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(Hypertension. 1998;31:125.)
© 1998 American Heart Association, Inc.

Scientific Contributions

Cyclic Strain–Induced Reactive Oxygen Species Involved in ICAM-1 Gene Induction in Endothelial Cells

Jing-Jy Cheng; Being-Sun Wung; Yeun-Jen Chao; Danny Ling Wang

From the Graduate Institute of Life Sciences (J.-J.C., B.-S.W.), National Defense Medical Center and Cardiovascular Division (Y.-J.C., D.L.W.), Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, ROC.

Correspondence to Danny Ling Wang, PhD, Cardiovascular Division, Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, ROC. E-mail lingwang{at}ibms.sinica.edu.tw

	Abstract

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Abstract—Vascular endothelial cells (ECs) are constantly subjected to pressure-induced strain. We have previously demonstrated that strain can induce intercellular adhesion molecule-1 (ICAM-1) expression in ECs. The molecular mechanisms of gene induction by strain, however, remain unclear. Recent evidence suggests that intracellular reactive oxygen species (ROS) may act as second messengers. The potential role of ROS in strain-induced ICAM-1 expression was examined. ECs grown on a flexible membrane base were deformed with various sinusoidal negative pressures to produce an average strain of 12%. Cyclic strain induced an increase in intracellular ROS measured by fluorescent intensity of dichlorofluorescein formed after peroxidation. Maximal levels of ROS were seen after 30 minutes. Levels subsequently decreased but remained elevated compared with unstrained groups. Concomitantly, a sustained increase of H₂O₂ decomposition activity was observed in strained ECs. Both ROS and H₂O₂ decomposition activity returned to basal levels after removal of the strain. ECs treated with an antioxidant (N-acetylcysteine or catalase) inhibited strain-induced ROS generation and ICAM-1 mRNA levels followed by decreased ICAM-1 expression on EC surfaces. This inhibition may account for the reduced monocytic cell adhesion in antioxidant-treated ECs but not in strained controls. Our findings indicate that cyclic strain–induced monocyte adhesion to ECs is mediated, at least in part, by an increase of ICAM-1 gene expression via the elevation of ROS levels in strained ECs. Our results support the importance of intracellular ROS in the modulation of hemodynamic force–induced endothelial responses.

Key Words: cyclic strain • reactive oxygen species • ICAM-1 • gene expression • monocyte • endothelial cell

	Introduction

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Vascular ECs, which play an important role in maintaining normal vascular homeostasis, are constantly exposed to mechanical forces, including blood flow–induced shear stress and pressure-induced strain. Effects of shear stress on ECs have been largely acknowledged^{1 2} and recognized to include modulation of immediate early genes, such as c-fos,³ and the release of many important substances such as prostacyclin,⁴ tissue plasminogen activator,⁵ ET-1,⁶ and nitric oxide.⁷ The effects of strain on ECs, however, remain relatively unclear. Studies have indicated that ECs subjected to pulsatile flow treatment express higher c-fos genes than those cells under steady flow treatment.³ Because rhythmic distension of the vessel wall is a component of pulsatile flow, cyclic strain on vessel walls may thus play an important role in modulation of gene expression. Previous studies from our laboratory as well as others have indicated that mechanical strain can modulate endothelial secretion of vasoactive materials, including ET-1,^{8 9 10 11} endothelium-derived nitric oxide,¹² and prostacyclin.^{11 13} Subsequent studies from this laboratory have shown that mechanical strain can stimulate the chemotaxis and adhesion of monocytes to ECs by increasing MCP-1^{14 15} and ICAM-1¹⁶ expression in ECs. Evidence showing that strain-induced gene modulation is mediated predominantly through the protein kinase C pathway and Ca²⁺ influx has also been provided.^{9 14} Although the effects of strain on gene regulation have recently been studied, the molecular mechanisms by which mechanical deformation leads to increased gene expression and protein release remain largely uncharacterized.

ECs producing various adhesion molecules, including ICAM-1 following stimulation with cytokines, have been studied intensively.¹⁷ During atherogenesis, ICAM-1 plays an important role in the recruitment and accumulation of monocytes/macrophages in the subendothelial space of the arterial wall. Recent studies indicate that, in addition to cytokines, fluid shear stress can also stimulate the expression of ICAM-1 gene in ECs. A shear-stress response element (ie, GAGACC) that appears in the 5' promoter region of the ICAM-1 gene as well as in other genes has been proposed to be responsible.¹⁸ Studies by Khachigian et al¹⁹ further indicate that NF-B interacts functionally with the shear-stress response element in ECs exposed to fluid shear stress. Shyy et al²⁰ recently identified a new hemodynamic regulatory element identical to phorbol ester–responsive element. The transcription factor that binds to the phorbol ester–responsive element is AP-1. Both NF-B and AP-1 are transcription factors involved in the induction of many genes, and their activation can be initiated by various stimuli. NF-B and AP-1 activation thus play important roles in cellular adaptation to environmental changes during pathophysiological states, including hypertension and atherogenesis.

