- Email: [email protected]
Potential pro-inflammatory action of resveratrol in vascular smooth muscle cells from normal and diabetic rats
Nutrition, Metabolism & Cardiovascular Diseases (2006) 16, 322e329
www.elsevier.com/locate/nmcd
Potential pro-inflammatory action of resveratrol in vascular smooth muscle cells from normal and diabetic rats Andrea Cignarella a,b,*, Claudia Minici a, Chiara Bolego a, Christian Pinna a, Paola Sanvito a,b, Rosa Maria Gaion b, Lina Puglisi a a
Department of Pharmacological Sciences, University of Milan, via G. Balzaretti 9, I-20133 Milan, Italy b Department of Pharmacology and Anaesthesiology, University of Padova, L.go Meneghetti 2, I-35121 Padova, Italy Received 10 January 2005; received in revised form 21 April 2005; accepted 26 May 2005
KEYWORDS Resveratrol; Smooth muscle cells; Aorta; Inflammation; Inducible NO synthase; Cyclooxygenase-2
Abstract Background and aim: Based on the reported cardioprotective effects of resveratrol, a polyphenolic antioxidant abundant in grapes that binds to estrogen receptors, and the well-characterized anti-inflammatory properties of 17bestradiol, the effects of resveratrol on the functional expression of inflammatory enzymes were assessed in vascular smooth muscle cells (SMC) from normoglycaemic and streptozotocin-diabetic rats. Methods and results: SMC were isolated from the aorta four weeks after treating rats with streptozotocin or its vehicle. In SMC exposed to a cytokine mixture for 24 h, unexpectedly, treatment with resveratrol (0.1e100 mM) as well as the structurally related isoflavone genistein (1 nMe1 mM) enhanced expression of inducible NO synthase (iNOS). Genistein failed to mimic the elevated iNOS activity induced by resveratrol. Inhibition of estrogen receptors by the pure antiestrogen ICI 182,780 reversed the action of resveratrol on iNOS. In addition, resveratrol failed to alter cyclooxygenase-2 protein levels but reduced the accumulation of prostaglandin E2 in the culture medium of SMC from normoglycaemic, but not diabetic rats. Conclusions: These results indicate that resveratrol, at concentrations approaching putative peak plasma levels in vivo, exhibited no anti-inflammatory properties in vascular SMC from normal and diabetic rats. By contrast, resveratrol displayed a potential pro-inflammatory activity in settings of vascular inflammation. ª 2005 Elsevier B.V. All rights reserved.
* Corresponding author. Dipartimento di Scienze Farmacologiche, Universita ` degli Studi di Milano, via G. Balzaretti 9, I-20133 Milano, Italy. Tel.: C39 02 5031 8304; fax: C39 02 5031 8284. E-mail address: [email protected] (A. Cignarella). 0939-4753/$ - see front matter ª 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.numecd.2005.05.010
Lack of vascular anti-oxidant activity by resveratrol
Introduction The inflammatory response in vascular tissue and plasma is an important mediator of cardiovascular disease [1]. Vascular dysfunction is typically found in diabetes and is thought to result primarily from sustained hyperglycaemia and increased oxidant burden [2,3]. As a result, marked alterations of vascular NO pathways have been described during diabetes and inflammation. Inducible nitric oxide synthase (iNOS) is expressed in blood vessels in response to inflammation and its expression is critically involved in vascular dysfunction during diabetes [4,5]. Another important inflammatory enzyme in vascular tissue is cyclooxygenase 2 (COX-2). This enzyme can be constitutively expressed in some tissues and generates proinflammatory substances from arachidonic acid, particularly prostaglandin D2 (PGD2) and prostaglandin E2 (PGE2) (reviewed in ref. [6]). Resveratrol, a plant polyphenolic compound, has revealed anti-oxidant, immunomodulating and chemopreventive effects in several in vitro biological systems, thereby showing potential as antitumour and cardioprotective agent [7,8]. It has been reported that resveratrol enhances expression and activity of endothelial NOS in human endothelial cells [9]. This naturally occurring bioflavonoid may exert anti-inflammatory effects by inhibiting COX-2 and iNOS expression in macrophages [10e12]. In addition, the generation of reactive oxygen species and the proliferation of bovine aortic smooth muscle cells (SMC) induced by oxidized low-density lipoprotein are inhibited by resveratrol and related compounds [13]. Since 17b-estradiol blocks iNOS expression in vascular SMC from control [14] but not from diabetic rats [15], and resveratrol can interact with estrogen receptors (ER) as a mixed agonistantagonist [16,17], we set out to assess the antiinflammatory properties of resveratrol in SMC from both non-diabetic and diabetic rats. Quite unexpectedly, it turned out that resveratrol enhanced cytokine-induced iNOS functional expression, at least in part, via an ER-dependent mechanism and did not affect COX-2 expression, while reducing COX-2 activity in SMC from control but not diabetic rats.
