doi: 10.1161/hq1201.099428
© 2001 American Heart Association, Inc.
Vascular Biology |
NO Modulates Monocyte Chemotactic Protein-1 Expression in Endothelial Cells Under Cyclic Strain
From the Cardiovascular Division, Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, ROC.
Correspondence to Dr Danny Ling Wang, Cardiovascular Division, Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan 11529. E-mail lingwang{at}ibms.sinica.edu.tw
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Abstract |
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Endothelial cells (ECs) under hemodynamic forces increase intracellular reactive oxygen species (ROS) that modulate gene expression. We previously showed that NO attenuated the shear flow–induced gene level. The present study explored the role of endothelial NO in cyclic strain–treated ECs. Treatment of ECs with S-nitroso-N-acetylpenicillamine (SNAP), an NO donor, reduced cyclic strain–induced monocyte chemotactic protein (MCP)-1 expression. Conversely, exposure of ECs to an NO synthase inhibitor augmented MCP-1 mRNA levels. NO attenuated the binding of activator protein-1 to the 12-O-tetradecanoylphobol-13-acetate–responsive element (TRE) in the MCP-1 promoter region. ECs overexpressed with endothelial NO synthase (eNOS) inhibited cyclic strain–induced MCP-1 expression and MCP-1 promoter (-540 bp) activity. Consistently, ECs treated with SNAP or infected with adenovirus carrying eNOS reduced strain-induced superoxide levels. These strain-induced superoxide and MCP-1 expressions were greatly blunted by treating ECs with an NADPH oxidase inhibitor, diphenyleneiodonium chloride or apocynine, but not with a xanthine oxidase inhibitor. ECs infected with adenovirus carrying the dominant-negative mutant of Rac (RacN17), a component of NADPH oxidase, reduced the strain-induced superoxide and MCP-1 expression. In contrast, ECs transfected with a constitutively active Rac (RacV12) increased MCP-1 and 4x TRE promoter activities. However, ECs cotransfected with eNOS and RacV12 reduced those promoter activities. Consistently, the increases of superoxide levels and MCP-1 expression by overexpression of RacV12 were abolished after infecting ECs with eNOS. Our results show that NO from eNOS-inhibiting redox-sensitive MCP-1 expression is mediated via Rac-dependent NADPH oxidase by reducing ROS. This study provides a molecular basis to support the notion that endothelial NO acts as an antioxidant by negatively regulating redox-sensitive gene expression in ECs constantly under hemodynamic influence.
Key Words: endothelial cells • cyclic strain • reactive oxygen species • Rac • NADPH oxidase
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Introduction |
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Endothelial cells (ECs) are constantly under the influence of hemodynamic forces, including flow-induced shear stress and pressure-triggered cyclic strain. Because vessel walls are constantly under rhythmic distension, a component of pulsatile flow, cyclic strain to vessel walls, plays a key role in the modulation of cellular signaling and gene expression that may contribute to vascular disorders, including atherosclerosis. ECs subjected to cyclic strain transmit the mechanical force into intracellular signals and induce cellular responses.1 Recent evidence has suggested that reactive oxygen species (ROS) play a pivotal role in growth factor– and hemodynamic force–induced endothelial responses.2 Increased intracellular ROS levels induce redox-sensitive genes that may be involved in vascular dysfunction, including atherosclerosis and hypertension-induced complications. ECs subjected to cyclic strain increase ROS, which result in an induction of various redox-sensitive genes, including monocyte chemotactic protein (MCP)-1,3–7 a protein that is believed to be involved in the recruitment of monocytes into the subendothelial space during atherogenesis. Removal of ROS production by antioxidant treatment alleviated this cyclic strain–induced MCP-1 expression.3 The origin of ROS produced in activated ECs is not yet defined. However, NADPH oxidase or its like has been suggested to be the major source of ROS production in ECs under hemodynamic conditions.8,9 Recent evidence further indicates that a gp91phox-containing NADPH oxidase is selectively expressed in ECs.10–12 Rac, a Rho family GTPase, along with other components is known to be required for NAPDH oxidase activity13–16 and is essential for superoxide production in ECs.17 Although the dominant-negative mutant of Rac (RacN17) reduced interleukin-1–stimulated nuclear factor-
