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(Circulation Research. 1997;81:1-7.)
© 1997 American Heart Association, Inc.

Articles

Cyclic Strain–Induced Monocyte Chemotactic Protein-1 Gene Expression in Endothelial Cells Involves Reactive Oxygen Species Activation of Activator Protein 1

B. S. Wung, J. J. Cheng, H. J. Hsieh, Y. J. Shyy, , D. L. Wang

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

Correspondence to Dr D.L. Wang, 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 Endothelial cells (ECs) are constantly exposed to blood pressure–induced mechanical strain. We have previously demonstrated that cyclic strain can induce gene expression of monocyte chemotactic protein-1 (MCP-1). The molecular mechanisms of gene induction by strain, however, remain unclear. Recent evidence indicates that intracellular reactive oxygen species (ROS) can act as a second messenger for signal transduction and thus affect gene expression. The potential role of ROS in strain-induced MCP-1 expression was investigated. ECs under cyclic strain induced a sustained elevated production of intracellular superoxide. ECs under strain or pretreated with either H₂O₂ or xanthine oxidase/hypoxanthine induced MCP-1 expression. Strain- or oxidant-induced MCP-1 mRNA levels could be inhibited by treating ECs with catalase or antioxidant N-acetyl-cysteine (NAC). Functional analysis of MCP-1 promoter and site-specific mutations indicates that the proximal tissue plasminogen activator–responsive element (TRE) in the -60-bp promoter region is sufficient for strain or H₂O₂ inducibility. Electrophoretic mobility shift assays demonstrated an increase of nuclear proteins binding to TRE sequences from ECs subsequent to strain or H₂O₂ treatment. NAC or catalase pretreatment of ECs inhibited the strain- or H₂O₂-induced AP-1 binding. These results clearly indicate that cyclic strain inducibility of MCP-1 in ECs uses the interaction of AP-1 proteins with TRE sites via the elevation of intracellular ROS levels in strained ECs. These findings emphasize the importance of intracellular ROS in the modulation of hemodynamic force–induced gene expression in vascular ECs.

Key Words: cyclic strain • reactive oxygen species • monocyte chemotactic protein-1 • activator protein 1/tissue plasminogen activator–responsive element

	Introduction

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Endothelial cells lining vascular walls are constantly under the influence of hemodynamic forces, including flow-induced shear stress and pressure-generated strain. These hemodynamic forces can modulate various gene expressions and protein release from ECs and consequently have profound effects on endothelial function. Effects of shear stress on vascular ECs include the induction of TPA,¹ prostacyclin,² Et-1,³ NO,⁴ MCP-1,⁵ and other substances. Recent studies, including our own work, have indicated that mechanical strain can also play an important role in the modulation of various gene expressions in ECs.^{6 7 8 9 10 11 12 13} Among these genes are Et-1,^{6 9 10} MCP-1,^{11 12} and ICAM-1.¹³

Although the gene expression modulated by hemodynamic forces has been well recognized,^{14 15} the intracellular mechanisms by which physical forces are transmitted from extracellular origins to intracellular signals and subsequently alter gene expression remain largely unknown. Signal pathways, including PKC activation and calcium mobilization, have been demonstrated to be involved in the cyclic strain–induced Et-1 and MCP-1 expression.^{10 11} Among transcriptional factors, NF-B and AP-1 have been shown to contribute to the hemodynamic force–induced gene expression. Recent evidence, however, suggests that ROS may act as second messengers in cells exposed to various stimuli.^{16 17 18 19 20 21} Since flow-dependent release of ROS²² and activation of transcriptional factors NF-B and AP-1 by ROS during gene induction have been reported,^{19 21} it is important to know whether hemodynamic forces can induce intracellular ROS production and what the functional role of ROS is in the hemodynamic force–induced gene expression. We have recently demonstrated that intracellular ROS levels can be elevated by cyclic strain treatment.²³ Cyclic strain–induced ROS appear to be involved in the induction of PAI-1 release²³ and gene expression of MCP-1 and ICAM-1 in ECs.²⁴ However, the molecular mechanisms of ROS production and their effects on gene expression in ECs have not been defined. In the present study, we further demonstrate that intracellular ROS levels are directly involved in cyclic strain–induced MCP-1 gene expression through the modulation of the AP-1 binding site in the MCP-1 promoter region. Our findings thus support the importance of intracellular ROS in the modulation of hemodynamic force–induced gene expression in vascular ECs.