Recent evidence suggests that ROS may act as second messengers and thus have an affect on gene expression of various proteins, including ICAM-1 during cytokine treatment.^{21 22 23} Although the detailed molecular mechanisms are not clear, ROS activation of the NF-B and AP-1 transcription factors has been reported.^{22 23 24} Because hemodynamic forces, including shear stress and strain, can upregulate genes that are believed to involve the activation of AP-1 and NF-B,^{19 25} it is of interest to know whether ROS participate in these hemodynamic force–induced cellular responses. The present study clearly demonstrates that mechanical strain can increase intracellular ROS levels and that these increased ROS are involved in the strain-induced ICAM-1 expression. These findings thus emphasize the importance of intracellular ROS as a modulator in the regulation of hemodynamic force–induced gene expression in vascular ECs.

	Methods

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Cell culture
Human umbilical vein ECs were isolated from human umbilical cord according to the procedure described previously.²⁶ ECs were cultured for 3 days in Petri dishes. Confluent primary ECs were removed by 1% trypsin and then seeded onto the silical membrane base of a Flexicell cell plate (Flexcell International Co) until the monolayer became confluent. The original culture medium was then replaced with medium 199 containing 2% FCS, and ECs were incubated overnight before each experiment.

In Vitro Cyclic Strain on Cultured ECs
The strain unit (Flexcell FX-2000, Flexcell International Co) consisted of a vacuum unit linked to a computerized valve control unit, described in detail elsewhere.^{27 28} This strain device provides a nonuniform stretch. Strain profile analysis²⁸ indicates that radial strain responds nonlinearly to increasing vacuum pressure. Peak radial strain occurs at the radial distance of 9.5 mm from the center. The flexible membrane supporting the cultured cells was deformed by a sinusoidal negative pressure with a peak level of -20 kPa, which produces a strain on cells ranging from minimal strain at the center of the membrane to a peak value of 25% at the periphery (maximal strain, 25%; average strain, 12%) at a frequency of 1 Hz (60 cycles/min) maintained at various intervals. The average strain was calculated by averaging radial strain over the total plate surface area.^{16 28} To simplify the description, we referred this group as the 12% average strain group. After the strain experiment, total RNAs were collected for analysis of mRNA levels of ICAM-1. For free radical analysis, strained ECs were trypsinized and resuspended at 3x10⁸ cells/L. For antioxidant experiments, NAC (20 mmol/L) or catalase (3.5x10⁵ U/L) was introduced into culture media 0.5 hour before the strain treatment.

Measurement of Intracellular ROS
Intracellular ROS were estimated by a method previously described.²⁹ Briefly, strained ECs, after trypsinization, were resuspended in PBS with a final density of 3x10⁸ cells/L immediately followed by incubation with 20 µmol/L of nonpolar 2',7'-DCFH (Serva, Germany) at 37°C for 30 minutes in the dark. Reaction was stopped by centrifugation at 200g for 5 minutes, and the cell pellets were resuspended in PBS. The relative fluorescence intensity of fluorophore DCF, which was formed by peroxide oxidation of its nonfluorescent precursor, was detected at an emission wavelength of 525 nm by use of an excitation wavelength of 475 nm with a Hitachi 4010 fluorescence spectrophotometer. DCFH with fresh culture medium was used as a blank control.

Measurement of H₂O₂ Decomposition Activity
H₂O₂ decomposition rate was measured as previously described.³⁰ Briefly, cells were removed from the plate by a rubber policeman. The strain-treated cells collected in 50 mmol/L potassium phosphate buffer (pH 7.0) were rapidly frozen at -70°C overnight. Cells were thawed and centrifuged, and aliquots of supernatant containing 50 µg protein were collected. H₂O₂ decomposition rate was determined spectrophotometrically by measurement of the decomposition of exogenously added H₂O₂ (10 mmol/L). The rate of disappearance of H₂O₂ was followed by observance of the rate of decrease in absorbance at 240 nm. One unit is defined as 1 µmol/min H₂O₂ decomposition at pH 7.0 at room temperature. In some experiments, ECs were pretreated for 0.5 hour with NAC (20 mmol/L), TNF (1x10⁵ U/L), H₂O₂ (0.1 mmol/L), or specific catalase inhibitor, ie, 3-amino-1,2,4-triazole (3-ATA, 20 mmol/L) before strain treatment.

Northern Blot Analysis
Total RNA isolation was obtained with guanidine isothiocyanate as previously described.¹⁴ RNA was transferred onto membrane and then hybridized with ³² P-labeled ICAM-1 cDNA. Autographic results were scanned and analyzed by a densitometer (Computing Densitomer 300S, Molecular Dynamics).