B binding via the inhibition of ROS production,18 RacN17 transfection protects cells from hypoxia/reoxygenation-induced injury.19 ECs constantly release NO via the activation of endothelial NO synthase (eNOS). Although eNOS is a constitutively expressed enzyme, eNOS expression and activity are modulated by a various stimuli, including shear stress and cyclic strain.20,21 The released NO regulates blood pressure and regional blood flow22,23 and inhibits vascular smooth muscle cell proliferation,24 platelet aggregation,25 and leukocyte adhesion.26 NO has been shown to attenuate cytokine-induced expression of MCP-1 and adhesion molecules in vitro.27,28 Decreased NO release aggravates vascular dysfunction, and local gene transfer of eNOS inhibits atherosclerotic lesions.29,30 Accumulating evidence suggests that endothelial NO enhances vascular resistance to oxidative stress.31 Conversely, the inhibition of eNOS increases vascular oxidative stress and, consequently, induces gene expression (including MCP-1)32 that eventually leads to vascular disorders. Despite those inhibitory effects of NO on endothelial responses, the detailed mechanisms of the effects of NO on vascular walls are not yet clear. Endothelial NO appears to serve as a negative regulator to modulate redox-sensitive gene expression. Our earlier data have indicated that ECs under hemodynamic forces increase ROS that alter the Ras/Raf/extracellular signal–regulated kinase (ERK) signaling pathway and gene expression.7,33 Furthermore, NO negatively regulates this ERK signaling pathway and inhibits shear-induced early growth response-1 (Egr-1) expression.33 All these results support the notion that endothelial NO inhibits redox-sensitive gene expression.
The present study sought to examine the role of NO and its inhibitory mechanism in ECs under cyclic strain. By using the gene transfer of eNOS, we found that endothelial NO acts as a negative regulator by inhibiting MCP-1 in ECs under cyclic strain. This inhibition is a transcriptional event. Furthermore, we demonstrated that Rac plays a key role in superoxide production and MCP-1 induction in ECs under cyclic strain. Finally, the induction of MCP-1 and ROS levels by overexpression of activated Rac was abolished by transfecting ECs with eNOS, suggesting that NO-inhibiting superoxide production is mediated via Rac-dependent NADPH oxidase. These results suggest that endothelial NO exerts its antioxidant effect by reducing ROS levels and, thus, acts as a negative regulator in ECs under hemodynamic influence.
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Methods |
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Materials
The MCP-1 cDNA probe and MCP-1 promoter (P540Luc) were gifts from Dr J.J. Shyy, University of California at San Diego, La Jolla, and were used previously.3 RasL61, RacN17, and RacV12 were kindly provided by Dr M. Kirin of the University of California at San Diego, La Jolla. Overexpression plasmids of mitogen-activated protein kinase/ERK 1 (MEK1) or MEK kinase (pFCMEK1 and pFCMEKK, respectively) were obtained from Stratagene. The eNOS expression plasmid was kindly provided by Dr P.F. Chen of the University of Texas, Houston. S-Nitroso-N-acetylpenicillamine (SNAP) and NG-nitro-L-arginine methyl ester (L-NAME) were purchased from Calbiochem.
rAd Production
Replication-defective adenoviruses were produced as described.34 The adenovirus shuttle plasmid vector pAd-CMV, kindly supplied by Dr S.H. Chen at Mount Sinai School of Medicine, New York, NY, contains a cytomegalovirus (CMV) promoter. pAd-PGK was constructed by replacing the CMV promotor in pAd-CMV with the promoter human phosphoglycerate kinase (PGK) gene. The recombinant adenovirus (rAd) was prepared by cotransfecting 293 cells with pAd-PGK containing the candidate cDNA in expression cassettes and with pJM17, kindly provided by Dr L. Chan at Baylor College of Medicine, Houston, Tex, by use of an Effectene (Qiagen) transfection system. Two to 3 weeks after transfection, rAd plaques were picked, propagated, and screened for specific cDNA sequence by polymerase chain reaction and protein expression. A large-scale production of high-titer rAd was performed as described.34 Monolayers of 293 cells infected with rAd viruses were harvested by centrifugation and resuspended in fresh medium. After a freezing and thawing, the supernatant was collected, and rAd was harvested by CsCl gradient ultracentrifugation. The viral particles collected were dialyzed against buffer (10 nmol/L Tris, pH 7.4, 1 mmol/L MgCl2, and 10% [vol/vol] glycerol). Virus stocks were divided into aliquots and stored at -80°C. Viral titers were determined by using a plaque-assay method. The numbers of plaques formed were determined as plaque-forming units (pfu).