Methods
323 182,270 was purchased from Tocris (Bristol, UK). The anti-iNOS polyclonal antibody was purchased from Transduction Laboratories, whereas the antiCOX-2 polyclonal antibody was from Santa Cruz. Peroxidase-coupled secondary antibodies were obtained from Vector. Resveratrol and genistein were freshly dissolved in DMSO.
Diabetes induction Diabetes was induced by injecting a single dose of STZ (65 mg/kg) freshly dissolved in citrate buffer (pH 4.5) to male SpragueeDawley rats weighing 200e225 g (Charles River, Calco, Italy). Control animals were injected with vehicle. At sacrifice animals had plasma glucose levels above 25 mM. The Principles of laboratory care as issued by the US National Institutes of Health (publication 85 23, revised 1985) were followed in this study.
Cell culture SMC were obtained from aortic intimal-medial layers of non-diabetic and 28-day diabetic rats according to Ross [18]. Cells were grown in medium 199 (M199) as previously described [14]. Experiments were carried out in phenol red-free M199. SMC were incubated for the indicated time with a cytokine mixture [14] comprising 10 ng/mL interleukin-1b (IL-1b), 10 ng/mL interferon-g (IFNg), 25 ng/mL tumour necrosis factor (TNF)-a and 1 mg/mL lipopolysaccharide (LPS). Such a mixture, which is likely to occur in vivo in a setting of vascular inflammation [1e3], consistently stimulated iNOS protein synthesis in SMC. Test compounds were added along with cytokines. Vehicle concentration was 0.1% in each well.
Western blot analysis At the end of incubations, cells were harvested in lysis buffer as described elsewhere [19]. At least 30 mg cell protein were loaded onto 10% SDSacrylamide gels. At the end of the run, proteins were transferred to nitrocellulose membrane and incubated with the primary antibodies (all 1:1000) overnight, then with the peroxidase-conjugated secondary antibodies for 1 h. Proteins were detected by chemiluminescence (Amersham Biosciences). Loading control was performed using tubulin immunodetection.
Materials Nitrite assay 17b-estradiol, resveratrol, genistein and streptozotocin (STZ) were obtained from Sigma (Milan, Italy). The estrogen receptor antagonist ICI
For nitrite measurement, 250 ml of medium samples were treated with 20 ml of 6.5 M HCl and 20 ml
324
A. Cignarella et al.
of 37.5 mM sulfanilic acid. After 10-min incubation, 20 ml of 12.5 mM N-(1-naphthyl)-ethylendiamine was added. Optical density was read at 550 nm after 15 min. Values were expressed as mmol nitrite/mg cell protein.
Determination of prostaglandin E2 After incubation, the culture medium was collected and centrifuged at 12,000 rpm for 5 minutes. PGE2 was measured with an ELISA kit (Cayman Chemical) according to the manufacturer’s instructions. Results were expressed as pg PGE2 /mg cell protein.
Statistical analysis Data were obtained from at least 3 independent experiments, each value representing meanGSEM of duplicate or triplicate determinations. Comparison between groups was performed by unpaired Student’s t-test. For multiple comparisons, oneway ANOVA with Bonferroni’s post-hoc test was used. Values of P!0.05 were considered statistically significant.