Flow Cytometry
After strain treatment, ECs were washed three times with M199, detached with PBS buffer containing EDTA, and centrifuged. Each pellet was washed with PBS containing 0.5% BSA and resuspended in 0.2 mL PBS containing monoclonal antibody to ICAM-1 at a saturating concentration (20 µg/mL). After incubation at 4°C for 30 minutes, cells were centrifuged (13 000 rpm for 5 minutes) and washed twice with PBS. ECs were then incubated in secondary antibody with FITC-labeled anti-mouse IgG (Sigma) for 30 minutes at 4°C. After a wash in PBS, the ECs were fixed in 4% paraformaldehyde in PBS and analyzed by a flow cytometer (FAC-Scan; Becton Dickinson).

Cell Adherence Measurements
The human monocytic cell line THP-1 was obtained from American Type Culture Collection. THP-1 cells were suspended in RPMI 1640 containing 0.1% FCS and labeled with 1 µCi [³H]thymidine (specific activity, 23 Ci/mmol; Amersham) overnight. Cells were washed three times in fresh RPMI 1640 culture medium, and 3x10⁵ cells were added to each well containing ECs and incubated for 1 hour. Nonadherent THP-1 cells were removed by washing with M199. ECs with adherent THP-1 cells were lysed with lysis buffer, and radioactivities were counted by a scintillation counter. For antioxidant experiments, ECs were pretreated with NAC for 0.5 hour before strain treatment. Strained or unstrained ECs were then washed with M199 three times before the introduction of THP-1 cells.

Statistical Analysis
Statistical analysis was performed with Student’s t test. Data are presented as mean±SEM. Statistical significance was defined as P<.05.

	Results

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Cyclic Strain Increases Intracellular ROS Levels in ECs
To analyze the intracellular ROS generated by strain, ECs were incubated with DCFH, and the fluorescent intensities of oxidized product DCF were measured. As shown in Fig 1, the intracellular ROS were maintained at basal levels in unstrained control cells. In contrast, ECs subjected to 12% average strain rapidly increased their ROS levels and reached a maximum by 30 minutes after strain (Fig 1A). The ROS levels then declined but still maintained a sustained elevated level as strain was maintained. ECs treated with TNF or LPS also showed a sustained but higher magnitude of increase in ROS levels. The strain-induced ROS production was basically serum independent, because strained ECs cultured in either 2% or 20% FCS showed similar ROS induction (Fig 1B) despite the lower basal ROS levels of ECs under a 20% serum condition. To further demonstrate that this ROS generation was strain dependent, ECs, after a 30-minute exposure to strain, were allowed to recover, and the ROS were measured. As shown in Fig 1B, ROS in previously strained ECs gradually returned to control basal levels after 2 hours of recovery. These strain-induced ROS could be inhibited by pretreatment of ECs with NAC (Fig 1C). The presence of catalase in the medium also attenuated the strain-induced ROS levels. In contrast, catalase exhibited no effect on unstrained control cells. These data indicate that this ROS generation is cyclic strain dependent but serum independent.

View larger version (19K): [in this window] [in a new window]   Figure 1. Cyclic strain increases reactive oxygen species in human ECs. Strained ECs were immediately removed from plates and incubated with nonpolar DCFH. Intracellular ROS were analyzed by fluorescence intensity of fluorophore DCF. A, Intracellular ROS levels from ECs strained at various time intervals. TNF and LPS-treated ECs were used as positive controls (C). B, ROS levels from ECs cultured either in 20% or 2% FBS. Cells were treated with 12% strain (S) for 0.5 hours followed by 0.5 hours (S+R0.5) or 2 hours (S+R2) of rest before ROS analysis. C, ROS were measured from unstrained or strained ECs (12%, 0.5 hour) pretreated with an antioxidant, NAC (20 mmol/L) or catalase (3.5x105 U/L), for 0.5 hours. All data are mean±SEM from three to five experiments. *P<.05 vs unstrained control cells. +P<.05 vs unstrained control ECs cultured with 20% FBS in B. #P<.05 vs strained ECs in B and in C.

Cyclic Strain Increases Antioxidant Activity in ECs
Because ECs under strain increased their ROS generation, the intracellular antioxidant activity, ie, H₂O₂ decomposition activity, in strained cells was analyzed. As shown in Fig 2A, H₂O₂ decomposition activities of ECs under strain treatment rapidly increased, and these increased levels were sustained as strain was maintained. This elevation of strain-induced H₂O₂ decomposition activity was comparable to the increase in ECs under H₂O₂ or TNF treatment. To demonstrate further that the increased activity was strain dependent, ECs, after 1 hour of exposure to strain, were allowed to recover, and these activities were measured. As shown in Fig 2B, elevated activities in previously strained cells gradually returned to control basal levels after 2 hours of recovery. The strain-induced H₂O₂ decomposition activity could be inhibited by pretreatment of ECs with the antioxidant NAC. To demonstrate that the increased H₂O₂ decomposition was due to the catalase in strained ECs, the ECs were pretreated with a specific catalase inhibitor, 3-ATA, before strain treatment. As shown in Fig 2B, the H₂O₂ decomposition activities in those strained cells were greatly inhibited. These results indicate that strain induces ROS generation in ECs accompanied by an increase in intracellular catalase activities.