	Materials and Methods

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EC Cultures
Human umbilical vein ECs were isolated from human umbilical cords according to the procedures described previously.²⁵ ECs were seeded on a flexible membrane base of a culture well (Flex 1, Flexcell Co) and grown for 3 days until confluence was attained. The medium was replaced with the same medium, but it contained only 2% fetal calf serum (GIBCO BRL); the cells were incubated overnight before the experiment. For transfection study, BAECs were used. BAECs were cultured in DMEM with 10% fetal calf serum, 1.0x10⁵ U penicillin, and 100 mg streptomycin/L.

In Vitro Cyclic Strain on Cultured ECs
The strain unit FX-2000 (Flexcell), which has been characterized and described in detail elsewhere,^{10 11 12 13 26 27} consisted of a vacuum unit linked to a valve controlled by a computer program. ECs cultured on the flexible membrane base were subjected to a cyclical strain produced by a sinusoidal negative pressure with a peak level of 20 kPa (average strain, 12%) at a frequency of 1 Hz (60 cycles/min) for various durations.

Chemiluminescence Assay of Superoxide Production
Superoxide production was measured by lucigenin-amplified chemiluminescence as previously described.²⁸ Briefly, ECs were lysed immediately after strain treatment with a lysis buffer containing lucigenin (200 µmol/L). Readings were begun immediately upon addition of lysis buffer. Samples with the addition of SOD (1.0x10⁵ U/L) were used as blank controls. Each reading was recorded as single photon counts by using a microplate scintillation counter (Topcount, Packard Instrument Co).

RNA Isolation and Northern Blot Analysis
Total RNA was isolated from ECs by the guanidinium isothiocyanate/phenol-chloroform method as described previously.¹¹ RNA (10 µg per lane) was separated by electrophoresis on a 1.2% agarose formaldehyde gel and transferred onto a nylon membrane (Nytran, Schleicher & Schuell Inc) by a vacuum blotting system (VacuGene XL, Pharmacia). After hybridization with the ³²P-labeled MCP-1 cDNA probes, the membrane was washed with 1x SSC containing 1% SDS at room temperature for 30 minutes and exposed to x-ray film (Kodak X-Omat-AR) at -70°C. Autoradiographic results were scanned and analyzed by a densitometer (Computing Densitometer 300S, Molecular Dynamics).

Reporter Gene Constructs
MCP-1 promoter constructs were as follows: P540Luc, P400Luc, P270Luc, P150Luc, and P73Luc, all containing the luciferase reporter gene (Luc) of plasmids pGL2 (Promega) and corresponding to the sequences in the fragment of 540, 400, 270, 150, and 73 bp of the 5' flanking region of the MCP-1 gene, respectively. These constructs share a common 3' end (starting from 20 bp downstream from the transcription initiation site) and differ only in the 5' end.²⁹ Mutants M1 and M2 correspond to the P540Luc sequences, with specific mutations in the distal TRE site beginning at -88 bp and the proximal TRE site beginning at -60 bp, respectively (starting from transcription initiation site). These reporter genes have been previously described.²⁹

Luciferase Assay
DNA plasmids, purified by a Wizard Maxipreps DNA purification system (Promega), were transfected into BAECs in their 60% confluence using the lipofectamine method (GIBCO-BRL). The pSV-ß-galactosidase plasmid, which contains a ß-galactosidase gene driven by the simian virus 40 promoter and enhancer, was cotransfected to normalize the transfection efficiency. After transfection, cells were incubated overnight with 10% FBS-DMEM to reach confluence. The cells were seeded on flexible membranes for cyclic strain. The cell extract was prepared and assayed for luciferase activity using the Promega Biotec assay system. To normalize the transfection efficiency for individual samples, the ß-galactosidase activity was assayed by adding the substrate, o-nitrophenyl-ß-D-galactopyranoside to 20 µL cell lysate and incubated at 37°C before recording at 420 nm.

