This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tsao, P. S. Articles by Cooke, J. P. Search for Related Content PubMed PubMed Citation Articles by Tsao, P. S. Articles by Cooke, J. P. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB NITRIC OXIDE

(Circulation. 1995;92:3513-3519.)
© 1995 American Heart Association, Inc.

Articles

Exposure to Shear Stress Alters Endothelial Adhesiveness

Role of Nitric Oxide

Philip S. Tsao, PhD; Neil P. Lewis, MD, PhD; Susan Alpert, PhD; John P. Cooke, MD, PhD

From the Section of Vascular Medicine, Division of Cardiovascular Medicine, Stanford University, Stanford, Calif.

Correspondence to John P. Cooke, MD, PhD, Director, Section of Vascular Medicine, Division of Cardiovascular Medicine Stanford University, 300 Pasteur Dr, Stanford, CA 94305-5246.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background Shear stress increases the release of nitric oxide (NO) by endothelial cells (ECs). We and others have provided evidence that endothelium-derived NO inhibits monocyte adhesion to the vessel wall. We therefore hypothesized that previous exposure to shear stress would inhibit endothelial adhesiveness for monocytes by virtue of its effect to increase NO release.

Methods and Results Confluent monolayers of bovine aortic endothelial cells, human aortic endothelial cells, or human venous endothelial cells were exposed to laminar fluid flow. Culture media were collected for measurement of NO (by chemiluminescence) and the prostacyclin metabolite 6-keto-prostaglandin F₁. NO_x and 6-keto-prostaglandin F₁ accumulated in the conditioned medium during laminar fluid flow from 30 minutes to 24 hours in a time-dependent fashion. In another set of studies, ECs previously exposed to flow or to static conditions were washed with Hanks' buffer and exposed to THP-1 cells for 30 minutes. Adherent cells were counted by microscopy. Previous exposure to flow reduced endothelial adhesiveness for monocytes by 50% (P<.05). The effect of flow on endothelial adhesiveness occurred within 30 minutes. This effect was abrogated by nitro-L-arginine (an antagonist of NO synthesis), as well as by tetraethylammonium ion (an antagonist of the flow-activated potassium channel); the effects of these inhibitors were reversed by the NO donor SPM-5185. Although the cyclo-oxygenase inhibitor indomethacin totally inhibited the flow-induced production of prostacyclin by ECs, it minimally affected adherence of THP-1 cells. The early effect of flow on endothelial adhesiveness was not mediated by alterations in the expression of the endothelial adhesion molecules VCAM-1 or ICAM-1 as assessed by fluorescent activated cell sorting.

Conclusions Shear stress alters endothelial adhesiveness for monocytes; at early time points, this effect is largely due to flow-stimulated release of NO and, to a lesser extent, prostacyclin. This effect of flow occurs within 30 minutes and is probably due to alterations in the signal transduction or activation state (rather than the expression) of endothelial adhesion molecules.

Key Words: atherosclerosis • endothelium-derived factors • leukocytes • flow

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
As blood flows through a conduit vessel, vasodilation occurs in proportion to the increment in endothelial shear stress.^{1 2} This phenomenon requires the integrity of the endothelium and in most conduit vessels is due to release of EDRF.^{3 4 5 6} EDRF is now known to be NO, derived from the metabolism of L-arginine NOS.^{7 8 9} In some circulations, other substances (eg, prostacyclin or endothelium-derived hyperpolarizing factor) may contribute to flow-mediated vasodilation.^{10 11 12}

Endothelium-derived NO not only is a potent vasodilator but also inhibits leukocyte-EC interaction.^{13 14 15} The adhesion of mononuclear cells to cultured BAECs is inhibited by the addition of genuine NO to the medium.¹⁴ The ex vivo adhesion of normal mononuclear cells to the endothelium of hypercholesterolemic rabbit thoracic aorta is modulated by changes in endothelium-derived NO activity; dietary arginine enhances NO activity and inhibits monocyte adhesion.¹⁶ In contrast, chronic administration of the NOS antagonist L-NA reduces NO synthesis in the normocholesterolemic rabbit thoracic aorta and increases the adhesiveness of the endothelium for monocytes.¹⁶ Accordingly, the present investigation was designed to test the hypothesis that flow-stimulated release of NO may modulate endothelial adhesiveness for monocytes and to investigate the mechanism of this effect.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
EC Culture
BAECs (passages 7 to 10) were grown to confluence on 150- or 60-mm-diameter culture dishes in DMEM with 10% calf serum (Life Technologies). DMEM was composed of (in mmol/L) NaCl 109, KCl 5.5, CaCl₂ 1.8, MgSO₄ 1.7, NaHCO₃ 51, NaH₂PO₄ 1.0, Fe(NO₃)·9H₂O 3.6x10^-3, glucose 30, sodium pyruvate 1.0, and phenolsulfonphthalein 4.2x10^-2. One day after achieving confluence, media were changed to DMEM plus 1% calf serum for 24 hours. Media were then changed to defined serum free media for the experiments outlined below.

HAECs and HUVECs (Clonetics) were grown to confluence in EGM (Clonetics) supplemented with 2% fetal calf serum in 35-mm dishes in a 5% CO₂ atmosphere at 37°C.

THP-1 cells (American Type Culture Collection) were cultured in RPMI media (Life Technologies) containing 10% fetal calf serum. Viability of cells used, as determined by trypan blue exclusion, was generally >95%.

Experimental Protocol
To expose ECs to shear stress, confluent monolayers grown on 150-mm-diameter culture dishes were placed in a cone-plate viscometer device.^{17 18} This shear stress device consists of a cone that rotates above a stationary base plate (the 150-mm culture dish) containing the cultured ECs. The Plexiglas cone makes an angle of 0.5° with the culture plate and is coupled to a variable motor. The angular velocity of the cone is precisely adjusted by a motor controller so as to expose the ECs to physiological levels of laminar fluid flow. Analysis of the flow field reveals that this cone-plate system induces a steady laminar flow, which can be precisely regulated in the range of 0 to 40 dyne/cm².

In subsequent experiments, we used a mixing table to induce shear stress. In comparison to the cone-plate viscometer, this technique induces qualitatively similar changes in cell alignment, NO production, and monocyte adhesion and has the added virtues of simplicity and the ability to conduct experiments with multiple conditions in parallel. Shear stress was induced by placing confluent monolayers grown on 60-mm culture dishes on the mixing table (Thermylene) rotating at 120 revolutions per minute for 0, 0.5, 1, 2, 4, 6, 12, or 24 hours. As in previous studies, shear stress induced ECs to align in the direction of flow, an effect clearly visible after 12 hours. The shear stress–induced generation of NO by this technique was compared with that of the cone-plate viscometer. Confluent endothelial monolayers grown on 150-mm-diameter culture dishes were placed in the cone-plate viscometer device. After exposure of BAECs to varying amounts of defined shear stress for 4 hours, medium was collected for measurement of NO_x. Fluid flow using the mixing table (120 revolutions per minute) caused BAECs to produce levels of NO_x comparable to those attained using the cone-plate viscometer to generate a shear stress of 12 dyne/cm².

