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J Appl Physiol 102: 1520-1527, 2007. First published December 28, 2006; doi:10.1152/japplphysiol.00881.2006
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Resveratrol attenuates oxLDL-stimulated NADPH oxidase activity and protects endothelial cells from oxidative functional damages

Shu-Er Chow,1 Ya-Ching Hshu,2 Jong-Shyan Wang,3 and Jan-Kan Chen2

1Center for General Studies; 2Department of Physiology, College of Medicine; and 3Graduate Institute of Rehabilitation Science, Chang Gung University, Taoyuan, Taiwan

Submitted 9 August 2006 ; accepted in final form 18 December 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
trans-Resveratrol (RSV) has been shown to have cardioprotective effect during ischemia-reperfusion through reactive oxygen species (ROS)-scavenging activity. Elevated ROS has been implicated in the initiation and progression of atherosclerosis. The nicotinamide adenine dinucleotide phosphate oxidase (NOX) is a major source of vascular ROS formation. In the present study, we show that exposure of vascular endothelial cells (EC) to oxidized low-density lipoproteins (oxLDL) results in elevations of NOX activity and cellular ROS levels. The oxLDL effects are effectively suppressed by RSV or astringinin (AST), either before or after oxLDL exposure. In this study, we show that RSV or AST treatment appears to suppress NOX activity by reducing the membrane association of gp91phox and Rac1, two protein species required for the assembly of active NOX complex. Exposure to RSV or AST protects EC from oxidative functional damages, including antiplatelet activity and mononucleocyte adhesion. In addition, ANG II-induced NOX activation is also attenuated. These results suggest that RSV or AST protects EC from oxLDL-induced oxidative stress by both direct ROS scavenging and inhibition of NOX activity.

oxidized low-density lipoprotein; nicotinamide adenine dinucleotide phosphate oxidase; reactive oxygen species

OXIDATIVE STRESS and superoxide anion generation are believed to promote the conversion of low-density lipoproteins (LDL) to oxidized LDL (oxLDL), contributing to the pathogenesis of atherosclerosis (16, 39). OxLDL may be internalized by endothelial cells (EC) and macrophages, leading to endothelial dysfunction and foam cell formation, respectively (18). Endothelial NADPH oxidase (NOX) is a major source of reactive oxygen species (ROS) of the vasculature, and atherogenic levels of LDL have been shown to induce a marked increase in NOX-generated ROS by the endothelium (35). In addition, it has been shown that overactive NOX accounts for a genetic predisposition to atherosclerosis (16, 17, 35). OxLDL affects the endothelium in several ways, including induction of adhesion molecules, stimulation of EC apoptosis, and inhibition of some vasodilator functions (5). The biological responses triggered by oxLDL are mediated by various intracellular signaling pathways, e.g., NF-{kappa}B, and by regulation of gene expression (34).

The NOX complex in phagocytic cells is a flavocytochrome composed of two membrane-bound proteins, gp91phox and p22phox, and two cytosolic proteins, p47phox and p67phox. The small-molecular-weight G protein Rac is also necessary for the assembly of active NOX complex (1, 33, 40). Although EC appear to express most of the components found in the phagocyte oxidase, the functional importance of these subunits is incompletely understood. Recent evidence showed that a gp91phox-containing NOX is the major source of ROS in vascular EC (14) and that Rac1 is involved in EC O2bullet– production (2). These findings strongly suggest that both gp91phox and Rac1 are critical components of the endothelial NOX (42). NOX activity has been found to be elevated in a number of cardiovascular pathologies (4, 16).