View larger version (19K): [in this window] [in a new window]   Figure 2. Cyclic strain increases antioxidant activity in ECs. A, ECs after strain (12%) treatment for 1, 3, 24, and 48 hours were removed from plates. The H2O2 decomposition activities were measured spectrophotometrically by the rate of disappearance of H2O2. ECs pretreated with H2O2 or TNF were used as positive controls. B, ECs were strained with 1 hour (S1) followed with 0.5 hour (S1+R0.5) or 2 hours (S1+R2) of rest before the H2O2 decomposition analysis. ECs were pretreated with NAC (20 mmol/L, NAC+S1) or 3-ATA (20 mmol/L, 3-ATA+S1) for 0.5 hour, respectively, before strain treatment. Results are represented as mean±SEM from four to six experiments. *P<.05 vs unstrained control ECs (C). #P<.05 vs strained ECs (S1) in B.

ROS Are Involved in Strain-Induced ICAM-1 mRNA Levels and Expression
Our previous studies have shown that ECs subjected to strain treatment induce ICAM-1 mRNA levels and ICAM-1 expression on the cell surface as well as an increase of secretion of soluble ICAM-1.¹⁶ To demonstrate that ROS are involved in strain-induced ICAM-1 gene expression in ECs, ECs were treated with an antioxidant, NAC or catalase, before strain treatment. As shown in Fig 3, NAC and catalase treatments significantly inhibited the strain-induced ICAM-1 mRNA levels. In contrast, NAC and catalase had little effect on basal levels from unstrained control cells. For comparison, thrombin-induced or platelet-derived growth factor–induced ICAM-1 gene expression was not significantly affected by this catalase pretreatment of ECs (data not shown). As a consequence of decreasing ICAM-1 mRNA levels, these NAC-treated strained ECs resulted in a decrease of ICAM-1 expression on the EC surface (Fig 4). NAC treatment had a minor effect (13% decrease) in ICAM-1 expression in unstrained controls. In contrast, NAC treatment of strained groups caused a significant decrease of 40% in ICAM-1 expression compared with strained but untreated ECs. Together, these results indicate that strain-induced ICAM-1 expression is modulated by intracellular ROS levels generated during strain.

View larger version (22K): [in this window] [in a new window]   Figure 3. Antioxidant inhibits strain-induced ICAM-1 mRNA levels. ECs were in unstrained control conditions (C) or subjected to strain (S), TNF, or H2O2 treatment for 3 hours. In some experiments, ECs were pretreated with NAC or catalase for 0.5 hour before the strain treatment (NAC+S or Cat+S). ECs pretreated with TNF or H2O2 were used as positive controls. Cells were collected for RNA extraction. Total RNAs were analyzed for ICAM-1 level by Northern blot hybridization with ICAM-1 probe. Equal amounts of RNA applied to each lane are demonstrated by the 18S RNA shown for each lane. Data are representative of duplicate experiments with similar results.

View larger version (17K): [in this window] [in a new window]   Figure 4. Antioxidant inhibits strain-induced expression of ICAM-1 on strained ECs. ECs were treated with antioxidant (NAC) 0.5 hour before strain treatment. After strain (12%) for 24 hours, the cells were removed. Washed ECs were incubated with monoclonal antibody to ICAM-1 followed by treatment with FITC-labeled anti-mouse IgG. These FITC-labeled ECs were then analyzed by flow cytometry. Results are represented as mean fluorescence intensity (MFI) per 104 cells. *P<.05 vs unstrained control cells. #P<.05 vs untreated strained cells.

Antioxidant Pretreated Strained ECs Reduced the Adhesion of Monocytic THP-1 Cells
Previous studies^{14 16} from this laboratory indicated that strained ECs increased the adhesion of monocytes or THP-1 cells because of the increase of ICAM-1 expression on the cell surfaces. Because antioxidant-treated cells were shown to have a reduced ICAM-1 expression, antioxidant-treated strained ECs were tested for their ability to attract monocytic THP-1 cells. As shown in Fig 5, NAC-treated ECs only slightly inhibited the adhesiveness of THP-1 cells to control unstrained ECs, whereas they showed a significant inhibition in the strained groups. These results clearly demonstrate that ROS are involved in the strain-induced ICAM-1 expression on ECs that further results in an increase of adhesiveness to monocytic cells.