In some experiments, cells were treated with the NOS inhibitor L-NA (10^-4 mol/L), the cyclo-oxygenase inhibitor indomethacin (10^-6 mol/L), the K⁺ channel blocker TEA (10^-5 mol/L), and/or the NO donor SPM-5185 for 2 hours before and during shear stress. Media were collected for measurement of NO_x and prostacyclin accumulation, and the ECs were harvested for isolation of total RNA.

Measurement of NO and Prostacyclin Production
Samples of the medium (100 µL) were collected from EC cultures during exposure to shear stress or static conditions and then analyzed for NO_x. NO_x levels were measured with a commercially available chemiluminescence apparatus (Dasibi model 2108) after sample reduction in boiling acidic V(III) (vanadium) at 98°C.^{16 19} Boiling acidic vanadium reduces NO₂^- and NO₃^- to NO, which reacts with ozone to generate light. Signals from the photomultiplier tube were analyzed by a computerized integrator and quantified as areas under the curve. Standard curves for NO₂/NO₃ were linear over the range of 50 pmol to 10 nmol.

Prostacyclin release into the conditioned medium was detected by measuring its stable breakdown product 6-keto-PGF₁ by commercially available ELISA (Amersham).

Northern Blot Analysis
ECs exposed to shear stress or static conditions were lysed in acid guanidinium isothiocyanate solution, and total RNA was isolated using phenol extraction. Total RNA was denatured by heating, size-fractionated on a 1.5% agarose/2.2 mol/L formaldehyde gel, and then transferred to a nylon membrane. Hybridizations were performed overnight at 42°C with a ³²P-labeled, random-primed full-length cDNA probe for bovine constitutive NOS (provided by Thomas Michel) or human GAPDH (American Type Culture Collection), in 50% formamide, 10 µg/mL sheared salmon sperm DNA in 6x SSC, 5x Denhardt's solution, and 0.5% SDS. Blots were then washed twice in 2x SSC/0.1% SDS for 15 minutes at room temperature and then twice in 0.4x SSC/0.1% SDS for 15 minutes at 65°C. Bands were visualized by standard autoradiography techniques.

Monocyte Adhesion Assay
In another set of experiments, BAECs, HAECs, or HUVECs previously exposed to flow or static conditions for 0.5, 2, or 4, 12, or 24 hours were washed with HBSS (Irvine Scientific) containing 2 mmol/L Ca²⁺, 2 mmol/L Mg²⁺, and 2 mmol/L HEPES. Culture dishes were then placed on a rocking platform, and endothelial adhesiveness for THP-1 cells (resuspended in HBSS at a concentration of 1.5x10⁶ mL^-1) was assessed. THP-1 cells were incubated with the ECs for 30 minutes, at which time the medium was aspirated and replaced by fresh binding medium to remove nonadherent cells. After a second washing, the dishes were returned to the rocker platform for an additional 5 minutes. Medium was removed and replaced with binding medium containing 2% gluteraldehyde (Sigma). After overnight fixing, adherent cells were quantified by microscopy.

FACS Analysis
To detect changes in the expression of glycoprotein adhesion molecules known to mediate monocyte-EC interaction, we performed fluorescence activated cell sorting using specific mAb against human VCAM-1 and human ICAM-1. Confluent HAECs or HUVECs were exposed to shear stress or static conditions for 2 hours. For flow cytometry analysis, HAECs and HUVECs were gently detached with 5 mmol/L EDTA. Subsequently, 10% fetal calf serum was added to the cell suspension. To assess the expression of endothelial adhesion molecules, cell suspensions (2x10⁵ mL^-1) were incubated for 25 minutes on ice with anti-human VCAM-1 mAb (1:100) (Endogen, Inc), anti-human ICAM-1 mAb (0.5 mg) (Genzyme), or an isotype-matched control antibody in HBSS. Nonspecific binding was blocked by incubation of the cells with human serum. Subsequently, the cells were stained with goat anti-mouse mAb conjugated with Texas red dye (0.5 mg/mL) (Molecular Probes). LPS (5 µg/mL)-treated cells were used as positive controls for VCAM-1 and ICAM-1 expression. Cells were processed for fluorescence analysis using a highly modified dual-laser FACS IV (Becton Dickinson Co). Nonviable cells were detected by the addition of 1 mg/mL PI. PI-positive cells were excluded by electronic gating. In general, viability was judged to be >95%.

Drugs
The following drugs were used: L-NA, indomethacin (Sigma), TEA chloride (Kodak), and nitro-L-amino-ornithine (donated by Dr Dan Pollart, SRI, Menlo Park, Calif). L-NA was dissolved in 50% ethanol and diluted for use in experiments to a final concentration of 10^-5 mol/L L-NA and 0.1% ethanol. Due to concern regarding the effect of the vehicle, we repeated experiments with this antagonist using L-NA that had been dissolved in water heated to 42°C overnight. All other agents were soluble in distilled water or physiological saline solution. SPM 5185 was generously donated by Dr Martin Feelisch (Schwarz-Pharma). SPM 5185 is a nitrovasodilator that is rapidly biotransformed (half-life of 5 minutes in vivo with intravenous administration). The active metabolites have considerably longer half-lives and are the likely precursors of NO formation in vascular tissue.

Statistical Analysis
Data are expressed as mean±SEM. Comparisons of multiple mean values were made by ANOVA followed by a Bonferroni test. P<.05 was accepted as statistically significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Effect of Flow on NO and Prostacyclin Production
In static conditions, BAECs produce a basal amount of NO, which is maintained over the observed 24-hour period. Exposure of BAECs to shear stress increased production of NO compared with cells exposed to static conditions (Fig 1A). We observed similar effects of flow to stimulate NO release from HAECs and HUVECs (n=3 for both cell types; data not shown). Shear stress also caused a time-dependent increase in the production of prostacyclin by BAECs (Fig 1B).

View larger version (18K): [in this window] [in a new window]   Figure 1. Plots showing that shear stress increases the elaboration of prostacyclin and NO. A, Samples of the conditioned medium from BAECs exposed to shear stress or static conditions were measured at time points between 0 and 24 hours. NOx levels were measured by chemiluminescence. NOx levels were increased by shear stress in a time-dependent fashion. B, Prostacyclin release into the conditioned medium was detected by measuring its stable breakdown product 6-keto-PGF1 by ELISA. Shear stress increased the elaboration of prostacyclin in a time-dependent fashion.

