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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3570-3577.)
© 1997 American Heart Association, Inc.

Articles

Reactive Oxygen Species Are Involved in Shear Stress-Induced Intercellular Adhesion Molecule-1 Expression in Endothelial Cells

J.J. Chiu; B.S. Wung; John Y.J. Shyy; H.J. Hsieh; ; D.L. Wang

From the Institute of Biomedical Sciences–Academia Sinica, and Graduate Institute for Life Sciences (B.S.W.), National Defense Medical Center, and Department of Chemical Engineering, National Taiwan University (H.J.H.), Taipei, Taiwan, ROC.

Correspondence to Dr Jeng-Jiann Chiu, Cardiovascular Division, Institute of Biomedical Sciences, Academia Sinica, Taipei, 11529, Taiwan, ROC.

	Abstract

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Abstract Vascular endothelial cells (ECs) are constantly subjected to flow-induced shear stress. Although the effects of shear stress on ECs are well known, the intracellular signal mechanisms remain largely unclear. Reactive oxygen species (ROS) have recently been suggested to act as intracellular second messengers. The potential role of ROS in shear-induced gene expression was examined in the present study by subjecting ECs to a shear force using a parallel-plate flow chamber system. ECs under shear flow increased their intracellular ROS as indicated by superoxide production. This superoxide production was maintained at an elevated level as shear flow remained. Sheared ECs, similar to TNF-, PMA-, or H₂O₂-treated cells, increased their intercellular adhesion molecule-1 (ICAM-1) mRNA levels in a time-dependent manner. Pretreatment of ECs with an antioxidant, N-acetyl-cysteine (NAC) or catalase, inhibited this shear-induced or oxidant-induced ICAM-1 expression. ROS that were involved in the shear-induced ICAM-1 gene expression were further substantiated by functional analysis using a chimera containing the ICAM-1 promoter region (-850 bp) and the reporter gene luciferase. Shear-induced promoter activities were attenuated by pretreating sheared ECs with NAC and catalase. Flow cytometric analysis and monocytic adhesion assay confirmed the inhibitory effect of NAC and catalase on the shear-induced ICAM-1 expression on ECs. These results clearly demonstrate that shear flow to ECs can induce intracellular ROS generation that may result in an increase of ICAM-1 mRNA levels via transcriptional events. Our findings thus support the importance of intracellular ROS in modulating hemodynamically induced endothelial responses.

Key Words: endothelial cells • ICAM-1 • reactive oxygen species • shear stress

	Introduction

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Vascular endothelial cells (ECs) are constantly subjected to the influence of hemodynamic forces including shear stress imposed by blood flow. Shear stress thus plays an active role in the processes of leukocyte/ECs interaction and the subsequent leukocyte extravasation into inflamed tissue.^{1 2} The effects of fluid shear stress on endothelial biology and gene expression have been extensively studied.^{3 4 5 6} In addition to its exertion as a physical force in preventing leukocytes from adhering to ECs, shear stress can further alter the adhesive properties of ECs by modulating the surface expression of adhesive proteins.^{7 8 9} Among the proteins that have been shown to be regulated by shear stress are intercellular adhesion molecule-1 (ICAM-1)^{9 10 11 12 13} and vascular cell adhesion molecule-1 (VCAM-1).^{9 10 13 14} Exposure of ECs to laminar flow increases the gene and protein expression of ICAM-1.^{9 10 11 12 13} In contrast, shear stress treatment of ECs reduces surface expression of VCAM-1.^{13 14} In addition to the shear effects on ECs, we have recently demonstrated that ECs that are subjected to pressure-induced cyclic strain can also increase their ICAM-1 and monocyte chemotactic protein-1 (MCP-1) mRNA levels.^{15 16}

