamic forces may influence the site of a lesion.

Early in the course of atherogenesis, hypercholesterolemia induces an alteration in endothelial function. After 8 weeks of hypercholesterolemia, there is a marked increase in superoxide anion generation by the endothelium of the rabbit thoracic aorta.27 Endothelial generation of superoxide anion may promote adhesion. Leukocyte adherence and emigration in cat mesenteric venules are enhanced by LPS, an effect that is significantly reduced by administration of SOD.28 Injection of human oxLDL elicits rolling and adhesion of circulating leukocytes in arterioles and postcapillary venules of hamsters.29 This adhesion is significantly attenuated when the animals are pretreated with bovine copper-zinc-SOD or heparin to release extracellular SOD. Increasing evidence indicates that endothelium-derived NO may act as an endogenous regulator of oxidative enzyme activity. In normocholesterolemic rabbit thoracic aorta, there is a basal production of superoxide anion by nonendothelial cells.24 Exogenous NO donors significantly reduce the generation of superoxide anion. Administration of an NO precursor, benzoyl-L-arginine ethyl ester, decreases alloxan-stimulated superoxide production by rabbit aorta, an effect that is reversed by the NOS antagonist NG-monomethyl-L-arginine.30

The mechanism by which NO reduces oxidative stress remains undefined. It is unlikely that NO exerts this effect by directly scavenging superoxide anion, because the product of this reaction, peroxynitrate, is a highly reactive free radical that itself can initiate lipid peroxidation.31 However, it is possible that NO reacts directly with lipid peroxyl radicals, thereby disrupting the chain of autocatalytic reactions involved in the oxidation of intracellular lipid. Indeed, NO donors inhibit copper-catalyzed oxidation of LDL cholesterol in vitro.32 Second, NO may directly inhibit the generation of oxygen-derived free radicals by oxidative enzymes. By virtue of its ability to nitrosylate proteins, NO may alter their behavior or activity. Clancy and colleagues19 found that when neutrophils are exposed to NO, their ability to generate superoxide anion is limited, apparently because of a direct effect of NO on the multimeric oxidative enzyme NADPH oxygenase, since prior incubation of its membrane-bound component with NO inhibits the assembly (and activation) of the enzyme. These observations are concordant with the observation of Yates and colleagues,33 who found that autocrine NO regulates macrophage oxidation of LDL cholesterol. When mouse peritoneal macrophages were exposed to TNF-{alpha} and interferon-{gamma} to induce NO elaboration, their ability to oxidize human LDL cholesterol was reduced; this effect was reversed by an antagonist of NOS. Similarly, in the present study, the effect of flow in reducing superoxide anion generation by endothelial cells was NO dependent. Taken together, these studies indicate that NO reduces oxidant stress. The critical role of NO in modulating endothelial generation of oxygen-derived free radicals has also been demonstrated by Niu and coworkers,20 who used intracellular fluorophores responsive to intracellular increase in oxygen-derived free radicals to demonstrate that antagonists of NOS precipitated oxidative stress. Similarly, in the present study, the effect of flow in reducing superoxide anion generation by endothelial cells was largely NO dependent.

The effect of NO in reducing the generation of reactive oxygen species may explain its repression of NF-{kappa}B–mediated gene expression. Antioxidants such as n-acetylcysteine and pyrrolidine dithiocarbamate are known to inhibit the dissociation of NF-{kappa}B from its inhibitor, I-{kappa}B{alpha}.34 35 In cultured human umbilical vein endothelial cells, interleukin-1–induced expression of VCAM-1 is selectively antagonized by exposure of the cells to pyrrolidine dithiocarbamate.15 NO donors mimic the effect of antioxidants in suppressing NF-{kappa}B–mediated gene expression. The activation of NF-{kappa}B by TNF-{alpha} in cultured human saphenous vein endothelial cells was blocked by exogenous NO, whereas NO donors had little effect on other nuclear binding proteins (activator protein–1 and GATA).36 Immunoprecipitation studies indicated that exogenous NO stabilized the NF-{kappa}B/I-{kappa}B{alpha} complex. Moreover, exogenous NO enhanced the transcription of I-{kappa}B{alpha} but not that of the NF-{kappa}B subunits p50 and p65. These observations most likely explain previous findings that exogenous NO donors inhibit interleukin-1–stimulated VCAM-1 expression and monocyte adhesion.37

We found that the effects of flow in inhibiting NF-{kappa}B–mediated VCAM-1 expression and endothelial adhesiveness could be accounted for by elaboration of endogenous NO. The effects of flow are mimicked by NO donors, whereas the cGMP analogue had only a partial effect. This implies that NO exerts its effects in part by cGMP-independent pathways, in agreement with the above studies. Similarly, Zeiher et al38 found that cGMP analogues did not mimic the effect of exogenous NO donors in inhibiting NF-{kappa}B activity and monocyte chemotactic protein-1 expression by NO. This is in contrast to the in vivo work of Kurose and colleagues,39 who found that the enhanced adhesion of leukocytes to vessels perfused by nitro-L-arginine methyl ester could be completely reversed by 8-Br-cGMP. This discrepancy may be due to other effects of the cGMP analogue in vivo (eg, vasodilation with attendant increases in flow or direct effects on monocyte adhesive proteins). However, Kuchan and Frangos40 have also demonstrated that the negative regulation of endothelin-1 by flow is dependent on NO and is mimicked by 8-Br-cGMP. The mechanism of action may differ from the present study, since the endothelin promoter does not appear to contain an NF-{kappa}B consensus sequence.