Northern blot analysis of mRNA from BAECs exposed to shear stress revealed an increase in the 4.8-kb message for endothelial NOS after 6 hours of shear stress (data not shown). Message levels for the constitutively expressed gene GAPDH remained unchanged. Exposing BAECs to shear stress led to an immediate increase in NO synthesis; in contrast, induction of NOS message occurred much later.

Effect of Flow on Endothelial Adhesiveness
THP-1 monocytes adhered to BAECs exposed to static conditions. In contrast, monocyte binding to ECs exposed to shear stress was significantly reduced (Fig 2). We observed similar effects of shear stress on the endothelial adhesiveness of HAECs and HUVECs (n=3 for both cell types; data not shown). This alteration in endothelial adhesiveness was observed in BAECs exposed to as little as 0.5 hour of shear stress. In comparison with ECs exposed to the static condition, those exposed to shear stress demonstrated a time-dependent reduction in endothelial adhesiveness; at 30 minutes, 1 hours, and 2 hours, there were reductions of 28%, 36%, and 53%, respectively, in THP-1 cells bound per high-power field. A duration of shear stress greater than 2 hours did not further reduce endothelial adhesiveness (Fig 2). Exposure of BAECs to 12 dyne/cm² using the cone-plate viscometer technique for 4 hours produced similar inhibition of monocyte adhesion.

View larger version (39K): [in this window] [in a new window]   Figure 2. Histogram illustrating alterations in endothelial adhesiveness with shear stress. ECs were exposed to static or shear stress conditions for 2 to 24 hours. Subsequently, cells were washed with HBSS and incubated with THP-1 cells (1.5x106 mL-1). After 30 minutes, the medium was replaced with fresh binding medium to remove nonadherent cells. Subsequently, the adherent cells were fixed and quantified by videomicroscopy. Exposure to shear significantly inhibited endothelial adhesiveness for THP-1 cells at all time points.

To determine whether the shear stress-induced alteration in endothelial adhesiveness was due to an autocrine effect of NO released from BAECs, in some experiments cells were preincubated with the NOS antagonist L-NA (10^-4 mol/L) and throughout the application of shear stress. L-NA completely reversed the effect of flow on endothelial adhesiveness (Fig 3). This effect was not specific to L-NA as another NOS antagonist, nitro-L-amino-ornithine, produced similar results (data not shown). The effect of L-NA was reversed by the NO donor SPM-5185 (Fig 3).

View larger version (25K): [in this window] [in a new window]   Figure 3. Bar graph showing that L-NA blocks the effects of shear stress on endothelial adhesiveness. BAECs were exposed to quiescent or shear stress conditions for 2 hours and subsequently incubated with THP-1 cells to assess endothelial adhesiveness in the presence of L-NA as described in Fig 2. L-NA blocks the effect of shear stress on endothelial adhesiveness. Cotreatment with the NO donor SPM-5185 reverses the effect of L-NA. Binding is expressed as percentage of the cell binding to control ECs exposed to static conditions (in the absence of an agonist or antagonist).

Pretreatment with the nonselective K⁺ channel blocker TEA (3 mmol/L) also reversed the effect of shear stress on endothelial adhesiveness (Fig 4). This concentration of TEA has previously been demonstrated to completely abrogate flow-induced release of NO. The effect of TEA was reversed by exposing the cells to the NO donor SPM 5185 (Fig 4). We observed similar effects of L-NA or TEA on shear stress–induced alterations of the adhesiveness of HAECs and HUVECs (n=3 for both cell types; data not shown).

View larger version (28K): [in this window] [in a new window]   Figure 4. Bar graph showing that TEA blocks the effects of shear stress on endothelial adhesiveness. BAECs were exposed to static or shear stress conditions for 2 hours and subsequently incubated with THP-1 cells to assess endothelial adhesiveness as described in Fig 2. TEA blocks the effect of shear stress on endothelial adhesiveness. Cotreatment with the NO donor SPM-5185 reverses the effect of TEA. Binding is expressed as percentage of the cell binding to control ECs exposed to static conditions (in the absence of an agonist or antagonist).

Prostacyclin has also been reported to decrease leukocyte-EC interactions. Therefore, in some experiments, BAECs were preincubated with the cyclo-oxygenase inhibitor indomethacin (10^-6 mol/L). Although indomethacin totally inhibited production of PGI₂, it only partially attenuated the effect of shear stress on endothelial adhesiveness (Fig 5).

View larger version (34K): [in this window] [in a new window]   Figure 5. Bar graph showing that indomethacin does not block the effects of shear stress on endothelial adhesiveness. BAECs were exposed to static or shear stress conditions for 2 or 4 hours and subsequently incubated with THP-1 cells to assess endothelial adhesiveness as described in Fig 2. At a concentration that nearly abolishes prostacyclin production, indomethacin (10-6 mol/L) has little effect on the ability of shear stress to reduce endothelial adhesiveness for monocytes. Binding is expressed as percentage of the cell binding to control ECs exposed to static conditions (in the absence of an agonist or antagonist). *Significantly different from control (static) condition.

Effect of Flow on Endothelial Adhesion Molecules
VCAM-1 and ICAM-1 are endothelial glycoprotein adhesion molecules that may mediate monocyte-EC interaction. To determine whether changes in the expression of these adhesion molecules were involved in the early effects of flow (ie, within 2 hours) on endothelial adhesiveness, we used FACS. Fig 6 shows a typical tracing derived from FACS analysis of cells exposed to static or shear conditions for 2 hours. The FACS analysis revealed no difference in the expression of VCAM-1 or ICAM-1 between HAECs exposed to static or shear conditions (Table). In contrast, cells stimulated with LPS expressed increased amounts of VCAM-1 and ICAM-1 (Fig 6).

View larger version (18K): [in this window] [in a new window]   Figure 6. Analysis by FACS of adhesion molecule expression by ECs exposed to shear stress or static conditions. Plots demonstrate the effect of shear stress (2 hours) on the basal expression of VCAM-1 and ICAM-1 in HAECs. Shear stress had no effect on expression of VCAM-1 and ICAM-1 at 2 hours. LPS (5 µg/mL) for 12 hours caused increased expression of both VCAM-1 and ICAM-1. Relative cell numbers (vertical) and fluorescence intensity on log10 scale (horizontal) are indicated. Similar results were obtained using HUVECs.