Despite the intensive studies on the effects of fluid shear stress on ECs, little is known about the mechanisms that transmit the mechanical stimuli to intracellular signals that ultimately regulate downstream gene expression. Various signals, including protein kinases,^{17 18} calcium influx,¹⁹ inositol triphosphate,^{20 21} cGMP,²² and G protein,^{22 23} have been demonstrated to be activated by shear stress. Whether these represent synergism or cross-talk among different signals remains unclear. Accumulating evidence suggests that reactive oxygen species (ROS) may function as second messengers in cells that are exposed to various stimuli.^{24 25 26} Whether ROS are involved in the cellular responses to mechanical stimuli has not been fully clarified. However, recent studies have demonstrated that the release of ROS from vascular ECs as a flow-dependent phenomenon.^{27 28} ROS that are involved in the activation of the transcriptional factors, nuclear factor-B (NF-B), and activator protein-1 (AP-1) have been demonstrated.^{29 30 31} The induction of genes in ECs that are exposed to hemodynamic forces is believed to involve the activation of NF-B and AP-1.^{32 33 34} Recent studies by Inoue et al³⁵ and Topper et al³⁶ have further shown that shear stress enhances the gene expression of superoxide dismutase (SOD) in ECs, ie, a defense mechanism to protect ECs from injuries by oxidative stress. Since ICAM-1 gene expression in ECs is inducible by chemicals that are known to increase oxidative stress^{37 38} and the ICAM-1 promoter region contains a number of AP-1-binding and NF-B-binding sites,³⁹ we postulate that shear-induced ICAM-1 gene expression may be mediated through intracellular ROS levels in sheared ECs. The present studies clearly demonstrate that ECs that are subjected to fluid flow induce intracellular ROS generation and that this ROS increase is involved in the upregulation of the ICAM-1 gene. Our findings thus provide a direct line of evidence for the involvement of intracellular ROS in modulating hemodynamically induced endothelial responses.

	Methods

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Endothelial Cell Culture
ECs were isolated from fresh human umbilical cords by means of the collagenase perfusion technique.⁴⁰ The cell pellet was resuspended in a culture medium consisting of medium 199 (M199, Gibco, Grand Island, New York) supplemented with 20% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (Gibco). ECs were grown in Petri dishes for 3 days and then seeded onto glass slides (75 by 38 mm, Corning, New York) precoated with fibronection (Sigma). Secondary cultures were used in experiments within 2 days subsequent to reaching confluence (~1-2 x10⁵ cells/cm²).

Flow Apparatus
The slide with cultured ECs was mounted in a parallel-plate flow chamber, which has been characterized and described in detail elsewhere.⁴¹ The chamber was connected to a perfusion loop system, kept in a constant-temperature controlled enclosure, and maintained at pH 7.4 by continuous gassing with a mixture of 5% CO₂ in air. The osmolality of the perfusate was adjusted to 285 to 295 mOsm/kg H₂O during the perfusion. The flow channel width (w) was 1 cm, and the channel height (h) was 0.025 cm. The Reynolds number, defined by the average inlet velocity and the channel height, was 30. The fluid shear stress generated on the ECs by flow was estimated as 20 dynes/cm², using the formula

where is the shear stress, µ is the dynamic viscosity of the perfusate, and Q is the flow rate. In some experiments, ECs were exposed to flow in the presence of an antioxidant, ie, N-acetyl-cysteine (NAC, 20 mmol/L, Sigma) or catalase (3.5x10⁵ U/L, Sigma). The static control cells were incubated and changed to the new culture medium, while the experimental cells were placed under flow conditions.

Chemiluminescence Assay of Superoxide Production
Superoxide production of ECs subjected to shear stress was measured by lucigenin-amplified chemiluminescence as previously described.⁴² Briefly, cells were immediately lysed after shear stress treatment with a lysis buffer containing lucigenin (200 µmol/L, Sigma). Readings were recorded immediately on the addition of lysis buffer. Samples with the addition of SOD (1.0x10⁵ U/L, Sigma) were used as blank controls. Each reading was recorded as a single photon count by using a microplate scintillation counter (Topcount, Packard Instrument Co. Meriden, Colorado).