EMSA
To prepare nuclear protein extracts, ECs were washed with cold PBS immediately removed by scraping in PBS. After centrifugation of the cell suspension at 2000 rpm, the cell pellets were resuspended in cold buffer A (10 mmol/L KCl, 0.1 mmol/L EDTA, 1 mmol/L dithiothreitol, and 1 mmol/L phenylmethylsulfonyl fluoride) for 15 minutes. The cells were lysed by adding 10% NP40 and then centrifuged at 6000 rpm to obtain a pellet of nuclei. The nucleic pellets were resuspended in cold buffer B (20 mmol/L HEPES, 0.4 mol/L NaCl, 1 mmol/L EDTA, 1 mmol/L dithiothreitol, and 1 mmol/L phenylmethylsulfonyl fluoride), vigorously agitated from time to time, and then centrifuged. The supernatant containing the nuclear proteins was used for the EMSA or stored at -70°C until used.

Two double-stranded oligonucleotides containing the AP-1 binding site were prepared: 5'-CGCTTGATGAGTCAGCCGGAA-3' (Promega) and 5'-TCCTGCTTGACTCCGCCCT-3' correspond to sequences at positions -73 to -55 bp, respectively, of the MCP-1 promoter as competitor. The oligonucleotides were end-labeled with [-³²P]ATP. Nuclear extract (10 µg) was incubated with 0.1 ng ³²P-labeled DNA for 15 minutes at room temperature in a final volume of binding buffer of 25 µL containing 1 µg poly dI-dC. The mixtures were electrophoresed on 7% nondenaturing polyacrylamide gels under high ionic strength. Gels were dried and imaged by autoradiography.

Statistical Analysis
Statistical analysis was performed by using Student's t test. Data were presented as mean±SEM. Statistical significance was defined as P<.05.

	Results

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Cyclic Strain Increases Intracellular ROS Levels
To study the effects of mechanical strain on intracellular ROS levels in ECs, ECs cultured on the flexible membrane were subjected to sinusoidal negative pressure to produce an average strain of 12%. ECs under this strain treatment for 0.5, 1, or 24 hours significantly increased their intracellular superoxide levels, as measured by lucigenin-amplified chemiluminescence, to 2.5±0.6-fold, 1.9±0.3-fold, or 1.7±0.3-fold, respectively, compared with unstrained control cells (Fig 1). In addition, ECs treated with PMA for 0.5 hour increased 4.9±0.4-fold. This strain-induced superoxide production could be abolished by coincubation of the cell lysate with SOD. These results demonstrate that cyclic strain can increase intracellular ROS levels.

View larger version (26K): [in this window] [in a new window]   Figure 1. Cyclic strain induces intracellular ROS levels in ECs. ECs under static (C) or cyclic strain (S, 12%) treatment were lysed, which was immediately followed by superoxide measurements with a lucigenin-amplified chemiluminescence method as described in "Materials and Methods." ECs treated with PMA (500 µg/L) for 0.5 hour were used as positive controls. In some experiments, SOD was added simultaneously into cell lysate as a blank control. Results are shown as mean±SEM from at least three separate experiments. *P<.05 vs unstrained control cells.