trans-Resveratrol (3,4',5-trihydroxystilbene; RSV), a phenolic component of the red wines, has been shown to have cardioprotective effect during ischemia-reperfusion through its ROS-scavenging activity (20, 36). A related compound named astringinin (3,3',4',5-tetrahydroxystilbene; AST) was also found to have antioxidant activity similar to that of RSV but with a higher radical-scavenging activity (10, 20). The beneficial effects of moderate red wine consumption on the incidence of cardiovascular disease (37) are generally attributed to its natural antioxidant content, especially the RSV. It has become clear that RSV possesses pleiotropic properties, including the activation or suppression of signaling pathways (37), regulation of enzyme activities through allosteric interaction (19), and gene expression (35). Thus the cardiovascular benefit of RSV may not simply be due to its antioxidant effect; it is an important issue and deserves further exploration. Recently, trans- and cis-isomers of RSV have been shown to reduce NOX activity in rat aortic homogenate and macrophages, respectively (25, 30). However, whether RSV acts on the subunit expression of NOX protein to influence NOX activity is currently not clear. Since endothelial NOX is the major player of the ROS production in vascular walls, and the oxidative stress is believed to be implicated in aberrant behavior of the vascular endothelium and the pathogenesis of cardiovascular disease, we therefore sought to examine how RSV regulates endothelial NOX activity and to examine if such regulation correlates with an effect on the functional end point of the EC.

In the present study, we show that in oxLDL-treated EC, there is a simultaneous increase of ROS and NOX activity, and oxLDL-elicited ROS elevation is effectively suppressed by RSV or AST treatment. RSV or AST effectively abrogates oxLDL-elicited NOX activity by suppressing at least the expression levels of the membrane associated gp91phox and Rac1. Furthermore, the prevention of oxLDL-induced NOX activation by RSV is correlated with preservation of several endothelial functions, including antiplatelet activity, monocyte adhesion, and ANG II-induced NOX activation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
EC culture.   Cell culture reagents and chemicals were purchased from Sigma Chemical (St. Louis, MO) except when otherwise specified. EC were isolated from human umbilical cord vein with 0.1% type IV collagenase. The cells were cultured in medium MCDB107 supplemented with 2% FBS and a FGF-enriched fraction of porcine brain extract (1 µg/ml) (7).

Preparation of oxLDL and cell treatments.   LDL was isolated by sequential ultracentrifugation from fresh human plasma. The oxidative modification of LDL with cupric ion was performed as previously described (7). In our hands, the unoxidized LDL had a thiobarbituric acid-reactive substance (TBARS) value of ~0.33 nmol/mg, and the oxLDL had TBARS values from 3.73 to 4.84 nmol/mg. Confluent cultures of EC were incubated with oxLDL (20 µg/ml) for 1 h, and the expression levels of NOX and superoxide release were determined. EC were also treated with RSV or AST (5–10 µM; 30 min) immediately before or after exposure to oxLDL. After treatment, the cells were collected, and the ROS formation, NOX activity, and NOX protein expression were examined.

ROS measurement.   Intracellular ROS was measured by spectrofluorometry using dihydrorhodamine 123 (DHR) fluorescent probe. The cells were incubated for 20 min at 37°C in the presence of 10 mol/l DHR with gentle agitation. The reaction was stopped by cooling on ice for 1 min with subsequent addition of 500 µl PBS followed by two washing steps. Fluorescent intensity was measured by flow cytometry on a FACScalibur instrument (BD PharMingen) with excitation at 488 nm and emission at 510–550 nm. Cellular fluorescence was quantitated by the geometric means of data distributions (GMean).

Isolation of membrane and cytosolic fractions.   The cells were treated with ice-cold hypotonic lysis buffer (10 mmol/l Tris, pH 7.4, 1.5 mmol/l MgCl2, 5 mmol/l KCl, 1 mmol/l DTT, 0.2 mmol/l sodium vanadate, 1 mmol/l PMSF, 1 g/ml aprotinin, 1 g/ml leupeptin) for 5 min. After drawing the lysate through a 25-gauge syringe needle with several rapid strokes, the samples were centrifuged at 2,000 g at 4°C for 5 min. The supernatant was recentrifuged at 100,000 g at 4°C for 90 min. The 100,000 g supernatant was referred to as cytosolic fraction, and the pellet was referred to as the membrane fraction.