View this table: [in this window] [in a new window]   Table 1. Effect of Shear Stress on Expression of Adhesion Molecules of HAECs

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The salient observations of the present study are that (1) exposure of bovine or human ECs to shear stress decreases endothelial adhesiveness for mononuclear cells, (2) this effect is largely due to shear stress-induced release of NO, (3) this effect occurs rapidly (within 30 minutes), and (4) this rapid effect is not due to alterations in the expression of the endothelial adhesion molecules VCAM-1 or ICAM-1.

We found that shear stress stimulates release of NO from cultured BAECs or HAECs. This effect is associated with decreased adhesiveness of the ECs for monocytes. When synthesis of NO is blocked by L-NA, the effect of shear stress on endothelial adhesiveness is abolished. This finding indicates that NO is the major autocrine effector by which flow acutely modulates endothelium-monocyte interaction. The shear stress–induced release of NO is also blocked by TEA, which is consistent with our previous observations that a flow-activated potassium channel is involved in the signal transduction of shear stress.^{18 20} It is therefore consistent with the hypothesis of this investigation that TEA prevents the effect of shear stress on endothelial adhesiveness. The effect of either inhibitor is completely reversed by administration of the NO donor SPM 5185, which adds further support to the hypothesis that NO is the major mediator of the acute effects of flow on endothelial adhesiveness.

In addition to activating an endothelial K⁺ current and releasing NO, flow modulates the release of other vasoactive factors, such as prostacyclin.²¹ Prostacylin is a potent vasodilator and inhibitor of platelet aggregation and adherence, as well as leukocyte adherence.^{14 22} In the present study, flow increased PGI₂ production. However, pretreatment with the cyclo-oxygenase inhibitor indomethacin had only a modest effect on shear stress–induced inhibition of monocyte adhesion.

The mechanisms by which NO modulates endothelial adhesiveness have not been completely delineated, but it is likely that endogenous NO has effects on signal transduction as well as transcriptional events that influence adhesion. Acute modulation of NO activity can directly alter the interaction of monocytes with ECs. The adhesion of mononuclear cells to ECs in culture is inhibited by genuine NO injected into the medium.¹⁴ Similarly, the increased adhesiveness of coronary endothelium for autologous neutrophils after ischemia-reperfusion is reversed within 30 minutes by administration of exogenous NO donors or the NO precursor L-arginine.^{13 23 24} The rapid effect of NO on adhesion may be mediated by cGMP-dependent protein phosphorylation. One candidate protein is VASP, which is present in ECs and circulating blood elements and is associated with actin filaments and focal contact areas. Studies indicate that phosphorylation of VASP is associated with the inhibition by NO of integrin-mediated platelet aggregation.^{25 26} NO may also exert its influence by inhibiting the elaboration of PAF.²⁷ In addition to its effects on platelet reactivity, PAF is a potent inducer of leukocyte adhesion via ICAM-1–dependent adhesion pathways, which may be involved in monocyte-endothelium interactions.^{28 29}

In the present investigation, we observed a rapid effect of NO on endothelial adhesiveness that is likely to be mediated by effects on the intracellular signaling of adhesion. The rapid time course (within 30 minutes) of the effect suggested that flow-stimulated NO affects the activation state or signal transduction of endothelial adhesion molecules rather than their expression. This notion is supported by our FACS analysis, which revealed no alteration in the expression of VCAM-1 or ICAM-1 at a time (2 hours) by which the effects of flow-stimulated NO on endothelial adhesiveness were maximal. Therefore, our observations support the existence of a mechanism by which flow alters endothelial adhesiveness with a rapid time course. Our finding does not exclude the possibility that chronic changes in flow may induce transcriptional events that lead to alterations in the expression of adhesion molecules or chemotactic proteins. Ohtsuka and colleagues³⁰ found that VCAM-1 expression of ECs (derived from mouse lymph nodes) was reduced by 75% after 24 hours of exposure to physiological levels (1.5 dyne/cm²) of shear stress. When ECs subjected to flow for 24 hours were reexposed to static conditions, the reduction in VCAM-1 expression was nearly reversed by 72 hours. Nagel and colleagues³¹ exposed HUVECs to laminar shear stress using methodology similar to ours and observed a time-dependent increase in ICAM-1 by immunohistochemistry over a time course of 48 hours. Northern blot analysis revealed an increase in ICAM message as early as 2 hours after the onset of shear stress. In contrast, E-selectin and VCAM-1 expressions were not affected in this study.³¹

Chronic changes in NO activity may influence endothelium-monocyte interaction in vivo. We have observed an increase in endothelial adhesiveness for mononuclear cells in the thoracic aorta of rabbits fed a 0.5% cholesterol diet for 2 weeks.¹⁶ This cholesterol-induced alteration in endothelial adhesiveness can be reversed by augmenting vascular NO activity via dietary supplementation with the NO precursor L-arginine. In contrast, when NO synthesis is reduced by dietary L-NA, the thoracic aorta of these animals manifest increased endothelial adhesiveness for monocytes.¹⁶ Administration of dietary arginine for a longer time period (10 weeks) inhibits the intimal accumulation of monocytes in the vessel wall of hypercholesterolemic rabbits^{32 33} ; in contrast, prolonged treatment with inhibitors of NOS accelerates monocyte accumulation.^{34 35}

NO may also influence leukocyte adhesion by inactivating superoxide anion or by inhibiting its generation.^{36 37 38 39} Oxidant stress induces ECs to express PAF and P-selectin (effects that occur within minutes of exposure to oxidants).⁴⁰ Oxidative stress promotes the transcription of VCAM-1 mediated in part by nuclear factor-B (or a related nuclear protein).³⁹ By virtue of its ability to inhibit the generation of oxygen-derived free radicals, NO could prevent the cascade of events triggered by nuclear factor-B.

Exposure of the endothelium to physiological levels of shear stress reduces the elaboration of endothelin, an effect that is mediated by NO through a cGMP-dependent mechanism.⁴¹ There is evidence that endothelins (ET-1, ET-2, and ET-3) can upregulate the expression of ICAM-1, VCAM-1, and E-selectin-1 on the endothelium and can stimulate neutrophil adhesion.^{41 42 43} Thus, NO may further exert its effects by modulating the expression of other autocrine factors that in turn regulate adhesion molecule expression.

Our findings indicate that an additional mechanism contributes to the preferential formation of atherosclerotic lesions in areas of low shear stress. In human carotid and coronary arteries, atherosclerotic plaques are found in the vicinity of arterial bifurcations and bends, where laminar flow is disturbed, and where recirculation establishes areas of low shear stress.^{44 45} Experimental reduction of flow in the rabbit carotid artery induces monocyte adherence and transmigration in vivo.⁴⁶ We propose that in areas of low shear stress, there is a reduced release of endothelium-derived NO that promotes monocyte-endothelium interaction.