RNA Isolation and Northern Blot Analysis
After the shear stress treatment, total RNA was isolated from ECs by the guanidium isothiocyanate/phenochloroform method as described previously.¹⁶ The RNA (10 µg/lane) was separated by electrophoresis on a 1% agarose formaldehyde gel and transferred onto a nylon membrane (Nytran, Schleicher & Schuell Inc., Germany) by a vacuum blotting system (VacuGene XL, Pharmacia, Piscataway, New Jersey). After hybridization with the ³²P-labeled ICAM-1 cDNA probes, the membrane was washed with 1x SSC containing 1% SDS at room temperature for 30 minutes and then exposed to X-ray film (Kodak X-Omat-AR, Rochester, New York) at -70°C. Autoradiographic results were scanned and analyzed by using a densitometer (Computing Densitometer 300S, Molecular Dynamics, Sunnyvale, California).

Reporter Gene Construct, Transfection, and Luciferase Assay
The ICAM-1 promoter construct contained 850 bp of ICAM-1 5'-flanking DNA linked to the firefly luciferase (LUC) reporter gene of plasmids pGL2 (Promega, Inc., Madison, Wisconsin). This fragment of ICAM-1 promoter contains various locations of binding sites for transcriptional factors such as AP-1, AP-3, NF-B, C/EBP, and Ets.³⁹ DNA plasmids, purified by a Wizard Maxipreps DNA purification system (Promega, Inc., Madison, Wisconsin), were transfected into bovine aortic ECs (BAECs) at their 60% confluence level by using the lipofectamine method (Gibco). The pSV-ß-galactosidase plasmid, which contains a ß-galactosidase (ß-gal) gene driven by SV40 promoter and enhancer, was cotransfected to normalize the transfection efficiency. After transfection, cells were incubated with Dulbecco's modified eagle medium (DMEM, Gibco) containing 10% FBS overnight to reach confluence. Transfected BAECs were seeded onto slides and subjected to shear flow treatment to assess the induction of promoter activities by shear stress. After flow treatment, a cell extract was prepared and assayed for luciferase activity using the Promega Biotec assay system. To normalize the transfection efficiency for each sample, the ß-gal activities were assayed by adding the substrate, o-nitrophenyl-ß-D-galactopyranoside (ONPG) to 20 µL of cell lysate and incubated at 37°C before recording at 420 nm.

Immunofluorescence With Flow Cytometry
The expression of ICAM-1 on the surface of ECs was measured by indirect immunofluorescence using flow cytometry. Subsequent to shear stress treatment, ECs were washed with M199 three times, detached with Versene buffer containing EDTA, and centrifuged. Each sample (4x10⁵ cells was washed with PBS containing 0.5% BSA and resuspended in 0.2 mL PBS containing monoclonal antibody to ICAM-1 (R&D, Minneapolis, MN) at a saturating concentration (20 mg/L). After incubation at 4°C for 30 minutes, cells were centrifuged at 1500 rpm for 5 minutes and washed twice with PBS to remove unbound antibody. ECs were then incubated with anti-mouse IgG (Cappel, West Chester, Pennsylvania) conjugated with FITC for 30 minutes at 4°C. After two final washes in PBS, the cells were resuspended in 0.5 mL of PBS containing 10% FBS and assayed within 1 hour. Fluorescein-labeled cells (~1.0x10⁴ cells/sample) were analyzed with a flow cytofluorometer (FACScan, Becton Dickinson). Cells incubated with FITC-conjugated antibody alone were used as negative controls.

Monocyte Cell Adherence Measurements
The human monocytic cell line THP-1 was obtained from the American Type Culture Collection (Rockville, Maryland) and maintained in culture medium RPMI 1640 (Gibco) supplemented with 10% FBS, L-glutamine, and penicillin. Before adhesion experiments, THP-1 cells were suspended in RPMI 1640 containing 0.1% FBS and labeled with 1 µCi [³H]thymidine (specific activity, 23 Ci/mmol; Amersham, Buckinghamshire, UK) overnight and then washed three times in fresh RPMI 1640 before being used. After exposure of ECs to flow for 24 hours, 2x10⁵ THP-1 cells were added to shear-treated ECs and incubated for 1 hour under static condition with replenished culture medium. After incubation, nonadherent THP-1 cells were quickly removed by washing with M199. ECs with adherent THP-1 cells were lysed and radioactivity was counted by a scintillation counter.