NOX assay.   Specific NADPH-dependent O2bullet– production was measured by lucigenin (5 µmol/l) chemiluminescence as described previously (16, 38). The NOX assay was performed with both cell homogenate and membrane fraction. The cells were exposed to oxLDL (20 µg/ml) immediately before or after RSV or AST treatment. After treatment, the cells were scraped into ice-cold Hanks balanced salt solution supplemented with 0.8 mM MgCl2 and 1.8 mM CaCl2, disrupted by rapid freezing in liquid nitrogen followed by sonication. Oxygen radical production was measured in the presence of 5 µM lucigenin, with or without NADPH (100 µM) for 20 min. The reaction was started by the addition of NADPH (100 mM), and the relative light units (RLU) of chemiluminescence were read in a microtiter luminometer (Dynatech ML2250, Dynatech Laboratories, Chantilly, VA.). The initial 1 min of enzyme activity was monitored; within this time period the luminescence generation was linear.

To examine if the observed luminescence was specific, lucigenin reduction was determined in reactions containing no NADPH or no membrane fraction. Luminescence levels under either of the "minus-one" conditions were below the background value. The background chemiluminescence value was subtracted from each reading before data calculations. The specificity of the superoxide production assay was demonstrated by inhibiting the reaction with superoxide dismutase (SOD) (120 U/ml). The SOD-inhibitable portion of the luminescence was considered as specific, and the data are expressed as RLU per minute per microgram cell protein.

Western blot analysis.   Immunoblotting was performed as described previously (26). The control and treated cells were lysed in lysis buffer (10 mM Tris·HCl, pH 8, 120 mM NaCl, 1% Nonidet P-40, 1 mM PMSF, 10 µg/ml pepstatin, and 20 µg/ml leupeptin) at 4°C for 20 min, and the lysate was then centrifuged for 10 min in a microcentrifuge. Aliquots of lysates equivalent to 6 x 104 cells were fractionated on a 12% SDS-PAGE. After electrotransfer, the nitrocellulose membranes were incubated with antibodies to gp91phox, p67phox, p47phox, or Rac1, respectively, followed by incubation with horseradish peroxidase-conjugated goat anti-mouse IgG (Amersham, Buckinghamshire, UK). The immunoreactive protein bands were visualized by enhanced chemiluminescence (ECL; Amersham International).

Platelet aggregation assay.   The Ethics Committee of Chang Gung Memorial Hospital reviewed and approved the protocol for this study. Venous blood was obtained from the volunteer donors, who provided written informed consent and who had not received any medication for at least 2 wk, by venipuncture and collected in a syringe containing sodium citrate (3.8 g/dl; 1 vol for 9 vol of blood). Platelet-rich plasma (PRP) was prepared by centrifugation of the blood sample at 120 g for 10 min at room temperature. The PRP was recentrifuged at 1,600 g for 10 min; the supernatant thus obtained is referred as platelet-poor plasma. Platelet aggregation was determined using an aggregometer (Payton, Series 10008). Three-tenths of a milliliter of PRP (2 x 108 platelets/ml) and 0.3 ml EC (2 x 106 cells/ml) were transferred to siliconized cuvettes, and the mixture was stirred at 900 revolutions/min at 37°C in the aggregometer for 1 min. Platelet aggregation was induced by 4 µM ADP in a light transmission aggregometer as described elsewhere (6).

Mononuclear cell-EC adhesion assay.   The assay was based on the stable cytoplasmic incorporation of a fluorescent dye, calcein AM (Molecular Probes, Eugene, OR). Mononuclear cells (1 x 107) were incubated with 1 ml of 10 µM calcein AM in PBS and activated with 10 µg/ml LPS for 30 min at 37°C. The cells were then washed twice with PBS to remove unincorporated dye and resuspended in MCDB107 medium at 1 x 106/ml. Mononuclear cells (200 µl) were incubated with EC grown in the 96-well plates for 30 min. Nonadherent mononuclear cells were removed by washing three times with basal medium, and the fluorescence was measured in a Denley Wellfluor fluorescence plate reader at an excitation wavelength of 485 nm and an emission wavelength of 530 nm.