EC Cultures and Gene Transfer
Human umbilical vein ECs were isolated as previously described.35 ECs were seeded on a flexible membrane base of a culture well and grown for 3 days until confluence. The medium was replaced with the same medium containing only 2% FCS, and ECs were incubated overnight before the experiment. For gene transfection study, bovine aortic ECs (BAECs) were cultured in DMEM medium supplement with 10% FCS, penicillin, and streptomycin. For adenovirus gene transfer study, human umbilical vein ECs (1.0x107 cells) were infected with 10 to 100 pfu of virus per cell (multiplicity of infection [moi] 10 to 100) in serum-free medium 199 (M199) for 2 hours, followed by an overnight incubation with M199 containing 10% FCS. The infected ECs were subcultured on a flexible membrane. Before the experiment, the medium was replaced with M199 containing 2% FCS.
In Vitro Cyclic Strain on Cultured ECs
The strain unit Flexcell FX-2000 (Flexcell), which we described in detail elsewhere,36 consists of a vacuum unit linked to a valve controlled by a computer program. ECs cultured on a flexible membrane base were deformed by a sinusoidal negative pressure with a peak level of -20 kPa, which produced 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) for various intervals.
RNA Isolation and Northern Blot Analysis
Total RNA isolated from the ECs with guanidinium isothiocyanate/phenol-chloroform was separated by electrophoresis on a 1.2% agarose formaldehyde gel and transferred onto a nylon membrane (Nytran, Schleicher & Schuell Inc) by a blotting system (VacuGene XL, Pharmacia). After hybridizing with the 32P-labeled cDNA probes, the membrane was washed and then exposed to x-ray film. The results were analyzed by a densitometer (Computing Densitometer 300S, Molecular Dynamics).
Electrophoretic Mobility Shift Assay
To extract nuclear protein, ECs were washed and scraped in PBS. After centrifugation, the cell pellets were resuspended in buffer A (10 mmol/L KCl, 0.1 mmol/L EDTA, 1 mmol/L dithiothreitol, and 1 mmol/L phenylmethylsulfonyl fluoride). The cells were lysed by adding 10% NP-40 and then centrifuged to obtain nucleus pellets. The nucleus pellets were resuspended in 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, and then centrifuged. The supernatant containing the nuclear proteins was ready for the electrophoretic mobility shift assay or stored at -70°C. The oligonucleotides corresponding to the activator protein-1 (AP-1) binding site were end-labeled with [
-32P]ATP. Nuclear extract (10 µg) was incubated with 0.1 ng 32P-labeled DNA for 15 minutes at a final volume of 25 µL of the binding buffer containing 1 µg poly(dI-dC). The mixtures were electrophoresed on 7% nondenaturing polyacrylamide gels. Gels were dried and imaged by autoradiography.
Luciferase Assay
For studies of gene transfection, DNA plasmids were transfected into BAECs at their 60% confluence by using the lipofectamine method (GIBCO-BRL). ECs were cotransfected with the pSV-ß-galatosidase plasmid to normalize the transfection efficiency. After transfection, ECs were incubated overnight until reaching confluence. ECs were then seeded on flexible membranes for cyclic strain. Luciferase activity was measured with the cellular extract by using the Biotec assay system (Promega) and was recorded with a microplate scintillation counter (Topcount, Packard Instrument Co). ß-Galactosidase activity was assayed by using the substrate, o-nitrophenyl-ß-D-galactopyranoside.
Chemiluminescence Assay of Superoxide Production
Superoxide was measured by lucigenin-amplified chemiluminescence. ECs were lysed after cyclic strain treatment with a lysis buffer containing lucigenin (200 µmol/L), as previously described.37 Reading was begun on addition of the lysis buffer. Each reading was recorded with a microplate scintillation counter (Topcount, Packard Instrument Co).