Statistical Analysis
Results were expressed as mean±SEM. Significance was determined by using the Student's t-test, and the level of statistical significance was defined as P<.05 from 4 or 5 separate experiments for all comparisons.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Shear Stress Increases Intracellular ROS Levels in ECs
Our previous studies have shown that cyclic strain-treated ECs increased their intracellular ROS levels as measured by fluorescent intensity of the peroxidative product⁴³ and lucigenin-amplified chemiluminescence of the superoxide production.¹⁶ In the present study, the shear-induced generation of ROS was examined by measuring the production of superoxide radicals in shear-treated ECs. As shown in Fig 1, ECs increased their intracellular ROS levels as early as 15 minutes after the onset of flow, reaching a maximal level about fourfold greater than that in static controls within 30 minutes. These ROS levels then declined but still maintained an elevated level of ECs after exposure to flow for 6 hours. When the cells exposed to flow for 30 minutes were then allowed to rest under static conditions, within 5 minutes, ROS levels rapidly returned to the basal control levels. ECs that were treated with phorbol 12-myristate 13-acetate (PMA), a PKC activator, also showed an increase of ROS production. Shear-induced superoxide production was inhibited by pretreating ECs with superoxide dismutase (SOD), a superoxide radical scavenger, a result confirming that superoxide radical was a major component in this ROS measurement. In contrast, SOD treatment of controls did not significantly affect their intracellular ROS levels.¹⁶ These results indicated that this ROS induction was a shear flow-dependent phenomenon.

View larger version (18K): [in this window] [in a new window]   Figure 1. Shear stress induces intracellular ROS in ECs. ECs were lysed immediately after flow treatment with a lysis buffer containing lucigenin, and the superoxide production in ECs was measured as described in the "Methods" section. ECs were exposed to shear stress for 15 minutes (S15'), 30 minutes (S30'), 1 hour (S1), 3 hours (S3), or 6 hours (S6). Sheared ECs were allowed to rest for 5 minutes (S30'+R5'). ECs treated with PMA (100 µg/L) for 30 minutes were used as positive controls. SOD (1x105 U/L) containing buffer was used to lyse the shear-treated or PMA-treated ECs (S30'+SOD and PMA+SOD). All data are represented as mean±SEM from 4 or 5 separate experiments. *P<.05 vs control unsheared ECs. #P<.05 vs respective sheared ECs. P<.05 vs PMA-treated ECs.

ROS Mediate the Shear Stress-Induced ICAM-1 Gene Expression
To demonstrate that the increased ROS were involved in the shear-induced ICAM-1 gene expression in ECs, we examined the effects of free radical scavenger and antioxidant enzyme, ie, NAC and catalase, on the induction of ICAM-1 mRNA by shear stress. As shown in Fig 2A, ECs exposed to flow for 3 hours or 6 hours remarkably increased their ICAM-1 mRNA expression, compared to that in static controls. The shear-induced mRNA levels, however, were attenuated by the pretreatment of ECs with an antioxidant, NAC. To further confirm that ROS were involved in this shear-induced ICAM-1 expression, ECs were first subjected to shear flow for 3 hours and then followed by the same flow for another 3 hours with NAC-containing culture medium. This treatment resulted in a decreased ICAM-1 expression, compared to that of ECs exposed to either 3 or 6 hours of continuous flow in the absence of NAC. To further substantiate that ROS play an important role in modulating shear-induced ICAM-1 expression, ECs were shear-treated with medium containing an antioxidant enzyme, ie, catalase. As shown in Fig 2B, the addition of catalase in the media abrogated the induced ICAM-1 gene expression in ECs exposed to flow for either 3 or 6 hours. ECs that were incubated in a static condition with culture medium collected from the cells exposed to shear stress for 6 hours did not significantly stimulate their ICAM-1 expression (Fig 2C). This rules out the possibility of an effect by released cytokine from ECs under flow on ICAM-1 gene induction. As positive controls, ECs treated with TNF, H₂O₂, and PMA increased their ICAM-1 mRNA expression. Treatment of these ECs with NAC or catalase also showed a partial inhibition in ICAM-1 expression. These results clearly indicate that mechanical stress enhances intracellular ROS production and that increased ROS are involved in the upregulation of ICAM-1 mRNA levels in ECs.