Statistical analyses.   Data are presented as means ± SD. The statistical differences were determined using Student-Newman-Keuls test and Dunn's Test (Sigma Stat Software Program, Jandel Scientific, San Rafael, CA). A P value of 0.05 or less was considered as significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of RSV and AST on oxLDL-stimulated ROS production.   The direct scavenging of 1,1-diphenyl-2-picrylhydrazyl free radical by RSV and AST has been reported by one of us (20). In the present study, we measure the effect of RSV or AST on oxLDL-stimulated ROS production in EC, and the concentrations of RSV or AST and oxLDL were adopted from our prior studies (6). Exposure of EC to oxLDL (20 µg/ml) for 1 h resulted in a substantial increase of ROS formation as measured by spectrofluorometry using DHR fluorescent probe. The oxLDL effect was abolished by a flavoprotein inhibitor, diphenylene iodonium (DPI) (Fig. 1A), suggesting that it is NOX dependent. The fluorescence intensity stimulated by oxLDL was also abolished by AST or RSV treatment either before or after oxLDL exposure (Fig. 1B). AST was a more potent inhibitor than RSV; at an AST concentration of 2.5 µg/ml, the oxLDL-stimulated ROS fluorescence intensity was completely inhibited.


Figure 1
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  		Fig. 1. trans-Resveratrol (RSV) or astringinin (AST) reduces superoxide anion production in oxidized low-density lipoprotein (oxLDL)-treated endothelial cells (EC). A: cells were treated with or without oxLDL (20 µg/ml) for 1 h, and the oxLDL-treated group was further treated with or without diphenylene iodonium (DPI) at 0.2 or 0.4 µM for 15 min. Reactive oxygen species (ROS) were quantified as described in text. DHR, dihydrorhodamine 123. B: inhibition of oxLDL-stimulated ROS production by RSV or AST. OxLDL-induced ROS production was dose dependently inhibited by RSV or AST (2.5–10 µM, 30 min). Data are presented as means ± SD; n = 5. *P < 0.05.

 
RSV and AST inhibit NOX activity.   Since NOX is the major enzyme catalyzing the oxidation of NADPH and the generation of O2bullet–, we therefore chose to examine if oxLDL-elicited O2bullet– radical production is mediated via the NOX system. As shown in Fig. 2A, oxLDL-stimulated O2bullet– production in EC was dependent on the presence of NADPH (5 µM), again strongly suggesting that the stimulation required NOX activity.


Figure 2
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  		Fig. 2. Suppression of oxLDL-elicited NADPH oxidase (NOX) activity by RSV or AST. A: EC were treated with or without oxLDL (10–20 µg/ml, 1 h) in the absence (–) or presence (+) of NADPH (5 µM). NADPH-dependent O2bullet– production was measured by lucigenin (5 µmol/l) chemiluminescence as described text (n = 5; *P < 0.005). B and C: cells were treated with RSV or AST (5 µM, 30 min) or DPI (0.2 µM, 30 min) either before or after oxLDL exposure (20 µg/ml, 1 h). NOX activity was assayed in intact cells (B) and cell homogenate (C). Data are presented as means ± SD; n = 3. *P < 0.05.

 
To examine if the antioxidant effect is due to inhibition of NOX activity, the NOX activity in intact cell and cell homogenate was measured as described. In the intact cell, oxLDL elicited a >5-fold increase of the NOX activity compared with that of the untreated cell. Exposure to RSV or AST at 5 µM for 30 min before or after oxLDL exposure significantly attenuated or abrogated oxLDL effect (Fig. 2B). DPI at 0.2 µM completely abrogated oxLDL-stimulated NOX activity. Similar results were obtained when the NOX activity was assayed with cell homogenate (Fig. 2C).

The cell homogenate was fractionated into membrane (100,000 g pellet) and cytosolic (100,000 g supernatant) fractions, and the NOX activity was examined. Figure 3 shows that in both control and oxLDL-treated cells, the majority of the NOX activity is present in the membrane fraction. Similar to that assayed in the intact cell and whole cell homogenate, the membrane-bound NOX activity was also inhibited by exposure to RSV or AST before or after oxLDL exposure (Fig. 4A). Apocynin, a specific inhibitor of NOX, has been shown to inhibit superoxide formation by preventing the assembly of the superoxide-generating enzyme NOX on activation (29). In this study, we also found that from 80% to 90% of the membrane-bound NOX activity was inhibited by 200 µM apocynin or 0.2 µM DPI (Fig. 4B), strongly suggesting that the stimulation of ROS production by oxLDL is mediated via activation of the NOX activity.