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Results |
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NO Regulates Cyclic Strain–Induced MCP-1 Gene Expression via the Transcriptional Event
To study the role of NO in MCP-1 expression from ECs under cyclic strain, ECs were preincubated with an NO donor, SNAP (100 µmol/L), and then subjected to cyclic strain for 2 hours. Consistent with our early report,3 ECs under cyclic strain treatment, compared with unstrained control cells, increased MCP-1 expression 2.2-fold. However, SNAP pretreatment of ECs suppressed cyclic strain–induced MCP-1 gene expression to near the control basal level (P<0.05 versus strained ECs). Conversely, ECs pretreated with an NO synthase inhibitor, L-NAME (500 µmol/L), augmented cyclic strain–induced MCP-1 gene expression to 3.1-fold the control basal level (P<0.05 versus strained ECs). SNAP or L-NAME pretreatment did not affect the basal MCP-1 expression in unstrained control ECs. We have shown that cyclic strain–induced MCP-1 gene expression involves an increase of AP-1 binding activity to 12-O-tetradecanoylphobol-13-acetate–responsive element (TRE) in the MCP-1 promoter region.3 To determine whether NO modulates cyclic strain–induced AP-1 binding activity, nuclear proteins were incubated with oligonucleotides corresponding to the AP-1 binding site and then subjected to electrophoretic mobility shift assay. As shown in Figure I (which can be accessed online at http://www.atvb.ahajournals.org), nuclear proteins from strain-treated ECs were shown to have an increased AP-1 binding activity. This increased binding activity was greatly blunted if ECs were preexposed to SNAP. In contrast, ECs pretreated with L-NAME slightly enhanced this AP-1 binding activity. The specificity of the AP-1 complexes was confirmed by competing with excess unlabeled oligonucleotide. These results indicate that the inhibitory effect of NO on cyclic strain–induced MCP-1 expression is a transcriptional event mediated at least partially via the decrease of AP-1 binding.
eNOS Gene Transfer Inhibits Strain-Induced MCP-1 Expression
In contrast to a constant endogenous release of NO in ECs, the NO donor has characteristics of simultaneous release and a short half-life. To further substantiate the effects of NO in ECs, adenovirus carrying endothelial NO synthase (Ad-eNOS) was used to infect ECs. The efficiency of Ad-eNOS infection into ECs was illustrated by the expression of eNOS protein in ECs with an infection dose–dependent manner (Figure 1A). These infected ECs were then seeded onto a flexible membrane, followed by cyclic strain or lipopolysaccharide (LPS) treatment. The results showed that in contrast to ECs infected with CMV empty control, the Ad-eNOS–infected ECs significantly inhibited cyclic strain–induced or LPS-induced MCP-1 expression. Consistently, if infected ECs were pretreated with L-NAME, the cyclic strain–induced MCP-1 expression was resumed (Figure 1B). To confirm that the inhibitory effect of NO derived from eNOS was a transcriptional event, eNOS plasmid was cotransfected with MCP-1/Luc into BAECs, and MCP-1 promoter activities were measured. MCP-1 promoter activity was induced by cyclic strain to 2-fold that of the ECs transfected with PSR
as an empty vector. In contrast, ECs infected with plasmids encoding eNOS driven by CMV greatly decreased cyclic strain–induced MCP-1 promoter activities to the level of those in static controls (P<0.05 versus ECs with PSR). These data confirm that eNOS is essential in maintaining endothelial integrity by inhibiting redox-sensitive MCP-1 expression in ECs.
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NO Reduces Superoxide Production and Leads to a Decrease of MCP-1 Expression in ECs
It has been reported that ECs treated with an eNOS inhibitor increase their ROS levels.38 NO may play an antioxidant role via the inhibition of ROS production.29 To assess this possibility, the superoxide levels in ECs treated with SNAP or infected with Ad-eNOS were determined. When ECs were treated with SNAP, the cyclic strain–increased superoxide levels were significantly reduced, in contrast to the levels in static control cells (Figure IIA, which can be accessed online at http://atvb.ahajournals.org). Similarly, ECs infected with Ad-eNOS released less superoxide than did ECs infected with an empty vector alone (online Figure IIB). Recent data suggest that NADPH oxidase is the major source of ROS.2,8,9,39,40 To assess whether NADPH oxidase is the key player of superoxide production in cyclic strain–treated ECs, ECs were pretreated with an NADPH oxidase inhibitor, diphenyleneiodonium chloride (DPI), which greatly reduced the cyclic strain–induced superoxide levels (Figure 2A). In contrast, pretreatment of ECs with a xanthine oxidase inhibitor, oxypurinol, did not have any effect on superoxide production. Consistently, DPI or another NADPH oxidase inhibitor, apocynine, but not xanthine oxidase inhibitor, was able to suppress cyclic strain–induced MCP-1 expression (Figure 2B). These results suggest that NADPH oxidase is the main source of cyclic strain–induced ROS production. Increased NO production decreased intracellular superoxide levels, which consequently led to MCP-1 gene suppression. These results suggest that the inhibitory effect of NO on MCP-1 expression involves the reduction of superoxide level and that NO exerts its inhibitory effect via NADPH oxidase.