View larger version (34K): [in this window] [in a new window]   Figure 2. ROS are involved in the shear stress-induced ICAM-1 gene expression. (A) Antioxidant NAC attenuates shear-induced and TNF-induced ICAM-1 mRNA levels. ECs were exposed to shear flow for 3 hours (S3) or 6 hours (S6). Incubation of ECs with NAC (20 mmol/L) for 1 hour was followed by exposure of ECs to shear flow for 3 hours in the presence of NAC (NAC+S3). In some experiments, ECs were subjected to shear flow for 3 hours and then followed by the same flow for another 3 hours with NAC-containing culture medium (S3+NAC+S3). ECs treated with TNF (1.2x105 U/L) for 6 hours were used as positive controls. ECs were preincubated with NAC for 1 hour before the addition of TNF for 6 hours (NAC+TNF). (B) Catalase inhibits shear-induced and TNF-induced ICAM-1 mRNA expression. ECs were preincubated with catalase (3.5x105 U/L) for 1 hour and then exposed to flow for 3 hours (CAT+S3) or 6 hours (CAT+S6) or to TNF for 6 hours (CAT+TNF) in the presence of catalase. ECs treated with TNF or H2O2 (100 µmol/L) for 6 hours were used as positive controls. (C) Incubation of ECs for 6 hours with culture medium collected from ECs preexposed to flow for 6 hours (C+Med) did not significantly stimulate their ICAM-1 mRNA expression. ECs treated with PMA (100 µg/L) for 6 hours were used as positive controls. ECs preincubated with NAC (NAC+PMA) or catalase (CAT+PMA) for 1 hour were followed with the addition of PMA and then incubated for 6 hours.

To further explore whether the transcriptional activity was involved in this shear-modulated or ROS-modulated ICAM-1 gene expression, a ICAM-1 promoter construct containing the ICAM-1 promoter region (-850 bp) and the reporter gene luciferase were used. ECs that were exposed to 1 hour of flow significantly increased ICAM-1 promoter activities, reaching a maximal level 2.5-fold greater than that in static cells by 3 hours, and then remained at an elevated level until 6 hours after the onset of flow (Fig 3). The addition of NAC or catalase to media completely abolished the induced ICAM-1 promoter activities in ECs that were exposed to flow for 3 hours. These results suggest that ROS-mediated ICAM-1 induction by shear is regulated at the transcriptional level.

View larger version (15K): [in this window] [in a new window]   Figure 3. ROS-mediated ICAM-1 induction by shear stress is a transcriptional event. Chimera containing 850 bp of ICAM-1 promoter region and reporter gene luciferase was transfected into BAECs and then subjected to shear flow for 1 (S1), 3 (S3), or 6 (S6) hours. Before flow treatment, transfected ECs were treated with NAC (20 mmol/L) (NAC+S3) or catalase (3.5x105 U/L) (CAT+S3) for 1 hour. ECs treated with TNF (1.2x105 U/L) or H2O2 (100 µmol/L) for 6 hours were used as positive controls. All data are represented as mean±SEM from 4 or 5 separate experiments. *P<.05 vs control unsheared ECs. #P<.05 vs respective sheared ECs.