Results Cytokine stimulation of cultured aortic SMC for 24 h resulted in the production of detectable levels of iNOS, which was virtually absent in unstimulated SMC (Fig. 1). Treatment with resver-
Control
atrol (0.1e100 mM) during cytokine stimulation enhanced iNOS expression in a concentrationdependent manner (up to 10 mM) in vascular SMC from normoglycemic rats (Fig. 1) but had no effect at 100 mM, showing a bell-shaped activity profile. In SMC from diabetic rats, iNOS up-regulation by resveratrol was greatest at 100 mM (Fig. 1), showing a lower sensitivity than in SMC from control rats. For comparison, we tested the effects of a structurally related compound, genistein (1 nMe 1 mM), on the same system. Like resveratrol, genistein increased cytokine-induced iNOS production by up to 70% in control and 40% in diabetic SMC (Fig. 2). Neither resveratrol nor genistein induced iNOS expression in the absence of the cytomix nor affected cell viability (data not shown). In previous studies, we demonstrated that 17b-estradiol reduces the functional expression of iNOS in cultured aortic SMC exposed to a cytokine mixture [14,15]. This finding was reproduced in SMC from control and, to a lesser extent, from diabetic rats under the experimental conditions selected for the present study (data not shown). Cytokine stimulation increased significantly nitrite levels in the medium of both SMC groups (Fig. 3). In partial agreement with protein results, resveratrol increased cytokine-induced nitrite levels, reaching a peak at 1 mM in control SMC (Fig. 3), while no effect was seen at 100 mM. In diabetic SMC, at concentrations of 1 mM and above resveratrol enhanced the effect of cytokines to a comparable extent (Fig. 3). By contrast, genistein failed to affect nitrite release into the medium after
Diabetic iNOS 2.50
iNOS protein (arbitrary units)
iNOS protein (arbitrary units)
2.50
* 2.00
* *
1.50 1.00 0.50 0
Cytomix Resveratrol
-
+ -
+ 10-7
+ 10-6
+ 10-5
+ 10-4 M
* 2.00
*
1.50 1.00 0.50
0 Cytomix Resveratrol
-
+ -
+ 10-7
+ 10-6
+ 10-5
+ 10-4 M
Figure 1 Effects of resveratrol on cytokine-induced iNOS protein synthesis in aortic SMC from control (panel A) and diabetic (panel B) rats. SMC were grown in phenol red-free M199 medium for two days, synchronized in 0.4% FCS for 24 h and stimulated with a cytokine cocktail (10 ng/ml IL-1b, 10 ng/ml IFN-g, 25 ng/ml TNF-a and 1 mg/ml LPS) for 24 h. Increasing concentrations of resveratrol (0.1e100 mM) were added at the same time as cytokines where indicated. The amount of iNOS in cell lysates was quantitated by scanning densitometry, whereby the intensity of the cytomix band was set arbitrarily to 1. Representative Western blots are shown. Data are expressed as meanGSEM of four to six independent experiments. )P!0.05 compared with cytokines alone.
Lack of vascular anti-oxidant activity by resveratrol
Control
325
Diabetic iNOS 2.50
*
2.00
* 1.50 1.00 0.50
iNOS protein (arbitrary units)
iNOS protein (arbitrary units)
2.50
2.00
*
1.50 1.00 0.50
0
Cytomix Genistein
* *
0 -
+ -
+
+
+
10-9
10-8
10-7
+ 10-6 M
Cytomix Genistein
-
+ -
+
+
+
+
10-9
10-8
10-7
10-6 M
Figure 2 Effects of genistein on cytokine-induced iNOS protein synthesis in aortic SMC from control (panel A) and diabetic (panel B) rats. SMC were grown and stimulated as described in the legend to Fig. 1. Increasing concentrations of genistein (1 nMe1 mM) were added at the same time as cytokines where indicated. The amount of iNOS in cell lysates was quantitated as described in the legend to Fig. 1. Representative Western blots are shown. Data are expressed as meanGSEM of five to seven independent experiments. )P!0.05 compared with cytokines alone.
cytokine stimulation in both control and diabetic SMC (data not shown), suggesting that iNOS function was affected by genistein also at a post-translational level. Neither resveratrol nor genistein induced any significant change in nitrite levels in the absence of cytomix (data not shown). The effects of resveratrol were also tested at an earlier time point of inflammatory challenge, when iNOS functional expression started to be consistently detectable. As shown in Fig. 4, there was no evidence of iNOS down-regulation in response to resveratrol when control SMC were stimulated with cytokines for 4 h. Conversely, the relative increase in iNOS synthesis following treatment with 10e100 mM resveratrol as compared with cytokines alone was significant after stimulation
Control
Diabetic 0.03
** Nitrite (µmol/mg protein)
Nitrite (µmol/mg protein)
0.03
** **
0.02
* 0.01
**
0.02
**
**
* 0.01
0
0
Cytomix Resveratrol
for 4 h, showing a linear rather than bell-shaped activity profile as was the case after stimulation for 24 h (see Fig. 1). In fact, no change in iNOS levels was observed with 0.1 and 1 mM resveratrol. The amount of iNOS after 4-h stimulation was increased only by 100 mM resveratrol treatment in diabetic SMC (Fig. 4). There was a general decrease in SMC sensitivity to resveratrol after 4-h with respect to 24-h stimulation. To assess whether the effects of resveratrol on iNOS expression were mediated by interaction with ER, SMC were preincubated for 30 min with the pure estrogen antagonist ICI 182,780 (1 mM). Preliminary experiments indicated that the ICI compound alone induced no change in iNOS protein synthesis (data not shown). Treatment with 0.1 mM resveratrol with
-
+ -
+
+
+
10-7
10-6
10-5
+ 10-4 M
Cytomix Resveratrol
-
+ -
+
+
+
10-7
10-6
10-5
+ 10-4 M
Figure 3 Effects of resveratrol on cytokine-induced nitrite accumulation in the culture medium of aortic SMC from control (panel A) and diabetic (panel B) rats. SMC were grown and stimulated as described in the legend to Fig. 1. Results are meanGSEM of four to six independent experiments performed in duplicate. )P!0.01 compared with untreated; ))P!0.05 compared with cytokines alone.