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ECs Infected With Ad-RacN17 Inhibit Superoxide Levels and MCP-1 Expression
Rac is required for NADPH oxidase activities.13–16 To assess the role of Rac-dependent NADPH oxidase in strain-treated ECs, cells were infected with adenovirus carrying the dominant-negative mutant of myc-tagged Rac (Ad-RacN17) for 3 days, followed with cyclic strain for 30 minutes. ECs infected with myc-tagged RacN17 showed an increased Rac protein in cells (Figure 3A and 3B). These Ad-RacN17–infected ECs not only reduced basal superoxide levels but also abolished cyclic strain–induced ROS generation (Figure 3D). Consistently, ECs infected with Ad-RacN17 significantly inhibited cyclic strain–induced MCP-1 gene expression (Figure 3C). Our results show that Rac plays an essential role in ROS production that is involved in cyclic strain–induced MCP-1 expression.
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NO Inhibition of MCP-1 Promoter Activity Is Rac Dependent
To assess whether Rac is the key signaling molecule in strained ECs affected by NO treatment, ECs were cotransfected with the constitutively active Rac (RacV12), the overexpression plasmid pFCMEK1 (downstream from Raf), or pFCMEKK (downstream from Rac), and MCP-1 promoter activities were measured. As shown in Figure 4A, only those ECs cotransfected with eNOS and RacV12 showed a significant inhibition on MCP-1 promoter activity, but ECs cotransfected with eNOS and pFCMEK1 or pFCMEKK did not. This indicates that the inhibitory effect of NO on MCP-1 promoter activity is mediated via Rac rather than via downstream signaling molecules. Similarly, the ECs transfected with eNOS inhibited their 4x TRE-Luc activity only in ECs cotransfected with RacV12 but not in cells cotransfected with pFCMEK1 or pFCMEKK (Figure 4B). Thus, Rac plays a key role in the inhibition of redox-sensitive MCP-1 expression by NO in strained ECs. To further confirm that NO from Ad-eNOS exerts an effect as an antioxidant. We analyzed the inhibitory effects of NO in Ad-RacV12–infected ECs. ECs either treated with SNAP or infected with Ad-eNOS significantly inhibited the Ad-RacV12–induced MCP-1 gene expression (Figure 4C). This inhibition appears to be mediated via the reduction of ROS, because ECs infected with Ad-eNOS inhibit ROS production induced by Ad-RacV12 (Figure 4D). Taken together, our results clearly indicate that NO produced from eNOS exerts its role as an antioxidant to attenuate the Rac-mediated endothelial responses.
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ECs under hemodynamic forces increase eNOS expression and its activity.20,21 NO has been shown to inhibit redox-sensitive genes, including cytokine-induced vascular cell adhesion molecule-1, intercellular adhesion molecule-1, monocyte chemotactic protein-1,41,42 and LPS-induced MCP-1,29 which is consistent with the role of NO in maintaining vascular integrity.43 The inhibitory effect of NO on cells is not all cGMP dependent.28,41 How NO exerts its inhibitory effects in target cells remains unclear. It may involve the interplay of reactive nitrogen and oxygen species with signaling molecules in cells. We indicated previously that cyclic strain to ECs induced MCP-1 expression that was alleviated by antioxidant treatment.3 NO-inhibiting shear-induced Egr-1 expression was mediated via the modulation of the Ras/Raf/ERK signaling pathway.33 In the present study, we further show that endothelial NO inhibits cyclic strain–induced MCP-1 expression. Several lines of evidence support this notion. First, a significant inhibition of strain-induced MCP-1 expression was shown in ECs treated with an NO donor. Second, the augmentation of this MCP-1 induction was illustrated in ECs treated with an NO synthase inhibitor. Third, NO attenuated the binding of AP-1 to the TRE required for MCP-1 gene induction in ECs. Fourth, ECs overexpressed with eNOS strongly inhibited MCP-1 expression and its promoter activity. These data indicate that NO from eNOS acts as a negatively regulator by inhibiting MCP-1 induction via reducing AP-1 binding.