Inhibitory Effects of Antioxidants on Shear-Induced ICAM-1 Expression and Monocytic Cell Adhesion on ECs
Since ECs that were pretreated with antioxidants were shown to have a reduced ICAM-1 gene expression under flow, we then investigated whether antioxidant-treated ECs also resulted in a reduction of surface expression of ICAM-1 on sheared ECs. As depicted in Fig 4, flow cytometric analysis showed that ECs that were exposed to flow for either 12 or 24 hours increased their ICAM-1 expression on ECs, as indicated by a shift of mean fluorescence intensity, compared to the static controls. These flow-induced shifts of mean fluorescence intensity were significantly reduced by pretreating ECs with NAC or catalase. As positive controls, ECs incubated with TNF or H₂O₂ increased their ICAM-1 expression. These results further confirm that ROS are involved in shear-induced ICAM-1 gene expression that ultimately affects ICAM-1 expression on ECs' surface. The consequence of this inhibitory effect of antioxidant on ICAM-1 expression in shear-treated ECs is the reduced adhesion of monocytic THP-1 cells to these ECs. As shown in Fig 5, ECs that were preexposed to flow for 24 hours increased the adhesion of THP-1 cells about twofold compared to that of static controls. NAC-pretreated or catalase-pretreated ECs significantly inhibited the adhesion of THP-1 cells to these sheared ECs. In contrast, these antioxidant treatments of control ECs did not significantly affect the basal adherence of THP-1 cells to ECs (data not shown). Significant inhibition of the monocytic adhesion on shear-treated ECs by preincubation of shear-treated ECs with antibody to ICAM-1 indicated that the ICAM-1 expressed on shear-treated ECs was a major adhesion protein contributing to increased monocytic adhesion. Similar to previous findings,¹⁵ ICAM-1 antibody produced only a minor effect in reducing THP-1 cell adherence to control ECs (data not shown). Thus, antioxidant pretreatment of ECs can inhibit shear-induced ICAM-1 expression that ultimately reduces endothelial interaction with circulating monocytes.

View larger version (25K): [in this window] [in a new window]   Figure 4. NAC or catalase attenuates shear-induced ICAM-1 expression on ECs. ECs exposed to shear flow for 12 or 24 hours were analyzed by flow cytometer. Before shear-stress treatment, ECs were pretreated with NAC (20 mmol/L) or catalase (3.5x105 U/L) for 1 hour and then subjected to shear flow in the presence of NAC or catalase. As positive controls, ECs were stimulated with TNF (1.2x105 U/L) or H2O2 (100 µmol/L). ECs incubated only with FITC-conjugated antibody were used as blank controls.

View larger version (16K): [in this window] [in a new window]   Figure 5. NAC or catalase inhibits the adhesion of monocytes to sheared ECs. ECs after shear flow for 24 hours were incubated with 3H-labeled monocytic THP-1 cells under static conditions with replenished medium. After removal of nonadherent THP-1 cells, ECs were lysed, and total radioactivity was counted (S24). ECs pretreated with NAC (20 mmol/L) or catalase (3.5x105 U/L) for 1 hour were then subjected to shear flow for 24 hours in the presence of NAC (NAC+S24) or catalase (CAT+S24). Anti-ICAM-1 (20 µg/mL) was incubated with sheared ECs before the introduction of THP-1 cells (S24+Ab). ECs treated with TNF (1.2x105 U/L) or H2O2 (100 µmol/L) for 24 hours were used as positive controls. Each column represents mean±SEM from 4 or 5 separate experiments. *P<.05 vs control unsheared ECs. #P<.05 vs respective sheared ECs.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Cells respond to various chemical agents, including cytokines and growth factors, by increasing intracellular ROS that may act as second messengers.^{24 25 26} These ROS have been indicated to modulate signaling pathways, including protein kinases,⁴⁴ phosphatases,⁴⁵ mitogen-activated protein kinase (MAPK),⁴⁶ and c-jun NH₂-terminal kinase (JNK).²⁶ Recent data even indicate that p21^ras is a common signaling target of reactive free radicals.⁴⁷ Not surprisingly, the downstream of signaling pathways, ie, the activation of transcriptional factors, including NF-B and AP-1, has also been shown to be modulated by intracellular ROS levels.^{29 30 31}