ROS Mediate Strain-Induced MCP-1 Gene Expression
In order to demonstrate that intracellular ROS were involved in the strain-induced MCP-1 gene expression, ECs were treated with different stimuli that were known to increase intracellular ROS. These stimuli include tumor necrosis factor, H₂O_2, and xanthine oxidase/hypoxanthine. After the application of stimuli, ECs increased their intracellular superoxide levels (data not shown). All these agents consistently induced MCP-1 mRNA levels (Fig 2A). To assess whether ROS were involved in the strain-induced MCP-1 expression, ECs were pretreated with an antioxidant, NAC, before strain treatment. As shown in Fig 2A, the strain-induced MCP-1 expression was significantly reduced by NAC pretreatment but not in the unstrained control ECs. To further confirm that intracellular ROS mediated strain-induced gene expression, ECs were strained in a medium containing either catalase, deferoxamine (an iron chelator), or DMTU (an OH radical scavenger). As shown in Fig 2B, the presence of catalase in the medium completely abolished the strain-induced MCP-1 expression. In contrast, the presence of catalase had no effect on unstrained control cells. Treatment with deferoxamine or DMTU also showed partial inhibition. These results strongly support the thesis that strain-induced MCP-1 gene expression is mediated through the elevated ROS levels triggered by cyclic strain.

View larger version (46K): [in this window] [in a new window]   Figure 2. ROS mediate strain-induced MCP-1 gene expression. A, ECs were in unstrained control conditions (C) or subjected to strain (S) for 3 hours. In some experiments, ECs were pretreated with NAC (20 mmol/L) for 0.5 hour before the strain treatment. ECs treated with tumor necrosis factor (TNF, 1.0x105 U/L), H2O2 (100 µmol/L), and xanthine oxidase/hypoxanthine (XO, 4 U/2 mmol/L) were used as positive controls for oxidative stress. B, ECs were strained for 3 hours in the presence of catalase (Cat, 3.5x105 U/L), deferoxamine (Def, 1 mmol/L), or DMTU (5 mmol/L). Total RNA was analyzed for MCP-1 levels by Northern blot hybridization with the MCP-1 probe. Equal amounts of RNA (10 µg) applied to each lane were demonstrated by the 18S RNA shown for each lane. Data are representative of duplicate experiments.

Proximal TRE Is the Element Responsible for Strain Inducibility
Previous study has shown that TRE in the 5' promoter region is the cis-regulatory element that responds to shear stress.²⁹ To test whether TRE is also involved in the MCP-1 induction by strain, functional analysis of MCP-1 promoter for cyclic strain inducibility in BAECs was performed. Five MCP-1–Luc chimeric genes containing sequentially deleted segments of the MCP-1 promoter region and the reporter gene luciferase were analyzed for the gene induction in response to cyclic strain. As indicated in Fig 3A, in chimeras (P150Luc) containing MCP-1 promoter regions at least 130 bp upstream from the transcription site, reporter gene activities were inducible, unlike static control cells. In contrast, the chimeric gene (P73Luc) lost not only its basal activity but also its strain inducibility. These data indicate that the putative cis element is located within the region between nucleotides -53 and -130, where two TREs are located: one with TGACTCC beginning at nucleotide -60 and the other with TCACTCA beginning at nucleotide -88. The proximal TRE site has been implicated in shear inducibility.²⁹ To demonstrate that this TRE site was also involved in strain response, several chimeric genes with site-specific mutagenesis at these two putative sequences were subjected to strain-induction assays. Fig 3B shows the induction of these mutants by strain. M1, which had a mutated distal site (TCACTCA replaced by GCACTTG) and a wild-type proximal site (TGACTCC), retained the strain inducibility. In contrast, M2, which had a mutated proximal site (TGACTCC replaced by GGACTTG), lost its strain inducibility. These data indicate that the proximal TRE (TGACTCC) is the element responsible for strain inducibility. This TRE site responded not only to strain but also to H₂O₂ treatment. As shown in Fig 4, chimera of the wild type (P540Luc) responded to the strain as well as H₂O₂ treatment. In contrast, M2, which has mutation in the proximal TRE site, lost its inducibility in response to strain and H₂O₂ exposure.