To summarize, we have found that the adhesiveness of ECs for monocytes is reduced by prior exposure to shear stress. The effect occurs rapidly and before any changes in the expression of the endothelial adhesion molecules VCAM-1 and ICAM-1. This action is largely due to the shear-induced release of endothelium-derived NO because the effect is blocked by inhibitors of NOS and by an antagonist of the flow-activated endothelial potassium channel. The preferential distribution of atherosclerotic plaque in areas of low shear stress may in part be explained by reduced elaboration of NO at these sites.

	Selected Abbreviations and Acronyms

 

6-keto-PGF1 = 6-keto-prostaglandin F1 BAECs = bovine aortic endothelial cells ECs = endothelial cells EDRF = endothelium-derived relaxing factor EGM = endothelial growth medium FACS = fluorescence-activated cell sorting GAPDH = glyceraldehyde-3-phosphate-dehydrogenase HAECs = human aortic endothelial cells HBSS = Hanks' balanced salt solution HUVECs = human umbilical vein endothelial cells ICAM-1 = intercellular adhesion molecule-1 L-NA = nitro-L-arginine LPS = lipopolysaccharide mAb = monoclonal antibody NO = nitric oxide NOS = nitric oxide synthase NOx = nitrogen oxides (NO and one-electron oxidation products of NO, NO2-, and NO3-) PAF = platelet-activating factor PGI2 = prostacyclin PI = propidium iodide SDS = sodium dodecyl sulfate SSC = standard saline citrate TEA = tetraethylammonium ion THP-1 = human monocytoid cells VASP = vasodilator-stimulated phosphoprotein VCAM-1 = vascular cell adhesion molecule-1

	Acknowledgments

 
This work was supported in part by grants from the National Heart, Lung, and Blood Institute (1P01-HL-48638-01), American Heart Association, University of California Tobacco-Related Disease Program IRT 215, Institute of Biological and Clinical Investigation of Stanford University, and Peninsula Community Foundation. Dr Tsao is the recipient of a National Service Research Award (1F32-HL-08779). Dr Lewis is the recipient of a British Heart Foundation International Fellowship. Dr Cooke is a recipient of the Vascular Academic Award from the National Heart, Lung, and Blood Institute (1K07-HC-02660). We gratefully acknowledge the valuable contributions of Bing-Yin Wang in the measurement of NO_x and Dan Pollart at Stanford Research Institute in the synthesis of L-NA.

Received August 25, 1994; revision received July 28, 1995; accepted August 1, 1995.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Hilton SM. A peripheral arterial conducting mechanism underlying dilation of the femoral artery and concerned in functional vasodilation in the skeletal muscle. J Physiol. 1959;149:93-111.

2. Lie M, Sejersted OM, Kiil F. Local regulation of vascular cross section during changes in femoral arterial blood flow in dogs. Circ Res. 1970;27:727-737. [Abstract/Free Full Text]

3. Hull SS, Kaiser L, Jaffe MD, Sparks HV. Endothelium-dependent flow induced dilation of canine femoral and saphenous arteries. Blood Vessels. 1986;23:183-198. [Medline] [Order article via Infotrieve]

4. Pohl H, Holtz J, Busse R, Bassenge E. Crucial role of endothelium in the vasodilator response to increased flow in vivo. Hypertension.. 1986;8:37-47. [Abstract/Free Full Text]

5. Rubanyi GM, Romero JC, Vanhoutte PM. Flow-induced release of endothelium derived relaxing factor. Am J Physiol. 1986;250:H1145-H1149. [Abstract/Free Full Text]

6. Cooke JP, Stamler JS, Andon N, Davies PF, Loscalzo J. Flow stimulates endothelial cells to release a nitrovasodilator that is potentiated by reduced thiol. Am J Physiol. 1990;28:H804-H812.

7. Furchgott RF. Studies on relaxation of rabbit aorta by sodium nitrite: the basis for the proposal that the acid-activatable inhibitory factor from retractor penis is inorganic nitrite and the endothelium-derived relaxing factor is nitric oxide. In: Vanhoutte PM, ed. Mechanics of Vasodilation. New York, NY: Raven Press; 1988:401-404.

8. Ignarro LJ, Burns RE, Buga GM, Wood KS. Endothelium-derived relaxing factor from pulmonary artery and vein possesses pharmacologic and chemical properties identical to those of nitric oxide radical. Circ Res. 1987;61:866-879. [Abstract/Free Full Text]

9. Palmer RMJ, Ashton DS, Moncada S. Vascular endothelial cells synthesized nitric oxide from L-arginine. Nature. 1988;333:664-666. [Medline] [Order article via Infotrieve]

10. Koller A, Kaley G. Prostaglandins mediate arteriolar dilation to increased blood flow velocity in skeletal muscle microcirculation. Circ Res. 1990;67:980-985.

11. Faraci FM, Heistad DD. Regulation of cerebral blood vessels by humoral and endothelium-dependent mechanism: update on humoral regulation of vascular tone. Hypertension. 1991;17:917-922. [Abstract/Free Full Text]

12. Feletou M, Vanhoutte PM. Endothelium-dependent hyperpolarization of canine coronary smooth muscle. Br J Pharmacol. 1988;331:168-170.

13. Tsao PS, Lefer AM. Time course and mechanism of endothelial dysfunction in isolated ischemic and hypoxic perfused rat hearts. Am J Physiol. 1990;259:C738-C745. [Abstract/Free Full Text]

14. Bath PMW, Hassall DG, Gladwin A-M, Palmer RMJ, Martin JF. Nitric oxide and prostacyclin: divergence of inhibitory effects on monocyte chemotaxis and adhesion to endothelium in vitro. Arterioscler Thromb. 1991;11:254-260. [Abstract/Free Full Text]

15. Kubes P, Suzuki M, Granger DN. Nitric oxide: an endogenous modulator of leukocyte adhesion. Proc Natl Acad Sci U S A. 1991;88:4651-4655. [Abstract/Free Full Text]

16. Tsao P, McEvoy LM, Drexler H, Butcher EC, Cooke JP. Enhanced endothelial adhesiveness in hypercholesterolemia is attenuated by L-arginine. Circulation. 1994;89:2176-2182. [Abstract/Free Full Text]

17. Dewey CF Jr, Bussolari SR, Gimbrone MA Jr, Davies PF. The dynamic response of vascular endothelial cells to fluid shear stress. J Biomech Eng. 1981;103:177-185. [Medline] [Order article via Infotrieve]

18. Ohno M, Gibbons GH, Dzau VJ, Cooke JP. Shear stress elevates endothelial cGMP: role of a potassium channel and G protein coupling. Circulation. 1993;88:193-197. [Abstract/Free Full Text]

19. Palmer RMJ, Ferridge AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature. 1987;327:524-526. [Medline] [Order article via Infotrieve]

20. Cooke JP, Rossitch E, Andon N, Loscalzo J, Dzau VJ. Flow activates an endothelial potassium channel to release an endogenous nitrovasodilator. J Clin Invest. 1991;88:1663-1671.