Involvement of ROS in cytokine-induced ICAM-1 expression in ECs has been shown.³⁸ Similarly, ECs treated with H₂O₂ also increased their ICAM-1 expression.³⁷ However, the molecular mechanisms by which intracellular ROS participate in hemodynamically induced endothelial response remain unclear. It has been reported that reperfusion-generated ROS are responsible for leukocyte adhesion to the endothelium.²⁷ Laurindo et al²⁸ have recently shown that the release of ROS from ECs is a flow-dependent phenomenon. Our studies have shown that ROS are involved in the cyclic strain-induced plasminogen activator inhibitor-1 secretion⁴³ and MCP-1 gene expression.¹⁶ Using a parallel-plate flow chamber system, we have also indicated that ROS contribute at least partially to the shear-induced c-fos expression.⁴⁸ Our present results further confirm that shear flow can increase ROS levels that are involved in ICAM-1 expression in ECs. Several lines of evidence support this notion. First, the production of superoxide radicals was increased in ECs that were exposed to shear flow. These ROS remained at an elevated level as shear flow continued. Second, shear-induced ICAM-1 expression was inhibited by pretreating ECs with an antioxidant, NAC. Third, the addition of catalase to cultured ECs to remove the intracellular generated H₂O₂ abolished shear-induced ICAM-1 expression. Fourth, antioxidant treatment reduced shear-increased ICAM-1 promoter activities. Finally, shear-induced ICAM-1 expression on the surface of ECs, as demonstrated by flow cytometry, was attenuated by pretreating ECs with NAC or catalase. These antioxidant treatments of ECs resulted in a decreased adhesion of monocytes to shear-treated ECs. In the present study, we have used NAC and catalase to treat ECs. The addition of NAC, a glutathione precursor, provided sufficient glutathione that reduced the intracellular ROS concentration via the glutathione peroxidase pathway.⁴⁹ Extracellularly added catalase, which is unlikely to have diffused into ECs, appears to rapidly clear the intracellularly generated H₂O₂, which is permeable to cell membranes.^{16 50} To demonstrate ROS effects, we treated ECs with H₂O₂. This treatment, however, elicited a lower ICAM-1 expression than that by shear or TNF. This could be due to the low concentration of H₂O₂ that we applied to ECs. Alternatively, the extrageneously added H₂O₂ may not completely reflect the effects from shear-induced ROS generated intracellularly. This ROS-mediated induction of the ICAM-1 gene by shear was unlikely a secondary effect of flow-induced release of cytokine or vasoactive substance from ECs due to the immediate response nature of ROS generation and promoter activities. In addition, treatment of ECs for 6 hours with culture medium collected from the cells that were preexposed to flow did not significantly stimulate ICAM-1 expression. This rules out the possibility of a secondary effect from the medium. Our findings clearly indicate that shear stress can induce ICAM-1 expression in ECs by using ROS as activating intermediates.

ECs adapt themselves to their hemodynamic environment by altering cellular physiology including gene modulation. Initial studies by Resnick et al⁵¹ defined a shear stress-responsive element (SSRE) in the promoter of the platelet-derived growth factor B-chain gene that was required for its induction by shear stress. Later studies by Khachigian et al³³ further indicated that the transcriptional factor that interacts with this core sequence was NF-B. Recent studies by Shyy et al³⁴ have identified another shear-responsive element in the shear-induced MCP-1 gene expression that corresponds to the AP-1 binding site. NF-B and AP-1 have been demonstrated to be involved in the regulation of ICAM-1 expression by various stimuli including cytokines.³¹ However, shear-stimulated transcriptional factors NF-B and AP-1 in ECs have also been reported.^{32 33 34} Since the ICAM-1 promoter region (-850 bp) contains various putative shear-responsive elements, including SSRE and a number of NF-B-specific and AP-1-specific binding sites,³⁹ it is very likely that these binding sites are activated by shear-induced ROS. Our recent studies of deletion constructs, however, indicate that different cis-acting elements in the promoter region of the ICAM-1 gene are involved in the cellular response to cytokine and shear flow (unpublished observation). The NF-B binding site in the distal promoter region is crucial for a cytokine or TNF effect. In contrast, the deletion of the NF-B binding site did not affect the shear inducibility. Although TNF-induced effects in cells involve ROS, this ROS induction apparently is not the sole mediator for this ICAM-1 induction, since antioxidants only partially inhibit TNF-induced ICAM-1. All of these results are consistent with previous reports, indicating that oxidant-induced ICAM-1 expression is mediated by a distinct pathway from that involved in the TNF response.³¹ Our data suggest that ROS act as important modulators for shear-induced gene expression. We have recently demonstrated that cyclic strain-induced MCP-1 gene expression involves the activation of AP-1 binding in the promoter region by ROS.¹⁶ The intracellular signals induced by mechanical forces are complex and unclear. However, other cis-acting elements involved in shear-induced gene expression cannot be excluded. The results of ICAM-1 promoter activities suggest that the induction of ICAM-1 by shear stress via ROS is mediated at the transcriptional level. These data provide further support for the notion that intracellular ROS may modulate signal pathways with subsequent alteration of gene transcription. The molecular mechanisms by which shear force leads to increased ROS and the subsequent alteration of gene expression in vascular ECs remain an important question that warrants further investigation.