View larger version (19K): [in this window] [in a new window]   Figure 3. TRE in the promoter region is functionally responsive to cyclic strain. A, Restriction map of the 540-bp MCP-1 5' upstream region is shown at the top. Solid boxes represent the shear stress–responsive element (SSRE; ie, 5-GGTCTC) and putative TRE sequences. ECs were cotransfected with the MCP-1–Luc chimera and pSV-ß-galactosidase plasmid for monitoring transfection efficiency. Reporter luciferase activities represent the folds of induction in the strained cells compared with those in unstrained control cells. B, Wild-type chimeric gene MCP-1–p540Luc and two mutants with site-specific mutation indicated by small letters were transiently transfected into ECs for the strain induction assay. Results are shown as folds of induction of the reporter luciferase activities from strained ECs to those of unstrained controls. Results are shown as mean±SEM from at least three separate experiments. All plasmids except p73Luc and M2 demonstrated a significant difference at P<.05.

View larger version (15K): [in this window] [in a new window]   Figure 4. TRE-containing sequence (TGACTACA) responds to cyclic strain and H2O2. Wild-type plasmid MCP-1–Luc-540 (P540Luc) or mutant (P540/mTRELuc) with site-specific mutation at proximal TRE sequence (GGACTTG) was transiently transfected into BAECs for cyclic strain and H2O2 (100 µmol/L) induction assay. Experimental conditions were the same as for Fig 3. Luciferase activities were normalized for the transfection efficiency. Results are shown as mean±SEM from at least three separate experiments. There was a significant difference (P<.05) for plasmid P540Luc.

ROS Mediates Strain-Induced AP-1 Binding Activity
Earlier studies^{20 30} have shown that oxidative stress or H₂O₂ can induce the rapid activation of AP-1 in ECs. We studied whether AP-1 was actually responsible for the strain-induced or oxidant agent–induced MCP-1 expression. By use of a gel mobility shift assay, the binding activities of AP-1 were determined to have increased 1 and 2 hours after strain treatment (Fig 5A). This binding could be inhibited by using unlabeled oligonucleotides corresponding to positions -73 to -55 bp of the MCP-1 promoter as competitor or preincubating the nuclear proteins with antibodies to c-fos and c-jun. NAC or catalase pretreatment of strained ECs reduced the AP-1 binding activities; this further indicated that intracellular ROS levels mediate these AP-1 binding activities (Fig 5B). H₂O₂ treatment also increased AP-1 binding. Consistent with the strain-dependent ROS induction and strain-induced gene expression as previously demonstrated,²⁴ nuclear proteins extracted from cells grown on the periphery of cultured wells where maximal strain is located showed a greater AP-1 binding activity (Fig 5B). These results clearly show that AP-1 binding activities induced by cyclic strain are mediated via the elevated intracellular ROS. These elevated ROS levels triggered by cyclic strain thus play an important role in the modulation of MCP-1 gene expression in vascular ECs.

View larger version (43K): [in this window] [in a new window]   Figure 5. The effects of cyclic strain, H2O2, catalase, and antioxidant NAC on AP-1 activation. A, ECs were treated with cyclic strain for 1 hour (S1) or 2 hours (S2). Total nuclear extracts were prepared and analyzed by EMSA using a 32P-labeled oligonucleotide probe containing a canonical TRE site. The specificity of the retarded complexes (AP-1) was assessed by preincubating the nuclear extracts with either excess unlabeled TRE-containing oligonucleotides corresponding to the MCP-1 promoter as a competitor (S1+250X) or with antibodies (Ab) to c-fos and c-jun (1 µg each, S1+Ab). B, EMSA was performed on ECs from static control (C) or under strain for 3 hours (S). Some ECs were pretreating with NAC (20 mmol/L, S+N) or catalase (cat, 3.5x105 U/L; S+cat) for 0.5 hour before strain treatment. The oxidant-inducing AP-1 activation was shown by using ECs treated with H2O2 (100 µmol/L) for 3 hours. The effect of strain-dependent ROS on AP-1 activity was further demonstrated by collecting nuclear extracts from ECs grown at the periphery (SP) of the culture well where maximal strain is located. Results are representative of duplicate experiments.