Figure 3
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  		Fig. 3. Stimulation of NOX activity by oxLDL. EC were stimulated with oxLDL (20 µg/ml) for 1 h, cell homogenate was fractionated into membrane (100,000 g pellet) and cytosolic (100,000 g supernatant) fractions, and NOX activity was then determined. RLU, relative light units. Data are presented as means ± SD; n = 3. *P < 0.05.

 

Figure 4
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  		Fig. 4. AST or RSV, or DPI/apocynin on the membrane-bound NOX in EC. The cells were exposed to oxLDL (20 µg/ml) for 1 h immediately before or after RSV or AST treatment (5 µM, 30 min) or DPI treatment (0.2 µM, 30 min) or apocynin treatment (200 µM, 30 min). The membrane fraction (100,000 g pellet) was prepared, and the NOX activity was measured as described. A: stimulation of EC with oxLDL after RSV or AST or DPI/apocynin treatment. B: stimulation of EC with oxLDL before RSV or AST or DPI/apocynin treatment. Data are presented as means ± SD; n = 3. *P < 0.05.

 
RSV or AST inhibits membrane targeting of gp91phox and Rac1.   Active NOX enzyme is assembled on cell membrane (3). Here we employ Western blotting analysis to examine the presence of the essential endothelial NOX components, including gp91phox, p47phox, p67phox, and Rac1 in the cell membrane. Our purpose is to see if the stimulation by oxLDL and the inhibition by RSV or AST of the NOX activity is correlated with a reduced membrane targeting of the NOX components. Figure 5A shows that oxLDL treatment increased membrane-bound gp91phox by ~2-fold compared with that of the untreated cells. The oxLDL effect was partially abolished by RSV or AST treatment before or after oxLDL exposure.


Figure 5
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  		Fig. 5. Western blot analysis of gp91phox and Rac1 in cytosol and membrane fraction. The cells were exposed to oxLDL (20 µg/ml) for 1 h immediately before or after RSV or AST treatment (5 µM, 30 min). Presence of gp91phox (A) and Rac1 (B) in membrane and cytosol fractions was detected with the respective antibodies. GAPDH protein was also blotted and served as an internal control (C) for gel loading. The experiment was repeated 3 times with consistent result. The immunoreactive bands were scanned with a densitometer, and the ratio of the oxLDL or RSV- or AST-treated cells to untreated cells was calculated (A and B). gp91phox or Rac1 in the untreated cells was set as 100%. Data are presented as means ± SD; n = 3. *P < 0.05.

 
It has been reported that during the NOX enzyme assembly, Rac is translocated to the membrane and becomes part of the active enzyme complex (15). The membrane-cytosol partitioning of Rac1 in treated and untreated cells was therefore examined. OxLDL did not induce significant Rac1 translocation to the membrane; however, RSV or AST treatment before or after oxLDL exposure greatly reduced membrane-associated Rac1 protein with a concomitant increase of the cytosolic Rac1 protein (Fig. 5B). The membrane-cytosol partitioning of other NOX complex components is not affected by RSV or AST treatments by Western blot (data not shown). Our result demonstrates that inhibition of NOX activity by RSV or AST may be mediated by both inhibition of gp91phox and Rac1 membrane targeting and by radical scavenging.

RSV or AST protects EC from oxLDL-induced functional damages.   The cell physiological relevance of RSV or AST treatment on oxLDL-exposed EC was examined. We compared the antiplatelet activity of the EC under various treatments. The EC were treated with ox-LDL (20 µg/ml) for 1 h with or without a 30-min preexposure to RSV or AST (5 µM) before they were coincubated with platelets and ADP (4 µM). The platelet aggregation was then determined by an aggregometer. Figure 6A shows that exposure to oxLDL reduced the ability of EC to stabilize platelets from ADP-induced aggregation. RSV or AST or DPI or apocynin treatment attenuated oxLDL-induced reduction of the EC antiplatelet activity.