21. Frangos JA, Eskin SG, McIntire LV, Ives CL. Flow effects prostacyclin production of cultured human endothelial cells. Science. 1984;227:1477-1479.

22. Bunting SR, Gryglewski R, Moncada S, Vane JR. Arterial walls generate from prostaglandin endoperoxides in a substance (prostaglandin X) which relaxes strips of mesenteric and coeliac arteries and inhibits platelet aggregation. Prostaglandins. 1976;12:897-913. [Medline] [Order article via Infotrieve]

23. Lefer DJ, Nakanishi K, Johnston WE, Vinten-Johanssen J. Antineutrophil and myocardial protecting actions of a novel nitric oxide donor following acute myocardial ischemia and reperfusion in dogs. Circulation. 1993;88:2337-2350. [Abstract/Free Full Text]

24. Weyrich AS, Ma XI, Lefer AM. The role of L-arginine in ameliorating reperfusion injury after myocardial ischemia in the cat. Circulation. 1992;86:279-288. [Abstract/Free Full Text]

25. Halbrügge M, Friedrioch C, Eigenthaler M, Schanzenbacher P, Walter U. Stoichiometric and reversible phosphoryation of a 46-KD protein in human platelets in response to cGMP- and cAMP-elevating vasodilators. J Biol Chem. 1990;265:3088-3093. [Abstract/Free Full Text]

26. Pohl U, Nolte C, Bunse A, Eigenthaler M, Walter U. Endothelium-dependent phosphorylation of vasodilator-stimulated protein in platelets during coronary passage. Am J Physiol. 1994;266:H606-H612. [Abstract/Free Full Text]

27. Heller R, Bussolino F, Ghigo D, Garbarino G, Pescarmona G, Till U, Boxia A. Nitrovasodilators inhibit thrombin-induced platelet-activating factor synthesis in human endothelial cells. Biochem Pharmacol. 1992;44:223-229. [Medline] [Order article via Infotrieve]

28. Zimmerman GA, McIntyre TM, Mehra M, Prescott SM. Endothelial cell-associated platelet-activating factor: a novel mechanism for signaling intercellular adhesion. J Cell Biol. 1990;110:529-540. [Abstract/Free Full Text]

29. Chihara J, Maruyama I, Yasuba H, Yasukawa A, Yamamoto T, Kurachi D, Mouri T, Seguchi M, Nakajima S. Possible induction of intercellular adhesion molecule-1 (ICAM-1) expression on endothelial cells by platelet-activating factor (PAF). J Lipid Mediators. 1992;5:159-162. [Medline] [Order article via Infotrieve]

30. Ohtsuka A, Ando J, Korenaga R, Kamiya A, Toyama-Sorimachi N, Miyasaka M. The effect of flow on the expression of vascular adhesion molecule-1 by cultured mouse endothelial cells. Biochem Biophys Res Commun. 1993;193:303-310. [Medline] [Order article via Infotrieve]

31. Nagel T, Resnick N, Atkinson WJ, Dewey CF Jr, Gimbrone MA Jr. Shear stress selectively upregulates intercellular adhesion molecule-1 expression in cultured human vascular endothelial cells. J Clin Invest. 1994;94:885-891.

32. Cooke JP, Singer AH, Tsao P, Zera P, Rowan RA, Billingham ME. Anti-atherogenic effects of L-arginine in the hypercholesterolemic rabbit. J Clin Invest. 1992;90:1168-1172.

33. Wang B, Singer A, Tsao P, Drexler H, Kosek J, Cooke JP. Dietary arginine prevents atherogenesis in the coronary artery of the hypercholesterolemic rabbit. J Am Coll Cardiol. 1994;23:452-458. [Abstract]

34. Cayatte AJ, Palacino JJ, Horten K, Cohen RA. Chronic inhibition of nitric oxide production accelerates neointima formation and impairs endothelial function in hypercholesterolemic rabbits. Arterioscler Thromb. 1994;14:753-759. [Abstract/Free Full Text]

35. Naruse K, Shimizu K, Muramatsu M, Toki Y, Miyazaki Y, Okumura K, Hasimoto H, Ito T. Long-term inhibition of NO synthesis promotes atherosclerosis in the hypercholesterolemic rabbit thoracic aorta: PGH2 does not contribute to impaired endothelium-dependent relaxation. Arterioscler Thromb. 1994;14:746-752. [Abstract/Free Full Text]

36. Niu X, Smith CW, Kubes P. Intracellular oxidative stress induced by nitric oxide synthesis inhibition increases endothelial cell adhesion to neutrophils. Circ Res. 1994;74:1113-1140.

37. Gaboury J, Woodman RC, Granger DN, Reinhardt P, Kubes P. Nitric oxide prevents leukocyte adherence: role of superoxide. Am J Physiol. 1993;265:H862-H867. [Abstract/Free Full Text]

38. Clancy RM, Leszczynska-Piziak J, Abramson SB. Nitric oxide, an endothelial cell relaxation factor, inhibits neutrophil superoxide anion production via a direct action on the NADPH oxidase. J Clin Invest. 1992;90:1116-1121.

39. Marui N, Offerman MK, Swerlick R, Kunsch C, Rossen CA, Ahmad M, Alexander RW, Medford RM. Vascular cell adhesion molecule-1 (VCAM-1) gene transcription and expression are regulated through an antioxidant-sensitive mechanism in human vascular endothelial cells. J Clin Invest. 1993;92:1866-1874.

40. Gaboury JP, Anderson DC, Kubes P. Molecular mechanisms involved in superoxide-induced leukocyte-endothelial cell interactions in vivo. Am J Physiol. 1994;266:H637-H642. [Abstract/Free Full Text]

41. Kuchan MD, Frangos JA. Shear stress regulates endothelin-1 release via protein kinase C and cGMP in cultured endothelial cells. Am J Physiol. 1993;264(pt 2):H150-H156.