	Discussion

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MCP-1, a potent chemoattractant for monocytes, has been suggested to play an important role in the recruitment and accumulation of monocytes and/or macrophages in the subendothelial space of the arterial wall during atherogenesis³¹ and tissue injury.¹⁸ The MCP-1 gene can be induced by various stimuli, including oxidized low-density lipoprotein³² and cytokines.¹⁸ Previous studies, including ours, have indicated that hemodynamic forces including shear flow and pressure-induced strain can stimulate MCP-1 gene expression, which subsequently affects monocyte/EC interaction.^{6 11 12 29} Shyy et al²⁹ further demonstrated that a proximal phorbol ester TRE in the promoter region of the MCP-1–encoding gene was critical for shear inducibility.²⁹ However, the molecular mechanisms by which physical forces are transmitted from extracellular origin to intracellular signals, which subsequently alter gene expression, remain largely unknown. Among those intracellular signals, calcium mobilization,^{33 34} PKC activation,^{35 36} and mitogen-activated protein kinase³⁷ may all be involved.

Recent studies provide evidence that ROS may act as second messengers in cells exposed to various stimuli.^{16 17 18 19 20 21} ROS contributing to the reperfusion-induced endothelial injuries have been recognized,³⁸ and the flow-dependent ROS release has been recently shown.²² Previous studies from this laboratory have shown that cyclic strain treatment of ECs can induce intracellular ROS generation.²³ This ROS generation was sustained at an elevated level as long as mechanical forces were maintained and returned to basal levels with the removal of forces.²³ Cyclic strain treatment evokes a rapid increase in ROS generation, followed by decreased but still elevated ROS levels compared with steady state control levels. This ROS generation was strain dependent, since ECs exposed to a genuine fluid agitation by using a rotary shaker (60 cycles/min) did not increase their ROS levels, nor did they increase MCP-1 expression (data not shown). A strain-dependent increase of catalase activities was concomitantly observed in the mechanical force–treated cells.²³ Since elevated ROS may induce oxidative stress, the increase of catalase activities may be crucial to protect cells from the potential damage of ROS. Thus, the strained cells remained morphologically and functionally intact as judged by the lack of DNA fragmentation as well as by no significant increase in lactate dehydrogenase activity in the culture media (data not shown). In addition, the quantity and the quality of the total RNA isolated from strained cells appeared to be normal and similar to those of unstrained control cells. Since these strain-induced intracellular ROS levels were less than the increase in PMA-treated ECs, we thus propose that a modest increase of ROS may act as a signal for endothelial adaptation to changes in the hemodynamic environment. We have shown that elevated ROS levels are involved in the release of PAI-1 from strained cells.²³ Our recent studies have indicated that strain-induced ROS generation correlated with strained-induced MCP-1 and ICAM-1 gene expression and that this gene induction could be attenuated by antioxidant treatment of strained cells.²⁴ In all, these results suggest that intracellular ROS production is involved in the mechanical force–induced cellular responses.

In the present study, we provide further data to support the thesis that ROS directly mediate the strain-induced MCP-1 gene expression. Several lines of evidence support this notion. First, strain-induced ROS generation, as analyzed previously by total peroxidative product of dichlorofluorescein diacetate^{23 24} or measured by superoxide production shown in the present study, correlated with MCP-1 induction, and this induction can be inhibited by treating the strained ECs with various antioxidants, ie, NAC, DMTU, and deferoxamine. Second, the antioxidant enzyme catalase, coincubated with strained ECs, abolished strain-induced MCP-1 gene expression but had no effect on the expression in unstrained control cells. Third, cyclic strain, similar to H₂O₂ treatment, induced AP-1 binding activity, and this binding could be attenuated by antioxidant pretreatment of strained ECs. Fourth, the promoter region containing the proximal TRE site, where AP-1 proteins bind, was sufficient for cyclic strain inducibility, and the mutation of this TRE site abolished this strain as well as H₂O₂ inducibility. In the present study, we treated ECs with NAC, a precursor of glutathione. The addition of NAC could provide glutathione levels sufficient to reduce the intracellular ROS concentration via a glutathione peroxide pathway. In contrast, catalase, a big molecule that is unlikely to diffuse into cells, appears to clear rapidly the intracellular generated H₂O₂, which is permeable to the cell membrane.^{39 40} However, both demonstrated a similar effect on the inhibition of AP-1 binding induced by strain. Our strain inducibility, which was not as great as that resulting from shear flow treatment,²⁹ might be due in part to the nonuniform stretch of this strain device. Thus, ECs near the center of membrane were not subjected to mechanical strain.²⁷ Nevertheless, our results clearly indicate that cyclic strain–induced MCP-1 gene expression involves the ROS activation on the AP-1 binding site in the MCP-1 promoter region.