Figure 6
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  		Fig. 6. Effect of RSV or AST on the antiplatelet activity and mononucleocyte adhesion to EC. EC were incubated with oxLDL for 1 h at 30 min before or after RSV or AST treatment (5 µM, 30 min). A: EC were coincubated with platelet and ADP (4 µM), and the platelet aggregation was measured as described in text. Apo, apocynin. B: mononuclear cells were activated with LPS (5 µM, 30 min) and labeled with calcein AM (5 µM, 30 min). Mononucleocytes (either with or without calcein AM preincubation) were added to the EC monolayers for 30 min, and the adherence was measured as described. Data are means ± SD; n = 4. The values obtained from controls in the absence of fluorescence dye were subtracted. *P < 0.05 vs. oxLDL-treated cells.

 
The effect of RSV or AST treatment on leukocyte endothelial adhesion was also examined. Figure 6B shows that treatment of EC with oxLDL promoted the adherence of leukocytes to EC monolayer by threefold. The increased leukocyte adhesion was greatly attenuated by incubation of EC with AST or RSV either before or after oxLDL exposure.

RSV or AST attenuates ANG II-induced NOX activation.   ANG II has been shown to play an important role in the pathophysiology of cardiovascular disease, including hypertension and atherosclerosis (26). It has been reported to activate NOX activity in EC via stimulation expression of gp91phox, leading to ROS production (23, 44). Here, we examined the effect of RSV or AST on the NOX activity in ANG II-treated EC. Figure 7 shows that ANG II (0.4 µM for 1 h) elicited a >3-fold increase of the NOX activity compared with that of the untreated cell. Exposure to RSV or AST at 5 µM for 30 min either before or after ANG II exposure significantly attenuated or abrogated the oxLDL effect.


Figure 7
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  		Fig. 7. RSV or AST inhibits ANG II-induced stimulation of NOX activity in EC. The cells were treated with ANG II (0.4 µM, 1 h) either before or after RSV or AST (5 µM, 30 min) exposure. NADPH-dependent O2bullet– production was measured as described in text. Data are means ± SD; n = 3. *P < 0.05 vs. ANG II-treated cells.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Elevated ROS is considered a major contributing factor to endothelial dysfunction, and antioxidants have been found to attenuate ROS effects, which has been suggested to be due primarily to their free radical-scavenging activity (8). In the present study, we show that antioxidant RSV or AST attenuates oxLDL-elicited oxidative stress and protects cultured EC from subsequent functional damages. The beneficial effect was due probably to suppression of the NOX activity and direct scavenging of ROS. RSV has been shown to have broad antioxidant activities in a number of biological systems (32). However, whether the effect of RSV is solely due to its antioxidant activity has not been clearly elucidated. Our finding here clearly indicates that the antioxidant effect of RSV or AST is not merely due to its free radical scavenging. The RSV effect on NOX activity appears to be mediated through influence of the NOX active enzyme complex assembly in the cell membrane, as is evidenced from a reduced membrane translocation of gp91phox and Rac1. More significantly, a functional end point evaluation showed that the decreased antiplatelet activity and increased mononucleocyte adhesion elicited by oxLDL were markedly attenuated by RSV or AST treatment. Moreover, the ANG II-induced NOX activation was also suppressed by RSV or AST. Our finding strongly supported a clear physiological relevance of the RSV or AST effect in protecting EC from oxidative stress-induced functional damages. The suppressive effect of RSV or AST on ANG II-induced NOX activation also suggested that inhibition of NOX activity by RSV or AST is not limited to oxLDL-related oxistress but rather could be a more broadly attributed effect.