42. McCarron RM, Wang L, Stanimirovic DB, Spatz M. Endothelin induction of adhesion molecule expression on human brain microvascular endothelial cells. Neurosci Lett. 1993;156:31-34. [Medline] [Order article via Infotrieve]

43. Lopez Farre A, Riesco A, Espinosa G, Digiuni E, Cernadas MR, Alvarez V, Monton M, Rivas F, Gallego MJ, Egido J, Casado S, Carmelo C. Effect of endothelin-1 on neutrophil adhesion to endothelial cells and perfused heart. Circulation. 1993;88:1166-1171. [Abstract/Free Full Text]

44. Glagov S, Zarins C, Giddens DP, Ku DN. Hemodynamics and atherosclerosis: insights and perspectives gained from studies of human arteries. Arch Pathol Lab Med. 1988;112:1018-1031. [Medline] [Order article via Infotrieve]

45. Asakura T, Karino T. Flow patterns and spatial distribution of atherosclerotic lesions in human coronary arteries. Circ Res. 1976;23:489-501.

46. Walpola PL, Gotlieb AI, Langille BL. Monocyte adhesion and changes in endothelial cell number, morphology, and F-actin distribution elicited by low shear stress in vivo. Am J Pathol. 1993;142:1392-1400.[Abstract]

This article has been cited by other articles:

				M. Asai, K. Takeuchi, M. Saotome, T. Urushida, H. Katoh, H. Satoh, H. Hayashi, and H. Watanabe Extracellular acidosis suppresses endothelial function by inhibiting store-operated Ca2+ entry via non-selective cation channels Cardiovasc Res, July 1, 2009; 83(1): 97 - 105. [Abstract] [Full Text] [PDF]

				N. Sud, S. Kumar, S. Wedgwood, and S. M. Black Modulation of PKC{delta} signaling alters the shear stress-mediated increases in endothelial nitric oxide synthase transcription: role of STAT3 Am J Physiol Lung Cell Mol Physiol, March 1, 2009; 296(3): L519 - L526. [Abstract] [Full Text] [PDF]

				J.-J. Chiu, L.-J. Chen, C.-I Lee, P.-L. Lee, D.-Y. Lee, M.-C. Tsai, C.-W. Lin, S. Usami, and S. Chien Mechanisms of induction of endothelial cell E-selectin expression by smooth muscle cells and its inhibition by shear stress Blood, July 15, 2007; 110(2): 519 - 528. [Abstract] [Full Text] [PDF]

				A. Rubaj, P. Rucinski, K. Rejdak, K. Oleszczak, D. Duma, P. Grieb, and A. Kutarski Biventricular versus right ventricular pacing decreases immune activation and augments nitric oxide production in patients with chronic heart failure Eur J Heart Fail, October 1, 2006; 8(6): 615 - 620. [Abstract] [Full Text] [PDF]

				A. Koller Flow-Dependent Remodeling of Small Arteries: The Stimuli and the Sensors Are (Still) in Question Circ. Res., July 7, 2006; 99(1): 6 - 9. [Full Text] [PDF]

				M. P. Burns and N. DePaola Flow-conditioned HUVECs support clustered leukocyte adhesion by coexpressing ICAM-1 and E-selectin Am J Physiol Heart Circ Physiol, January 1, 2005; 288(1): H194 - H204. [Abstract] [Full Text] [PDF]

				S. A. Spier, M. D. Delp, C. J. Meininger, A. J. Donato, M. W. Ramsey, and J. M. Muller-Delp Effects of ageing and exercise training on endothelium-dependent vasodilatation and structure of rat skeletal muscle arterioles J. Physiol., May 1, 2004; 556(3): 947 - 958. [Abstract] [Full Text] [PDF]

				J.-J. Chiu, P.-L. Lee, C.-N. Chen, C.-I Lee, S.-F. Chang, L.-J. Chen, S.-C. Lien, Y.-C. Ko, S. Usami, and S. Chien Shear Stress Increases ICAM-1 and Decreases VCAM-1 and E-selectin Expressions Induced by Tumor Necrosis Factor-{alpha} in Endothelial Cells Arterioscler Thromb Vasc Biol, January 1, 2004; 24(1): 73 - 79. [Abstract] [Full Text] [PDF]

				Y. C. Boo and H. Jo Flow-dependent regulation of endothelial nitric oxide synthase: role of protein kinases Am J Physiol Cell Physiol, September 1, 2003; 285(3): C499 - C508. [Abstract] [Full Text] [PDF]

				G. P. Sorescu, M. Sykes, D. Weiss, M. O. Platt, A. Saha, J. Hwang, N. Boyd, Y. C. Boo, J. D. Vega, W. R. Taylor, et al. Bone Morphogenic Protein 4 Produced in Endothelial Cells by Oscillatory Shear Stress Stimulates an Inflammatory Response J. Biol. Chem., August 15, 2003; 278(33): 31128 - 31135. [Abstract] [Full Text] [PDF]

				M. E. Davis, H. Cai, L. McCann, T. Fukai, and D. G. Harrison Role of c-Src in regulation of endothelial nitric oxide synthase expression during exercise training Am J Physiol Heart Circ Physiol, April 1, 2003; 284(4): H1449 - H1453. [Abstract] [Full Text] [PDF]

				B. Coles, A. Bloodsworth, S. R. Clark, M. J. Lewis, A. R. Cross, B. A. Freeman, and V. B. O'Donnell Nitrolinoleate Inhibits Superoxide Generation, Degranulation, and Integrin Expression by Human Neutrophils: Novel Antiinflammatory Properties of Nitric Oxide-Derived Reactive Species in Vascular Cells Circ. Res., September 6, 2002; 91(5): 375 - 381. [Abstract] [Full Text] [PDF]

				M. P Vallely, P. G Bannon, C. F Hughes, and L. Kritharides Endothelial Cell Adhesion Molecules and Cardiopulmonary Bypass Asian Cardiovasc Thorac Ann, December 1, 2001; 9(4): 349 - 355. [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]

				T. Osanai, N. Akutsu, N. Fujita, T. Nakano, K. Takahashi, W. Guan, and K. Okumura Cross talk between prostacyclin and nitric oxide under shear in smooth muscle cell: role in monocyte adhesion Am J Physiol Heart Circ Physiol, July 1, 2001; 281(1): H177 - H182. [Abstract] [Full Text] [PDF]

				F. C. Blumberg, K. Wolf, P. Sandner, C. Lorenz, G. A. J. Riegger, and M. Pfeifer The NO donor molsidomine reduces endothelin-1 gene expression in chronic hypoxic rat lungs Am J Physiol Lung Cell Mol Physiol, February 1, 2001; 280(2): L258 - L263. [Abstract] [Full Text] [PDF]