View larger version (18K): [in this window] [in a new window]   Figure 5. Antioxidant inhibits strain-induced adhesion of monocytic cells to ECs. ECs were treated with antioxidant (NAC) 0.5 hour before and during the strain (12%) treatment for 24 hours. Wells containing washed ECs were then incubated with [3H]-labeled THP-1 monocytic cells for 1 hour. Adherent THP-1 cells were lysed, and radioactivity was counted. *P<.05 vs unstrained control cells. #P<.05 vs untreated strained cells.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
ROS, including H₂O₂, are constantly produced in cells during electron transfer reaction.³¹ An increase in secretion of H₂O₂ has been observed in cells stimulated with cytokines, phorbol esters, and growth factors.^{31 32 33} Studies suggest that H₂O₂ may act as a signaling molecule in stimulated ECs²³ and in platelet-derived growth factor–treated smooth muscle cells.²⁹ ROS, thus, have been proposed as second messengers in cell signaling.^{21 22} During atherogenesis, oxidative stress has been suggested to be involved in endothelial activation.³⁴ When ECs are under oxidative stress, the transcriptional factors NF-B and AP-1 can be activated to induce certain redox-sensitive genes, including ICAM-1 and vascular cell adhesion molecule.^{22 23 34 35 36 37 38} Later studies have shown that antioxidant inhibits NF-B activation and oxidative stress–inducing effects on ECs and thus support the premise that ROS act as second messengers in ECs.^{22 39 40} However, the molecular mechanisms of the release of ROS and their effects on cellular integrity remain largely unclear.

Studies indicate that shear stress and strain can activate transcriptional factors NF-B and AP-1.^{19 25} In addition, NF-B and AP-1 can be activated by H₂O₂.^{23 24 41} ROS have been suggested to be involved in activating NF-B in atherosclerotic lesions.³⁴ Hemodynamic forces involved in atherogenesis are well recognized. However, neither the intracellular ROS levels nor the role of ROS in hemodynamic force–induced gene expression has been fully defined. Recent ex vivo and in vivo evidence indicates a flow-dependent release of free radicals from ECs.⁴² Our results demonstrate that ROS can be induced in cultured ECs under strain treatment. This ROS generation is strain dependent, because ECs exposed to a genuine fluid agitation by a rotary shaker (60 cycles/min) did not increase their ROS levels (data not shown). These strain-induced ROS are involved in the strain-induced ICAM-1 expression, because antioxidant treatment of ECs inhibits both the ROS and ICAM-1 inductions. The inhibitory effect of antioxidants on the adhesion of strain-induced monocytic cells to ECs further supports the notion that induced ROS can have a profound effect on monocytes/EC interaction.

Cyclic strain induces a rapid ROS generation that is maintained at elevated levels as strain remains. Elevated ROS do not appear to harm cells, because ECs remained not only morphologically but also functionally intact, as demonstrated by the lack of DNA fragmentation and no significant increase in lactate dehydrogenase activity in the culture medium (data not shown). In addition, the quantity and the quality of total RNA isolated from strained ECs appear to be normal and similar to those of unstrained ECs. However, because of the damaging potential of ROS, cells depend on elaborate defense mechanisms to rapidly metabolize these toxic intermediates to prevent significant free radical injury. Among these defense mechanisms, catalase plays a crucial role in removing H₂O₂ from cells. Under unstrained conditions, catalase activities in ECs have been estimated to be 19 U/mg protein, a value comparable to those in previous studies.^{43 44} Strain treatment rapidly induced catalase activities: an 15-fold increase compared with unstrained control cells. Because the increase of catalase activity was a rapid response, this increase could not be a transcriptional event, although we cannot rule out the possibility of transcriptional effects after longer strain treatment. Considering the excess intracellular catalase, the rapid increase of H₂O₂ decomposition may indicate enzyme relocation or access to a substrate. Nevertheless, these increases are comparable to those induced in ECs treated with H₂O₂ or TNF. This rapid induction of catalase activities is strain dependent, because catalase activities return to basal levels after strain is removed, a pattern that corresponds to ROS levels. In addition, these strain-induced catalase activities can be inhibited by pretreating strained ECs with antioxidant or catalase inhibitor. Our studies clearly indicate that the strain can specifically induce intracellular ROS levels and that their potential damaging effects can be prevented or attenuated by the concomitant increase of catalase activities.