Recent studies indicated that vascular NOX-driven O2bullet– production may play roles in the pathophysiology of the vascular disorders (16, 28, 43). A major attribute of nonphagocytic NOX is that it is constitutively active, and its activity is sensitively influenced by a wide variety of pathophysiological stimuli (4). A rapid posttranslational activation and/or an increased expression of oxidase subunits can be involved in the upregulation of NOX activity (14, 35). Endothelial NOX activity is increased by oscillatory shear stress (21, 22), ANG II (26), ischemia (9, 41), and oxLDL (35). During NOX's activation, the NOX components in the cytosol translocate to the plasma membrane and become associated with cytochrome b558 (27). OxLDL has been shown to induce proatherosclerotic NOX expression by increasing gp91phox mRNA expression (35). In this study, we show that endothelial NOX activity is increased on exposure to oxLDL, and the effect is correlated with an increased gp91phox and Rac1 membrane translocation.

A role of Rac1-mediated O2bullet– production in DNA synthesis and cytoskeleton reorganization has been reported in fibroblasts and EC, respectively (21, 27). Rac is normally maintained in EC in an inactive form in the cytosol complexing with the GDP dissociation inhibitor Rho-GDI (1). Stimulation of the cell by VEGF disrupts the binding of Rac with Rho-GDI, leading to Rac membrane translocation via its geranylgeranyl tail and NOX activation (15). In the present study, we show that in untreated EC, Rac1 is approximately evenly distributed in membrane and cytosol (Fig. 5). Exposure to oxLDL promotes the membrane translocation of Rac1, and such translocation is attenuated by RSV or AST treatment either before or after oxLDL exposure. Furthermore, we clearly show that an increased Rac1 membrane association is accompanied with an increased NOX activity and vice versa. Our result is consistent with a recent report showing that inhibition of Rac membrane targeting by geranylgeranyl transferase inhibitor suppressed NOX activation and ROS generation (31).

Some hydroxymethylglutaryl coenzyme A reductase-inhibiting statins that possess antioxidant activity have been shown to downregulate gp91phox mRNA expression in internal mammary arteries (35). In contrast, our study shows that the RSV or AST effect appears to be mediated through disrupting gp91phox and Rac1 membrane targeting. Intriguingly, treatment of cell with RSV or AST either before or after oxLDL exposure was shown to inhibit NOX activity equally well, yet the inhibition of gp91phox and Rac1 membrane association was more prominent in the latter. The exact mechanism underlying the RSV or AST effect is currently not clear.

The potential health benefits of RSV depend, in part, on its absorption, metabolism, and bioavailability (24). RSV was found to be absorbed by human subjects after moderate red wine consumption. The concentration of RSV in red wine, on average, was estimated to be ~5 mg/l (12, 32). Assuming a daily intake of 2 glasses of red wine (~375 ml), a person weighing 70 kg would receive a daily dose of ~27 µg/kg body wt. RSV has a rather short half-life and is metabolized extensively in the body. RSV glucuronides are the major metabolites detected in the plasma, raising the speculation that its metabolites might retain some bioactivity. The peak plasma concentration of RSV and its metabolites for a person ingesting a RSV dose of 25 mg/kg body wt was reported to be approximately 37 nM and 2.1 µM, respectively (13). The concentration of RSV used in the present study is ~1.2 µg/ml (5 µM), a dose ~2.5-fold higher than that which may be achieved by moderate drinking. The in vivo minimal bioactive concentration of RSV for cardiovascular benefit is currently not clear. However, the cardiovascular benefit from moderate red wine drinking seems to suggest that the concentration used for the in vitro study may be unnecessarily higher than that required for the in vivo effects.

To ameliorate the oxidative stress of a cell, reducing the ability to form ROS should be a more effective way than to scavenge them on their formation. Considering the therapeutic potential for NOX inhibitors (11), it is noteworthy that in addition to acting as ROS scavenger, the polyphenolic RSV, and especially AST, can also reduce ROS formation via inhibition of the NOX activity.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Science Council of Taiwan Grant NSC94–2320-B-182–021 to J.-K. Chen.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J.-K. Chen, Dept. of Physiology, College of Medicine, Chang Gung Univ., 259 Wen-Hwa 1st Rd., Kwei-Shan, Taoyuan 333, Taiwan (e-mail: jkc508{at}mail.cgu.edu.tw)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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