326
A. Cignarella et al.
Control
Diabetic iNOS
*
2.00
*
1.50 1.00 0.50
2.50
iNOS protein (arbitrary units)
iNOS protein (arbitrary units)
2.50
* 1.50 1.00 0.50 0
0
Cytomix Resveratrol
2.00
-
+ -
+
+
+
10-7
10-6
10-5
+ 10-4 M
Cytomix Resveratrol
-
+ -
+
+
+
10-7
10-6
10-5
+ 10-4 M
Figure 4 Effects of resveratrol on cytokine-induced iNOS protein synthesis in SMC from control (panel A) and diabetic (panel B) rats after short cytokine stimulation. SMC were grown and stimulated for 4 h as described in the legend to Fig. 1. The amount of iNOS in cell lysates was quantitated as described in the legend to Fig. 1. Representative Western blots are shown. Data are expressed as meanGSEM of three independent experiments. ) P!0.01 vs cytokines alone.
or without ICI 182,780 did not affect iNOS immunoreactivity (Fig. 5). By contrast, ICI 182,780 prevented the rise in cytokine-induced iNOS protein levels elicited by 10 mM resveratrol in control as well as diabetic SMC (Fig. 5), consistent with an action mediated through ER pathways. Therefore, resveratrol controlled iNOS expression in vascular SMC, at least in part, via ER-dependent mechanisms. The pro-inflammatory potential of resveratrol was tested on another inflammatory enzyme expressed by vascular SMC, COX-2. Under the present
Control
experimental conditions, COX-2 turned out to be constitutively expressed in both control and diabetic SMC, as shown in Fig. 6. Stimulation with the cytokine mixture did not lead to significant changes in COX-2 protein levels in either group. Resveratrol was ineffective on COX-2 levels in control SMC but increased them by w25% at the highest concentration tested (100 mM) in diabetic SMC. For comparison, 17b-estradiol elicited no effect whatsoever on COX-2 protein levels (data not shown). Despite detectable levels of COX-2 protein, PGE2 release into the medium was close to
Diabetic iNOS
*
1.50
#
1.00
0.50
0
Cytomix Resveratrol ICI 182,780
iNOS protein (arbitrary units)
iNOS protein (arbitrary units)
1.50
* # 1.00
0.50
0 -
+ -
+ 10-7 -
+ 10-7
+ 10-5
+ 10-5 M
10-6
-
10-6 M
Cytomix Resveratrol ICI 182,780
-
+ -
+ 10-7 -
+ 10-7
+ 10-5
+ 10-5 M
10-6
-
10-6 M
Figure 5 Effects of resveratrol and the estrogen receptor antagonist ICI 182,780 on cytokine-induced iNOS protein synthesis in aortic SMC from control (panel A) and diabetic (panel B) rats. SMC were grown and stimulated as described in the legend to Fig. 1. Preincubation with 1 mM ICI 182,780 for 30 min was performed before adding resveratrol (0.1 and 10 mM) at the same time as cytokines. The amount of iNOS in cell lysates was quantitated as described in the legend to Fig. 1. Representative Western blots are shown. Data are expressed as meanGSEM of three independent experiments. )P!0.05 compared with cytomix, #P!0.05 compared with 10 mM resveratrol alone.