This strain-dependent induction of ROS returned to basal levels 2 hours after the strain was removed. We have previously shown that strain-induced mRNAs of ET-1 and MCP-1 returned to their basal expression level 2 to 3 hours after the strain was removed.^{9 14} The strong correlation in pattern between gene induction-regression and intracellular ROS levels suggests that strain-induced gene expression is mediated through ROS. The involvement of ROS in ICAM-1 induction is supported by the H₂O₂ treatment of ECs.^{23 45} It is suggested that H₂O₂ activation of ICAM-1 transcription is mediated through AP-1 elements.²³ Similarly, we have recently shown that strain-induced MCP-1 gene expression involves ROS activation of AP-1 binding sites.⁴⁶ Although different methods were used to measure ROS levels, the ROS induction by strain is confirmed, because ROS levels measured by peroxidative product in the present study are in total agreement with the superoxide levels in our previous study⁴⁶ in terms of magnitude of the increase and the induction pattern. Studies indicate that antioxidant treatment of ECs attenuates the agonist-induced or oxidized LDL–induced effects on ECs.^{38 47 48} Our recent data suggest that antioxidant treatment of ECs can inhibit the strain-induced ICAM-1 promoter activities (unpublished data, 1997). In addition, results of the inhibitory effects of catalase on strain-induced MCP-1⁴⁶ and ICAM-1 expression support the importance of ROS in the gene induction. All these findings are consistent with our theory that ROS are involved in the expression of these strain-inducible genes. Nevertheless, the present study strongly suggests that ROS act as common signals in the response of ECs to either chemicals or mechanical stimuli.

In summary, the present study demonstrates clearly that cyclic strain to ECs can induce ROS and that this increased ROS level is involved in the strain-induced ICAM-1 expression. Our results thus emphasize the importance of intracellular ROS in the modulation of hemodynamic force–induced gene expression in vascular ECs. The underlying mechanisms by which mechanical deformation leads to increased ROS and subsequent modulation of gene expression remain an important question and warrant further studies.

	Selected Abbreviations and Acronyms

 

3-ATA = 3-amino 1,2,4,-triazole DCF = 2',7'-dichlorofluorescein DCFH = 2',7'-dichlorofluorescein diacetate EC = endothelial cell ET-1 = endothelin-1 ICAM-1 = intercellular adhesion molecule-1 LPS = lipopolysaccharide MCP-1 = monocyte chemotactic protein-1 NAC = N-acetylcysteine NF-B = nuclear factor-B ROS = reactive oxygen species TNF = tumor necrosis factor

	Acknowledgments

 
This work was supported in part by Academia Sinica and by a grant (NSC86–2314-B-001–004-M26) from the National Science Council, Taiwan, ROC.