ROS stimulation of NF-B and AP-1 activation, which will affect those redox-sensitive genes such as ICAM-1²⁰ and VCAM-1,⁴¹ has been recognized. Satriano et al¹⁸ have demonstrated that ROS act as second messengers for cytokine-induced MCP-1 expression. Shear flow–activated transcriptional factors NF-B and AP-1 have also been reported.^{29 42 43 44} In addition, Sumpio et al⁴⁵ have shown that cyclic strain to ECs induces the expression of immediate-early genes, c-fos and c-jun. Recent studies have shown the gene induction of Cu/Zn SOD and mitochondrial manganese SOD by the fluid mechanical stimuli.^{46 47} Furthermore, the inhibitory effect of antioxidant on NF-B and AP-1 mobilization has also been documented.^{39 48 49} Our results of strain-induced MCP-1 induction mediated through intracellular ROS generation and the inhibitory effect of MCP-1 gene induction by antioxidants are in complete agreement with these findings.

ROS, including H₂O₂, are constantly released in cells during electron transfer reactions.⁵⁰ How cells under mechanical force treatment increase their ROS generation, which subsequently alters gene expression, remains unclear. However, recent studies have shown that some antioxidants, eg, vitamin E, exert their inhibitory effect on ROS release via PKC inhibition.⁵¹ Although the relationship between PKC activation and ROS generation has not been clearly established, the importance of PKC in gene induction is supported by our previous report that showed the PKC involvement in strain-induced gene expression, including Et-1¹⁰ and MCP-1,¹¹ and more recently by our observation that strain-induced superoxide production can be inhibited by treating strained ECs with PKC inhibitors (authors' unpublished data, 1997). It has been reported that the released ROS activate mitogen-activated protein kinase⁵² and c-jun NH₂-terminal kinase.¹⁶ Recent data even indicate that p21^ras is a common signaling target of reactive free radicals and cellular redox stress.¹⁷ The molecular mechanisms by which hemodynamic forces lead to increased ROS and subsequent modulation of gene expression remain an important question that warrants further investigation.

In summary, the present study clearly indicates that cyclic strain can induce intracellular ROS generation, which acts as a second messenger to stimulate the MCP-1 gene expression via AP-1 binding. Our data thus support the importance of intracellular ROS as modulators for hemodynamic force–induced cellular responses. The imbalance or disorder of this intracellular ROS generation and/or antioxidant defense mechanism may contribute to the pathogenesis of various cardiovascular diseases, including atherosclerosis and hypertension. This hemodynamic force–induced ROS generation may also provide some insight into the basis of reperfusion-induced vascular injuries.

	Selected Abbreviations and Acronyms

 

AP-1 = activator protein 1 BAEC = bovine aortic EC DMTU = 1,3-dimethyl-2-thiourea EC = endothelial cell EMSA = electrophoretic mobility shifting assay Et-1 = endothelin-1 ICAM-1 = intercellular adhesion molecule-1 M1, M2 = mutants 1 and 2 MCP-1 = monocyte chemotactic protein-1 NAC = N-acetyl-cysteine NF-B = nuclear factor-B PAI-1 = plasminogen activator inhibitor-1 PKC = protein kinase C PMA = phorbol 12-myristate 13-acetate ROS = reactive oxygen species SOD = superoxide dismutase TPA = tissue plasminogen activator TRE = TPA-responsive element

	Acknowledgments

 
This study was supported in part by grant NSC86-2314-B001-004-M26 from the National Science Council, Taiwan, ROC.

Received December 15, 1996; accepted April 7, 1997.

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