ECs under hemodynamic forces increase ROS that contribute to signaling activities and gene expression.3–7 In the present study, ECs treated with an inhibitor to flavoprotein-containing NADPH oxidase, but not xanthine oxidase, significantly reduced their cyclic strain–induced ROS production, indicating the presence of NADPH oxidase for this ROS generation. ECs infected with Ad-RacN17 abolished cyclic strain–induced ROS production and, as a result, decreased MCP-1 expression. These data suggest that Rac-dependent NADPH oxidase acts as an upstream activator for cyclic strain–induced MCP-1 expression in ECs. The involvement of Rac in MCP-1 expression was further supported in ECs transfected with the constitutively active Rac (RacV12). RacV12 transfection into ECs led to an increase in MCP-1 promoter activity and gene expression. However, ECs cotransfected with eNOS and RacV12 inhibited MCP-1 expression. In contrast, ECs that overexpressed MEK1 and MEKK, downstream of Raf and Rac, respectively, abolished the inhibitory effect of eNOS on MCP-1 promoter activity. These results suggest that the inhibitory effect of NO is mediated via a pathway involving Rac and is upstream from MEK1 or MEKK. Cyclic strain induces transcriptional activity of AP-1, a heterodimer of c-Jun and c-Fos,3 that binds to the TRE in the 5' promoter region of MCP-1. Consistent with the enhancement of MCP-1 promoter activity by RacV12 transfection, RacV12 increased 4x TRE promoter activities that were attenuated by eNOS cotransfection. Overexpression of MEK1 and MEKK in ECs again abolished the inhibitory effect of eNOS. These results indicate a direct involvement of Rac in the inhibitory effect of eNOS. Because RacN17 transfection reduced superoxide levels in strained ECs, it appears that Rac plays a key role in ROS production caused by cyclic strain. In concert with the data showing ROS reduction after treatment of ECs with an NADPH oxidase inhibitor, the present data indicate that the inhibition of NO on ROS production is mediated via a Rac-dependent NADPH oxidase.
In the present study, the fact that ECs either treated with NO donor or infected with Ad-eNOS inhibited the Ad-RacV12–induced ROS production and MCP-1 expression supports the notion that NO exerts its role as an antioxidant. The negative-feedback mechanism of NO provides an underlying protective mechanism for NO in ECs under various stimuli, including hemodynamic forces. The detailed mechanism of NO inhibition on Rac-dependent ROS production in strained ECs has not been defined. NO may reduce superoxide production via a direct interaction on NADPH oxidase,44 which is in agreement with earlier reports that the inhibitory effect of NO is due to the inhibition of ROS generation.18,19 NO may inactivate NADPH oxidase by inhibiting its assembling process.45 Alternatively, NO may reduce NADPH oxidase activity via the inhibition of protein kinase C.46 The inhibitory effect of NO on MCP-1 induction is not mediated via cGMP because ECs treated with KT5823, a cGMP-dependent protein kinase inhibitor, did not attenuate the inhibitory effect of NO on cyclic strain–induced MCP-1 expression (data not shown). This is consistent with a previous observation that cGMP is not involved in the suppressive effect of NO on shear-induced Egr-1 expression.33 Nevertheless, our results support the hypothesis that NO attenuates redox-sensitive MCP-1 gene induction by cyclic strain via the inhibition of Rac-dependent ROS production. Shear stress–induced Egr-1 expression is modulated by NO via the ERK signaling pathway in ECs.33 The decrease of ROS production by NO attenuates the ERK1/2 activity, as was previously reported.47 Our results support the notion that NO acts as a negative regulator in endothelial responses to hemodynamic forces.
Atherogenesis or inflammation of the vascular wall is frequently associated with an increase of redox-sensitive genes, including MCP-1. It is conceivable that a decrease of Rac-dependent NADPH oxidase activity by NO attenuates, at least in part, vascular disorders. Study of the signaling modulation and gene alteration by NO in ECs provides us an insight into the functional role of NO in ECs under hemodynamic conditions. Because hemodynamic forces and/or reperfusion to the vascular wall tends to increase ROS that without proper surveillance may result in vascular disorders, vessel walls with eNOS gene transfer may provide a beneficial effect by attenuating these redox-induced responses.
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Acknowledgments |
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This work was supported in part by grant NSC88-2316-B001-004-M26 from the National Science Council, Taiwan, ROC.
Received June 18, 2001; accepted September 7, 2001.
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