Received January 29, 1997; first decision April 7, 1997; accepted July 11, 1997.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Davies PF. Flow-mediated endothelial mechanotransduction. Physiol Rev. 1995;75:519–560.[Abstract/Free Full Text]
2. Frangos JA, ed. Physical Forces and the Mammalian Cell. San Diego, Calif: Academic Press; 1993.
3. Hsieh HJ, Li NQ, Frangos JA. Pulsatile and steady flow induces c-fos expression in human endothelial cells. J Cell Physiol. 1993;154:143–151.[Medline] [Order article via Infotrieve]
4. Frangos JA, Eskin SG, McIntire LV, Ives CL. Flow effects on prostacyclin production by cultured human endothelial cells. Science. 1985;227:1477–1479.[Abstract/Free Full Text]
5. Diamond S, Eskin SG, McIntire LV. Fluid flow stimulates tissue plasminogen activator secretion by cultured human endothelial cells. Science. 1989;243:1483–1485.[Abstract/Free Full Text]
6. Kuchan MJ, Frangos JA. Shear stress regulates endothelin-1 release via protein kinase C and cGMP in cultured endothelial cells. Am J Physiol. 1993;264:H150–H156.[Medline] [Order article via Infotrieve]
7. Ranjan V, Xiao Z, Diamond ST. Constitutive NOS expression in cultured endothelial cells is elevated by fluid shear stress. Am J Physiol. 1995;269:H550–H555.[Medline] [Order article via Infotrieve]
8. Wang DL, Tang CC, Wung BS, Chen HH, Hung MS, Wang JJ. Cyclic strain increases endothelin-1 secretion and gene expression in human endothelial cells. Biochem Biophys Res Commun. 1993;195:1050–1056.[Medline] [Order article via Infotrieve]
9. Wang DL, Wung BS, Peng YC, Wang JJ. Mechanical strain increases endothelin-1 gene expression via protein kinase C pathway in human endothelial cells. J Cell Physiol. 1995;163:400–406.[Medline] [Order article via Infotrieve]
10. Sumpio BE, Widmann MD. Enhanced production of endothelium-derived contracting factor by endothelial cells subjected to pulsatile stretch. Surgery. 1990;108:277–281.[Medline] [Order article via Infotrieve]
11. Carosi JA, Eskin SG, McIntire LV. Cyclical strain effects on production of vasoactive materials in cultured endothelial cells. J Cell Physiol. 1992;151:29–36.[Medline] [Order article via Infotrieve]
12. Awolesi MA, Sessa WC, Sumpio BE. Cyclic strain upregulates nitric oxide synthase in cultured bovine aortic endothelial cells. J Clin Invest. 1995;96:1449–1454.[Medline] [Order article via Infotrieve]
13. Sumpio BE, Banes AJ. Prostacyclin synthetic activity in cultured aortic endothelial cells undergoing cyclic mechanical deformation. Surgery. 1988;104:383–389.[Medline] [Order article via Infotrieve]
14. Wang DL, Wung BS, Shyy YJ, Lin CF, Chao YJ, Usami S, Chien S. Mechanical strain induces monocyte chemotactic protein-1 gene expression in endothelial cells. Circ Res. 1995;77:294–302.[Abstract/Free Full Text]
15. Wung BS, Cheng JJ, Chao YJ, Lin J, Shyy YJ, Wang DL. Cyclical strain increases monocyte chemotactic protein-1 secretion in human endothelial cells. Am J Physiol. 1996;270:H1462–H1468.[Medline] [Order article via Infotrieve]
16. Cheng JJ, Wung BS, Chao YJ, Wang DL. Cyclic strain enhances adhesion of monocytes to endothelial cells by increasing intercellular adhesion molecule-1 expression. Hypertension. 1996;28:386–391.[Abstract/Free Full Text]
17. Carlos TM, Harlan JM. Leukocyte-endothelial adhesion molecules. Blood. 1994;84:2068–2101.[Abstract/Free Full Text]
18. Resnick N, Collins T, Atkinson W, Bonthron DT, Dewey CF, Gimbrone MA. Platelet-derived growth factor B chain promoter contains a cis-acting fluid shear-stress-responsive element. Proc Natl Acad Sci U S A. 1993;90:4591–4595.[Abstract/Free Full Text]
19. Khachigian LM, Resnick N, Gimbrone MA Jr, Collins T. Nuclear-factor-B interacts functionally with the platelet-derived growth factor B-chain shear-stress response element in vascular endothelial cells exposed to fluid shear stress. J Clin Invest. 1995;96:1169–1175.[Medline] [Order article via Infotrieve]
20. Shyy JY, Lin MC, Han J, Lu Y, Petrime M, Chien S. The cis-acting phorbol ester ’12-O-tetradecanoylphorbol 13-acetate’-responsive element is involved in shear stress-induced monocyte chemotactic protein-1 gene expression. Proc Natl Acad Sci U S A. 1995;92:8069–8073.[Abstract/Free Full Text]
21. Satriano JA, Shuldiner M, Hora K, Xing Y, Shan Z, Schlondorff D. Oxygen radicals as second messengers for expression of the monocyte chemoattractant protein, JE/MCP-1, and the monocyte colony-stimulating factor, CSF-1, in response to tumor necrosis factor- and immunoglobin G. J Clin Invest. 1993;92:1564–1571.[Medline] [Order article via Infotrieve]
22. Schreck R, Rieber P, Baeuerle PA. Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-B transcription factor and HIV-1. EMBO J. 1991;10:2247–2258.[Medline] [Order article via Infotrieve]
23. Roebuck KA, Rahman A, Lakshminarayanan V, Janakidevi K, Malik AB. H₂O₂ and tumor necrosis factor- activate intercellular adhesion molecule-1 (ICAM-1) gene transcription through distinct cis-regulatory elements within the ICAM-1 promoter. J Biol Chem. 1995;270:18966–18974.[Abstract/Free Full Text]
24. Li Y, Jaiswal AK. H₂O₂ induces c-fos and c-jun gene expression and increases AP-1 activity. Eur J Biochem. 1994;226:31–39.[Medline] [Order article via Infotrieve]