Lack of vascular anti-oxidant activity by resveratrol
Control
327
Diabetic COX-2 protein (arbitrary units)
COX-2 protein (arbitrary units)
COX-2 1.50
1.00
0.50
0
Cytomix Resveratrol
-
+ -
+
+
+
10-7
10-6
10-5
+ 10-4 M
1.50
*
1.00
0.50
0
Cytomix Resveratrol
-
+ -
+
+
+
10-7
10-6
10-5
+ 10-4 M
Figure 6 Effects of resveratrol on COX-2 protein synthesis in aortic SMC from control (panel A) and diabetic (panel B) rats. SMC were grown and stimulated as described in the legend to Fig. 1. The amount of COX-2 in cell lysates was determined by scanning densitometry, whereby the intensity of the cytomix band was set arbitrarily to 1. Representative Western blots are shown. Data are expressed as meanGSEM of three to five independent experiments. )P!0.05 compared with cytokines alone.
detection limit in the basal state and was enhanced by cytokine stimulation in both SMC groups (Table 1). Treatment with resveratrol reduced PGE2 levels in the medium of SMC from normoglycaemic rats only at 100 mM but had no effect in SMC from diabetic rats as compared with cytokines alone (Table 1). For comparison, 17b-estradiol treatment did not affect significantly PGE2 levels in both SMC groups (data not shown).
Table 1 Effect of increasing concentrations of resveratrol on prostaglandin E2 (PGE2) levels released into the incubation medium by SMC after inflammatory challenge with cytokines for 24 h PGE2 (pg/mg cell protein) Basal Cytokines CytokinesCresveratrol 0.1 mM CytokinesCresveratrol 1 mM CytokinesCresveratrol 10 mM CytokinesCresveratrol 100 mM
Control
Diabetes
197G71 3715G1301a 3622G1262
16G1 4197G1476a 5130G1834
3395G1197
5885G1112
2470G847
6274G1251
186G62b
5299G1149
Data are expressed as meanGSEM of three independent experiments each performed in triplicate. a P!0.01 vs. basal. b P!0.01 vs. cytokines (ANOVA and Bonferroni’s post-hoc test).
Discussion The naturally occurring polyphenol resveratrol has recently attracted considerable interest because of the appreciation of its antioxidant and antiinflammatory effects, which could possibly lead to cardioprotective and chemiopreventive actions [7,8]. This compound, abundant in grapes, may contribute to the reduced risk of coronary heart disease in humans who have consumed moderate amounts of alcohol from red wine regularly [20]. With this background, it was expected that resveratrol would reduce the functional expression of inflammatory enzymes such as iNOS in vascular SMC from non-diabetic and also from diabetic rats, which feature a vascular dysfunction of inflammatory nature [2,3]. By contrast, resveratrol enhanced cytokine-mediated iNOS activation in both SMC groups at concentrations that could be achieved in vivo, at least in part via an ERdependent mechanism. In SMC from normoglycaemic rats, however, resveratrol had no effect on iNOS functional expression at pharmacological concentrations (100 mM), possibly due to interactions with additional and rather aspecific molecular targets such as protein kinase C [21,22] that apparently were not activated in SMC from diabetic rats. Furthermore, the duration of inflammatory challenge affected iNOS regulation by resveratrol, suggesting that multiple molecular targets ultimately control its biological effects. Thus, two major questions arise. First, why did resveratrol fail to exert an anti-inflammatory effect in our experimental system? Second, what
328 kind of interaction with ER could account for the observed pro-inflammatory effects? Inhibition of iNOS function by resveratrol has been reported to occur particularly in macrophage cells [10,12,24e26]. In vascular SMC, however, the molecular pathways mediating the anti-proliferative properties of resveratrol [13,27e29], but not its potential anti-inflammatory activity, have been reported in detail. Most of the anti-inflammatory and growth inhibitory properties of resveratrol in different cell types have been ascribed to impaired activation of NF-kB [23]. It is conceivable that the lack of anti-inflammatory action of resveratrol in SMC from both non-diabetic and diabetic rats was accounted for by the use of a cytokine mix, which more closely resembles inflammatory settings in vivo than a single mediator. Even if we assume that resveratrol effectively down-regulated NF-kB, other transcription factors might have fostered iNOS induction. It is important to note, though, that our findings do not provide information concerning