The effect of NO on NF-{kappa}B and I-{kappa}B{alpha}, however, may not totally explain the effects of flow on VCAM-1 expression or monocyte binding. VCAM-1 promoter activity has been demonstrated to be under the complex control of both stimulatory and inhibitory transactivating factors. Moreover, deletion analysis has demonstrated that NF-{kappa}B–binding activity is important for optimal stimulation of the VCAM-1 promoter but is not essential for VCAM-1 transcription. In addition, NF-{kappa}B activity does not fully explain the effect on adhesion molecule expression, since ICAM-1 expression was only minimally affected by NO. Other nuclear binding proteins may dominate in cytokine- or lipid-induced ICAM-1 expression. Furthermore, the ICAM-1 promoter contains the SSRE GAGACC first defined by Resnick et al.41 This SSRE may be responsible for the positive regulation by shear stress of ICAM-1 that we observed in this study. This same SSRE is found in the promoter region of a number of genes regulated by flow, including endothelial cell NOS.42 43 Therefore, mechanisms independent of NO may contribute to the effects of flow on endothelial adhesiveness. For example, flow enhances the release of prostacyclin, which might also contribute to the flow-induced inhibition of endothelial adhesion.44 45

It is interesting to note that the effects of fluid flow seen in the present study persist even after cessation of flow. In contrast, NO production quickly falls to baseline levels after cessation of fluid flow. Kanai et al46 used a porphyrinic microsensor to detect NO elaborated by cultured endothelial cells in response to fluid flow. With cessation of the flow stimulus, NO concentration in the conditioned medium declined at a rate that would be predicted by oxidative degradation of the NO released into the medium during flow. This observation suggests that NO elaboration ceases instantaneously with discontinuation of the flow stimulus. However, in the present study, the effect on endothelial adhesiveness of flow-induced NO release persisted for at least 4 hours. We speculate that this persistent effect may be due to the inactivation by NO of oxidative enzyme activity, which probably has a long time constant, given the extraordinary affinity of NO for heme proteins.

Flow-stimulated NO also has acute effects on monocyte–endothelial cell interaction, as well as the more chronic effects on gene expression. Monocyte adherence to endothelial cells in culture is inhibited by administration of NO with a time course that implies an effect of adhesion pathways on signal transduction.44 We have also shown that the adherence of monocytoid cells to bovine aortic endothelial cells is inhibited by brief (ie, 15-minute) exposure to NO donors or increases in endogenous NO in the absence of any changes in VCAM-1 or ICAM-1 expression.23 The acute and chronic effects of NO on endothelial adhesiveness may play an important role in atherogenesis. The effect of habitual exercise in inhibiting atherosclerotic lesion formation47 48 may be due in part to the effect of exercise in enhancing the vascular expression of endothelial cell NOS and elaboration of NO.49 Similarly, the predisposition to lesion formation at sites of branching may be due in part to the reduced elaboration of NO at these sites.50 This speculation is supported by our previous observations that enhancement of vascular NO activity in the hypercholesterolemic rabbit inhibits endothelial adhesiveness for monocytes and reduces lesion formation.51 52 53 In contrast, chronic administration of NOS antagonists enhances endothelial adhesiveness and increases monocyte accumulation in the vessel wall.53 54 55 Similar effects of NO on endothelial cell–neutrophil interactions have been observed in hypercholesterolemia.56 Taken together, these studies implicate NO as an endogenous antiatherogenic molecule, which exerts its effects in part via its modulation of oxidant-responsive transcriptional pathways.

To summarize, this investigation reveals that exposure to flow decreases cytokine- and lipoprotein-stimulated endothelial cell superoxide production, VCAM-1 expression, and monocyte binding. The effects of flow are abolished by the NOS antagonist L-NA, whereas in the absence of flow, an exogenous NO donor mimics the effect of endothelial shear stress. Flow-mediated NO-dependent regulation of oxidant-responsive transcription may be a critical determinant of the site of a lesion.


*    Selected Abbreviations and Acronyms
 
HAEC = human aortic endothelial cell
ICAM-1 = intercellular adhesion molecule-1
L-NA = nitro-L-arginine
LPS = lipopolysaccharide
NF-{kappa}B = nuclear factor-{kappa}B
NO = nitric oxide
NOS = NO synthase
oxLDL = copper-oxidized LDL
PAPA-NONOate = 1-propanamine,3-(2-hydroxy-2-nitroso-propylhydrazino)
SOD = superoxide dismutase
SSRE = shear stress–responsive element
TBARS = thiobarbituric acid–reactive substances
THP-1 = human monocytoid cells
TNF-{alpha} = tumor necrosis factor-{alpha}
VCAM-1 = vascular cell adhesion molecule-1


*    Acknowledgments
 
This work was supported in part by a grant from the National Heart, Lung, and Blood Institute (1R01-HL-58638) and was done during the tenure of a Grant-in-Aid Award from the American Heart Association and Sanofi Winthrop. Dr Tsao is the recipient of a National Service Research Award (1F32-HL-08779). Dr Cooke is a recipient of the Vascular Academic Award from the National Heart, Lung, and Blood Institute (1K07-HC-02660) and is an Established Investigator of the American Heart Association. The authors appreciate the advice and assistance of Jack Sun and Susan Alpert (flow cytometry experiments), Jay Oakes and Andrew Weyrich (electrophoretic mobility shift assay), and Mike Gillette (manuscript preparation).

Received August 21, 1995; revision received April 11, 1996; accepted April 16, 1996.


*    References
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
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 *References
 

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