25. Sumpio BE, Du W, Wu W. Exposure of endothelial cells to cyclic strain induces c-fos, fosB and c-jun but not junB or junD and increases the transcription factor AP-1. Endothelium. 1994;2:149–156.
26. Gimbrone MA. Culture of vascular endothelium. In: Spaet TH, ed. Progress in Hemostasis and Thrombosis. New York, NY: Grune and Stratton; 1976;3:1–28.
27. Gilbert JA, Banes AJ, Link GW, Jones GL. Video analysis of membrane strain: an application in cell stretching. Exp Techniques. 1990;14:43–45.
28. Gilbert JA, Weinhold PS, Banes AJ, Link GW, Jones GL. Strain profiles for circular cell culture plates containing flexible surface employed to mechanically deform cells in vitro. J Biomech. 1994;27:1169–1177.[Medline] [Order article via Infotrieve]
29. Sundaresan M, Yu Z, Ferrans VJ, Irani K, Finkel T. Requirement generation of H₂O₂ for platelet-derived growth factor signal transduction. Science. 1995;270:296–299.[Abstract/Free Full Text]
30. Aebi H. Catalase in vitro. Methods Enzymol. 1984;105:121–126.[Medline] [Order article via Infotrieve]
31. Halliwell B, Gutteridge JMC. Role of free radicals and catalytic metal ions in human disease: an overview. Methods Enzymol. 1990;186:1–85.[Medline] [Order article via Infotrieve]
32. Krieger-Brauer HI, Kather H. Human fat cells possess a plasma membrane-bound H₂O₂-generating system that is activated by insulin via a mechanism bypassing the receptor kinase. J Clin Invest. 1992;89:1006–1013.[Medline] [Order article via Infotrieve]
33. Ohba M, Shibanuma M, Kuroki T, Nose K. Production of hydrogen peroxide by transforming growth factor-ß₁ and its involvement in induction of egr-1 in mouse osteoblastic cells. J Cell Biol. 1994;126:1079–1088.[Abstract/Free Full Text]
34. Collins T. Biology of disease: endothelial nuclear factor-kB and the initiation of the atherosclerotic lesion. Lab Invest. 1993;68:499–508.[Medline] [Order article via Infotrieve]
35. Schulze-Osthoff K, Beyaert R, Vandevoorde V, Haegeman G, Friers W. Depletion of the mitochondrial electron transport abrogates the cytotoxic and gene-inductive effects of TNF. EMBO J. 1993;12:3095–3104.[Medline] [Order article via Infotrieve]
36. Devary Y, Gottlieb RA, Laus LF, Karin M. Rapid and preferential activation of the c-jun gene during the mammalian UV response. Mol Cell Biol. 1991;11:2804–2811.[Abstract/Free Full Text]
37. Abate C, Patel L, Rauscher FJ, Curran T. Redox regulation of fos and jun DNA-binding activity in vitro. Science. 1990;249:1157–1161.[Abstract/Free Full Text]
38. Marui N, Offermann MK, Swerlick R, Kunsch C, Rosen CA, Ahmad M, Alexander RW, Medford RM. Vascular cell adhesion molecule-1 gene transcription and expression are regulated through an antioxidant-sensitive mechanism in human vascular endothelial cells. J Clin Invest. 1993;92:1866–1874.[Medline] [Order article via Infotrieve]
39. Khan AU, Wilson T. Reactive oxygen species as cellular messengers. Chem Biol. 1995;2:437–445.[Medline] [Order article via Infotrieve]
40. Schenk H, Klein M, Erdbrugger W, Droge W, Schulze-Osthoff K. Distinct effects of thioredoxin and antioxidants on the activation of transcription factors NF-B and AP-1. Proc Natl Acad Sci U S A. 1994;91:1672–1676.[Abstract/Free Full Text]
41. Barchowsky A, Munro SR, Morana SJ, Vincenti MP, Treadwell M. Oxidant-sensitive and phosphorylation-dependent activation of NF-B and AP-1 in endothelial cells. Am J Physiol. 1995;269:L829–L836.[Medline] [Order article via Infotrieve]
42. Laurindo FRM, Pedro MA, Barbeiro HV, Pileggi F, Carvalho MHC, Augusto O, daLuz PL. Vascular free radical release: ex vivo and in vivo evidence for a flow-dependent endothelial mechanism. Circ Res. 1994;74:700–709.[Abstract/Free Full Text]
43. Hart DH, Hobson JE, Walker DC, Autor AP. Antioxidant enzyme content of pulmonary artery endothelial cells: effects of subculture. Free Radic Biol Med. 1985;1:429–435.
44. Kinnula VL, Everitt JI, Whorton AR, Crapo JD. Hydrogen peroxide production by alveolar type II cells, alveolar macrophages and endothelial cells. Am J Physiol. 1991;261:L84–L91.[Medline] [Order article via Infotrieve]
45. Lo SK, Janakidevi K, Lai L, Malik AB. Hydrogen peroxide-induced increase in endothelial adhesiveness is dependent on ICAM-1 activation. Am J Physiol. 1993;264:L406–L412.[Medline] [Order article via Infotrieve]
46. Wung BS, Cheng JJ, Hsieh HJ, Shyy YJ, Wang DL. Cyclic strain-induced monocyte chemotactic protein-1 gene expression in endothelial cells involves reactive oxygen species activation of activator protein-1. Circ Res. 1997;81:1–7.[Abstract/Free Full Text]
47. Faruqi RC, de la Motte C, DiCorleto PE. -Tocopherol inhibits agonist-induced monocytic cell adhesion to cultured human endothelial cells. J Clin Invest. 1994;94:592–600.[Medline] [Order article via Infotrieve]
48. Belcher JD, Balla J, Balla G, Jacobs DR, Gross M, Jacob HS, Vercellotti GM. Vitamin E, LDL, and endothelium: brief oral vitamin supplementation prevents oxidized LDL-mediated vascular injury in vitro. Arterioscler Thromb. 1993;13:1779–1789.[Abstract/Free Full Text]

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