endothelial function, which can be stimulated by resveratrol via ER resulting in MAPK and eNOS activation [30]. The estrogenic component of resveratrol action was involved in the findings herein reported. ERa and ERb are expressed in vascular SMC of rats [14,15] and humans [31]. Resveratrol exhibits variable degrees of ER agonism in different test systems [17] and is known to act as a mixed agonist/antagonist for ERa and ERb according to cell type, response element sequence and coactivator/corepressor protein expression [16]. Our working hypothesis is that resveratrol, as much as the phytoestrogen genistein, enhanced iNOS activation in vascular SMC by virtue of preferential ERb agonist activity. Several lines of evidence support this notion. First, it is well established that genistein has remarkable affinity for ERb [32]; second, the synthetic selective ERb agonist 2, 3-bis(4-hydroxyphenyl)-propionitrile, unlike 17bestradiol, up-regulated iNOS function in aortic SMC from both control and diabetic rats (unpublished observations); third, the anti-inflammatory activity of 17b-estradiol in the brain has been shown to be mediated by ERa [33]. Post-translational modulation of iNOS activity by genistein may occur via different mechanisms including interaction with PPARs [34], while tyrosine kinase inhibition is unlikely to occur below mM concentrations. Under the present experimental conditions COX-2 protein was found to be constitutively expressed in vascular SMC. There have been reports of constitutive COX-2 expression in vascular cells [35], yet it cannot be ruled out that COX-2 expression as observed in the present study was
A. Cignarella et al. triggered by serum [36]. However, COX-2 protein in the basal state was virtually inactive, possibly due to impaired activity of the 85-kD phospholipase A2 in the absence of inflammatory stimuli [37]. Resveratrol at high concentration (100 mM) reduced COX-2 activity but not protein levels, in contrast to previous reports in other cell types [11,12,38]. Conversely, the apparent increase in PGE2 levels induced particularly by 10-mM resveratrol treatment in SMC from diabetic rats, to the best of our knowledge, was not reported previously. The different response to resveratrol in terms of COX-2 protein levels and activity suggests that COX-1 is a major source of inflammatory PGE2 in the rat [39]. In conclusion, using iNOS and COX-2 as surrogate endpoints, resveratrol showed no significant antiinflammatory properties in vascular SMC from normoglycaemic and diabetic rats at concentrations approaching putative peak plasma levels in vivo [40,41]. Conversely, a potential pro-inflammatory activity might be associated with this compound.
References [1] Blake GJ, Ridker PM. Novel clinical markers of vascular wall inflammation. Circ Res 2001;89:763e7. [2] De Vriese AS, Verbeuren TJ, Van de Voorde J, Lameire NH, Vanhoutte PM. Endothelial dysfunction in diabetes. Br J Pharmacol 2000;130:963e74. [3] Creager MA, Luscher TF, Cosentino F, Beckman JA. Diabetes and vascular disease: pathophysiology, clinical consequences, and medical therapy e part I. Circulation 2003;108:1527e32. [4] Bardell AL, MacLeod KM. Evidence for inducible nitricoxide synthase expression and activity in vascular smooth muscle of streptozotocin-diabetic rats. J Pharmacol Exp Ther 2001;296:252e9. [5] Gunnett CA, Heistad DD, Faraci FM. Gene-targeted mice reveal a critical role for inducible nitric oxide synthase in vascular dysfunction during diabetes. Stroke 2003;34: 2970e4. [6] Warner TD, Mitchell JA. Cyclooxygenases: new forms, new inhibitors, and lessons from the clinic. FASEB J 2004;18: 790e804. [7] Fre ´mont L. Biological effects of resveratrol. Life Sci 2000; 66:663e73. [8] Pervaiz S. Resveratrol: from grapevines to mammalian biology. FASEB J 2003;17:1975e85. [9] Wallerath T, Deckert G, Ternes T, et al. Resveratrol, a polyphenolic phytoalexin present in red wine, enhances expression and activity of endothelial nitric oxide synthase. Circulation 2002;106:1652e8. [10] Chan MM, Mattiacci JA, Hwang HS, Shah A, Fong D. Synergy between ethanol and grape polyphenols, quercetin, and resveratrol in the inhibition of the inducible nitric oxide synthase pathway. Biochem Pharmacol 2000;60:1539e48. [11] Martinez J, Moreno JJ. Effect of resveratrol, a natural polyphenolic compound, on reactive oxygen species and prostaglandin production. Biochem Pharmacol 2000;59: 865e70.