				R. H. Boger, S. M. Bode-Boger, P. S. Tsao, P. S. Lin, J. R. Chan, and J. P. Cooke An endogenous inhibitor of nitric oxide synthase regulates endothelial adhesiveness for monocytes J. Am. Coll. Cardiol., December 1, 2000; 36(7): 2287 - 2295. [Abstract] [Full Text] [PDF]

				M. R Andersen and S. Stender Endothelial nitric oxide synthase activity in aorta of normocholesterolemic rabbits: regional variation and the effect of estrogen Cardiovasc Res, July 1, 2000; 47(1): 192 - 199. [Abstract] [Full Text] [PDF]

				L. W. Kraiss, A. S. Weyrich, N. M. Alto, D. A. Dixon, T. M. Ennis, V. Modur, T. M. McIntyre, S. M. Prescott, and G. A. Zimmerman Fluid flow activates a regulator of translation, p70/p85 S6 kinase, in human endothelial cells Am J Physiol Heart Circ Physiol, May 1, 2000; 278(5): H1537 - H1544. [Abstract] [Full Text] [PDF]

				J. P Cooke The endothelium: a new target for therapy Vascular Medicine, February 1, 2000; 5(1): 49 - 53. [Abstract] [PDF]

				T. Osanai, N. Fujita, N. Fujiwara, T. Nakano, K. Takahashi, W. Guan, and K. Okumura Cross talk of shear-induced production of prostacyclin and nitric oxide in endothelial cells Am J Physiol Heart Circ Physiol, January 1, 2000; 278(1): H233 - H238. [Abstract] [Full Text] [PDF]

				J. Niebauer, J.o. Dulak, J. R. Chan, P. S. Tsao, and J. P. Cooke Gene transfer of nitric oxide synthase: Effects on endothelial biology J. Am. Coll. Cardiol., October 1, 1999; 34(4): 1201 - 1207. [Abstract] [Full Text] [PDF]

				J. J. Chiu, B. S. Wung, H. J. Hsieh, L. W. Lo, and D. L. Wang Nitric Oxide Regulates Shear Stress–Induced Early Growth Response-1 : Expression via the Extracellular Signal–Regulated Kinase Pathway in Endothelial Cells Circ. Res., August 6, 1999; 85(3): 238 - 246. [Abstract] [Full Text] [PDF]

				J. P Cooke The 1998 Nobel prize in Medicine: clinical implications for 1999 and beyond Vascular Medicine, May 1, 1999; 4(2): 57 - 60. [PDF]

				P. Clarkson, H. E. Montgomery, M. J. Mullen, A. E. Donald, A. J. Powe, T. Bull, M. Jubb, M. World, and J. E. Deanfield Exercise training enhances endothelial function in young men J. Am. Coll. Cardiol., April 1, 1999; 33(5): 1379 - 1385. [Abstract] [Full Text] [PDF]

				M. Gosling, J. Golledge, R. J. Turner, and J. T. Powell Arterial Flow Conditions Downregulate Thrombomodulin on Saphenous Vein Endothelium Circulation, March 2, 1999; 99(8): 1047 - 1053. [Abstract] [Full Text] [PDF]

				A. Lerman, J. C. Burnett Jr, S. T. Higano, L. J. McKinley, and D. R. Holmes Jr Long-term L-Arginine Supplementation Improves Small-Vessel Coronary Endothelial Function in Humans Circulation, June 2, 1998; 97(21): 2123 - 2128. [Abstract] [Full Text] [PDF]

				O. Traub and B. C. Berk Laminar Shear Stress : Mechanisms by Which Endothelial Cells Transduce an Atheroprotective Force Arterioscler Thromb Vasc Biol, May 1, 1998; 18(5): 677 - 685. [Abstract] [Full Text] [PDF]

				J. T Powell Vascular damage from smoking: disease mechanisms at the arterial wall Vascular Medicine, February 1, 1998; 3(1): 21 - 28. [Abstract] [PDF]

				S. Chien, S. Li, and J. Y-J. Shyy Effects of Mechanical Forces on Signal Transduction and Gene Expression in Endothelial Cells Hypertension, January 1, 1998; 31(1): 162 - 169. [Abstract] [Full Text] [PDF]

				G. Theilmeier, J. R. Chan, C. Zalpour, B. Anderson, B.-y. Wang, A. Wolf, P. S. Tsao, and J. P. Cooke Adhesiveness of Mononuclear Cells in Hypercholesterolemic Humans Is Normalized by Dietary L-Arginine Arterioscler Thromb Vasc Biol, December 1, 1997; 17(12): 3557 - 3564. [Abstract] [Full Text]

				J.J. Chiu, B.S. Wung, J. Y.J. Shyy, H.J. Hsieh, and D.L. Wang Reactive Oxygen Species Are Involved in Shear Stress-Induced Intercellular Adhesion Molecule-1 Expression in Endothelial Cells Arterioscler Thromb Vasc Biol, December 1, 1997; 17(12): 3570 - 3577. [Abstract] [Full Text]

				J.-L. Balligand and P. J. Cannon Nitric Oxide Synthases and Cardiac Muscle : Autocrine and Paracrine Influences Arterioscler Thromb Vasc Biol, October 1, 1997; 17(10): 1846 - 1858. [Abstract] [Full Text]

				S. P. Schwarzacher, T. T. Lim, B. Wang, R. S. Kernoff, J. Niebauer, J. P. Cooke, and A. C. Yeung Local Intramural Delivery of L-Arginine Enhances Nitric Oxide Generation and Inhibits Lesion Formation After Balloon Angioplasty Circulation, April 1, 1997; 95(7): 1863 - 1869. [Abstract] [Full Text]

				J. P. Cooke and P. S. Tsao Arginine: A New Therapy for Atherosclerosis? Circulation, January 21, 1997; 95(2): 311 - 312. [Full Text]

				P. S. Tsao, R. Buitrago, J. R. Chan, and J. P. Cooke Fluid Flow Inhibits Endothelial Adhesiveness: Nitric Oxide and Transcriptional Regulation of VCAM-1 Circulation, October 1, 1996; 94(7): 1682 - 1689. [Abstract] [Full Text]

				H. Matsushita, E. Chang, A. J. Glassford, J. P. Cooke, C.-P. Chiu, and P. S. Tsao eNOS Activity Is Reduced in Senescent Human Endothelial Cells: Preservation by hTERT Immortalization Circ. Res., October 26, 2001; 89(9): 793 - 798. [Abstract] [Full Text] [PDF]

This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tsao, P. S. Articles by Cooke, J. P. Search for Related Content PubMed PubMed Citation Articles by Tsao, P. S. Articles by Cooke, J. P. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB NITRIC OXIDE