Exposure to Shear Stress Alters Endothelial Adhesiveness
Role of Nitric Oxide
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 F1α. NOx and 6-keto-prostaglandin F1α 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.
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.
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 3.6×10−3, glucose 30, sodium pyruvate 1.0, and phenolsulfonphthalein 4.2×10−2. 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%.
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 NOx. Fluid flow using the mixing table (120 revolutions per minute) caused BAECs to produce levels of NOx 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 NOx 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 NOx. NOx 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-PGF1α 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 6× SSC, 5× Denhardt’s solution, and 0.5% SDS. Blots were then washed twice in 2× SSC/0.1% SDS for 15 minutes at room temperature and then twice in 0.4× 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 1.5×106 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.
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 (2×105 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%.
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.
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.
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⇓).
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.
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⇓).
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).
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⇓).
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⇓).
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 rabbits32 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-κB (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-κB.
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
|BAECs||=||bovine aortic endothelial cells|
|EDRF||=||endothelium-derived relaxing factor|
|EGM||=||endothelial growth medium|
|FACS||=||fluorescence-activated cell sorting|
|HAECs||=||human aortic endothelial cells|
|HBSS||=||Hanks’ balanced salt solution|
|HUVECs||=||human umbilical vein endothelial cells|
|ICAM-1||=||intercellular adhesion molecule-1|
|NOS||=||nitric oxide synthase|
|NOx||=||nitrogen oxides (NO and one-electron oxidation products of NO, NO2−, and NO3−)|
|SDS||=||sodium dodecyl sulfate|
|SSC||=||standard saline citrate|
|THP-1||=||human monocytoid cells|
|VCAM-1||=||vascular cell adhesion molecule-1|
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 NOx 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.
- Copyright © 1995 by American Heart Association
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