Studies by Tsao et al^{52 53} indicated that ECs that were preexposed to shear stress reduced endothelial adhesiveness to monocytes. They concluded that this phenomenon was contributed by flow-induced NO rather than alterations in the expression of adhesion molecules. In contrast, other studies^{10 12} indicated that ECs that were exposed to shear increased their surface expression of ICAM-1, which subsequently promoted leukocyte adhesion. De Caterina et al⁵⁴ recently demonstrated that NO could inhibit cytokine-induced expression of adhesion molecules in ECs such as VCAM-1 and E-selectin but not ICAM-1. Moreover, Khan et al⁵⁵ further indicated that this inhibitory effect was NO concentration dependent and regulated at transcriptional level by a redox-sensitive mechanism in ECs. The discrepancy between our results and those in studies by Tsao et al^{52 53} regarding increasing or inhibiting monocyte adhesion to sheared ECs may be due to the locally effective NO concentration generated by different shear devices. Although the intracellular NO and ROS and their interplay are important in determining the endothelial responses, the effect of flow-induced NO on intracellular ROS production and ICAM-1 expression remains unclear. Our present study clearly indicates that the increase in adhesion of monocytic THP-1 cells on ECs preexposed to flow for 24 hours is the result of increased ICAM-1 expression on sheared ECs.

Recent evidence has suggested that hypertension accelerates atherosclerosis in part because of synergy between elevated blood pressure and other atherogenic stimuli to induce oxidative stress on the arterial wall.⁵⁶ Involvement of ROS in the development of atherosclerosis and hypertension has been implicated by the finding that SOD attenuates endothelial dysfunction⁵⁷ and decreases blood pressure in spontaneously hypertensive rats.⁵⁸ Recent studies^{35 36} have suggested that ECs provide a potential atheroprotective mechanism against fluid mechanical stimulation by upregulation of superoxide dismutase enzymes. These reports together with our present results of ROS generation by shear flow and shear-modulated or oxidant-modulated ICAM-1 expression strongly suggest that intracellular ROS may play an important role in shear-induced gene expression. Imbalance of these ROS levels caused by impaired or insufficient antioxidative mechanisms in ECs may contribute to the pathogenesis of atherosclerosis and hypertension. The inhibitory effect of antioxidant on shear-induced or oxidant-induced ICAM-1 expression may offer some clues for the prevention of cardiovascular disorders. This shear flow-induced ROS generation may also provide some insights into the basis of reperfusion-induced vascular injuries.

In summary, our present studies clearly demonstrate that intracellular ROS levels in ECs can be induced by shear stress and that these increased ROS are involved in shear-induced ICAM-1 expression. Our results thus emphasize the importance of intracellular ROS in the modulation of hemodynamically induced endothelial responses.

	Acknowledgments

 
This work was supported in part by grant NSC 86-2314-B001-004 M26 from the National Science Council, Taiwan, ROC.

Received February 26, 1997; accepted August 20, 1997.

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