Lack of vascular anti-oxidant activity by resveratrol [12] Tsai SH, Lin-Shiau SY, Lin JK. Suppression of nitric oxide synthase and the down-regulation of the activation of NFkB in macrophages by resveratrol. Br J Pharmacol 1999; 126:673e80. [13] Liu Y, Liu G. Isorhapontigenin and resveratrol suppress oxLDL-induced proliferation and activation of ERK1/2 mitogen-activated protein kinases of bovine aortic smooth muscle cells. Biochem Pharmacol 2004;67:777e85. [14] Zancan V, Santagati S, Bolego C, Vegeto E, Maggi A, Puglisi L. 17b-estradiol decreases nitric oxide synthase II synthesis in vascular smooth muscle cells. Endocrinology 1999;140:2004e9. [15] Maggi A, Cignarella A, Brusadelli A, Bolego C, Pinna C, Puglisi L. Diabetes undermines estrogen control of inducible nitric oxide synthase function in rat aortic smooth muscle cells through overexpression of estrogen receptor-b. Circulation 2003;108:211e7. [16] Bowers JL, Tyulmenkov VV, Jernigan SC, Klinge CM. Resveratrol acts as a mixed agonist/antagonist for estrogen receptors a and b. Endocrinology 2000;141:3657e67. [17] Gehm BD, McAndrews JM, Chien PY, Jameson JL. Resveratrol, a polyphenolic compound found in grapes and wine, is an agonist for the estrogen receptor. Proc Natl Acad Sci USA 1997;94:14138e43. [18] Ross R. The smooth muscle cell II - growth of smooth muscle in culture and formation of elastic fibers. J Cell Biol 1971;103:2787e96. [19] Idel S, Ellinghaus P, Wolfrum C, et al. Branched chain fatty acids induce nitric oxide-dependent apoptosis in vascular smooth muscle cells. J Biol Chem 2002;277:49319e25. [20] Wu JM, Wang ZR, Hsieh TC, Bruder JL, Zou JG, Huang YZ. Mechanism of cardioprotection by resveratrol, a phenolic antioxidant present in red wine. Int J Mol Med 2001;8:3e17. [21] Ferriola PC, Cody V, Middleton Jr E. Protein kinase C interaction by plant flavonoids: kinetic mechanism and structure activity relationships. Biochem Pharmacol 1989; 38:1617e24. [22] Slater SJ, Seiz JL, Cook AC, Stagliano BA, Buzas CJ. Inhibition of protein kinase C by resveratrol. Biochim Biophys Acta 2003;1637:59e69. [23] Manna SK, Mukhopadhyay A, Aggarwal BB. Resveratrol suppresses TNF-induced activation of nuclear transcription factors NF-kB, activator protein-1, and apoptosis: potential role of reactive oxygen intermediates and lipid peroxidation. J Immunol 2000;164:6509e19. [24] Cho DI, Koo NY, Chung WJ, et al. Effects of resveratrolrelated hydroxystilbenes on the nitric oxide production in macrophage cells: structural requirements and mechanism of action. Life Sci 2002;71:2071e82. [25] Wadsworth TL, Koop DR. Effects of the wine polyphenolics quercetin and resveratrol on pro-inflammatory cytokine expression in RAW 2647 macrophages. Biochem Pharmacol 1999;57:941e9. [26] Bi XL, Yang JY, Dong YX, et al. Resveratrol inhibits nitric oxide and TNF-a production by lipopolysaccharide-activated microglia. Int Immunopharmacol 2005;5:185e93.
329 [27] Haider UG, Sorescu D, Griendling KK, Vollmar AM, Dirsch VM. Resveratrol suppresses angiotensin II-induced Akt/protein kinase B and p70 S6 kinase phosphorylation and subsequent hypertrophy in rat aortic smooth muscle cells. Mol Pharmacol 2002;62:772e7. [28] Haider UG, Sorescu D, Griendling KK, Vollmar AM, Dirsch VM. Resveratrol increases serine15-phosphorylated but transcriptionally impaired p53 and induces a reversible DNA replication block in serum-activated vascular smooth muscle cells. Mol Pharmacol 2003;63:925e32. [29] Zou J, Huang Y, Cao K, et al. Effect of resveratrol on intimal hyperplasia after endothelial denudation in an experimental rabbit model. Life Sci 2000;68:153e63. [30] Klinge CM, Blankenship KA, Risinger KE, et al. Resveratrol and estradiol rapidly activate MAPK signaling through estrogen receptors a and b in endothelial cells. J Biol Chem 2005;280:7460e8. [31] Hodges YK, Tung L, Yan XD, Graham JD, Horwitz KB, Horwitz LD. Estrogen receptors a and b: prevalence of estrogen receptor b mRNA in human vascular smooth muscle and transcriptional effects. Circulation 2000;101: 1792e8. [32] Kuiper GG, Lemmen JG, Carlsson B, et al. Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor b. Endocrinology 1998;139:4252e63. [33] Vegeto E, Belcredito S, Etteri S, et al. Estrogen receptora mediates the brain antiinflammatory activity of estradiol. Proc Natl Acad Sci USA 2003;100:9614e9. [34] Dang ZC, Audinot V, Papapoulos SE, Boutin JA, Lowik CW. Peroxisome proliferator-activated receptor g (PPARg) as a molecular target for the soy phytoestrogen genistein. J Biol Chem 2003;278:962e7. [35] Parfenova H, Parfenov VN, Shlopov BV, et al. Dynamics of nuclear localization sites for COX-2 in vascular endothelial cells. Am J Physiol 2001;281:C166e78. [36] Rimarachin JA, Jacobson JA, Szabo P, Maclouf J, Creminon C, Weksler BB. Regulation of cyclooxygenase-2 expression in aortic smooth muscle cells. Arterioscler Thromb 1994;14:1021e31. [37] Mitchell JA, Warner TD. Cyclo-oxygenase-2: pharmacology, physiology, biochemistry and relevance to NSAID therapy. Br J Pharmacol 1999;128:1121e32. [38] Subbaramaiah K, Chung WJ, Michaluart P, et al. Resveratrol inhibits cyclooxygenase-2 transcription and activity in phorbol ester-treated human mammary epithelial cells. J Biol Chem 1998;273:21875e82. [39] Giuliano F, Warner TD. Origins of prostaglandin E2: involvements of cyclooxygenase (COX)-1 and COX-2 in human and rat systems. J Pharmacol Exp Ther 2002;303: 1001e6. [40] Gescher AJ, Steward WP. Relationship between mechanisms, bioavailibility, and preclinical chemopreventive efficacy of resveratrol: a conundrum. Cancer Epidemiol Biomarkers Prev 2003;12:953e7. [41] Walle T, Hsieh F, Delegge MH, Oatis Jr JE, Walle UK. High absorption but very low bioavailability of oral resveratrol in humans. Drug Metab Dispos 2004;32:1377e82.
www.elsevier.com/locate/nmcd
Potential pro-inflammatory action of resveratrol in vascular smooth muscle cells from normal and diabetic rats Andrea Cignarella a,b,*, Claudia Minici a, Chiara Bolego a, Christian Pinna a, Paola Sanvito a,b, Rosa Maria Gaion b, Lina Puglisi a a
Department of Pharmacological Sciences, University of Milan, via G. Balzaretti 9, I-20133 Milan, Italy b Department of Pharmacology and Anaesthesiology, University of Padova, L.go Meneghetti 2, I-35121 Padova, Italy Received 10 January 2005; received in revised form 21 April 2005; accepted 26 May 2005
KEYWORDS Resveratrol; Smooth muscle cells; Aorta; Inflammation; Inducible NO synthase; Cyclooxygenase-2
Abstract Background and aim: Based on the reported cardioprotective effects of resveratrol, a polyphenolic antioxidant abundant in grapes that binds to estrogen receptors, and the well-characterized anti-inflammatory properties of 17bestradiol, the effects of resveratrol on the functional expression of inflammatory enzymes were assessed in vascular smooth muscle cells (SMC) from normoglycaemic and streptozotocin-diabetic rats. Methods and results: SMC were isolated from the aorta four weeks after treating rats with streptozotocin or its vehicle. In SMC exposed to a cytokine mixture for 24 h, unexpectedly, treatment with resveratrol (0.1e100 mM) as well as the structurally related isoflavone genistein (1 nMe1 mM) enhanced expression of inducible NO synthase (iNOS). Genistein failed to mimic the elevated iNOS activity induced by resveratrol. Inhibition of estrogen receptors by the pure antiestrogen ICI 182,780 reversed the action of resveratrol on iNOS. In addition, resveratrol failed to alter cyclooxygenase-2 protein levels but reduced the accumulation of prostaglandin E2 in the culture medium of SMC from normoglycaemic, but not diabetic rats. Conclusions: These results indicate that resveratrol, at concentrations approaching putative peak plasma levels in vivo, exhibited no anti-inflammatory properties in vascular SMC from normal and diabetic rats. By contrast, resveratrol displayed a potential pro-inflammatory activity in settings of vascular inflammation. ª 2005 Elsevier B.V. All rights reserved.
* Corresponding author. Dipartimento di Scienze Farmacologiche, Universita ` degli Studi di Milano, via G. Balzaretti 9, I-20133 Milano, Italy. Tel.: C39 02 5031 8304; fax: C39 02 5031 8284. E-mail address: [email protected] (A. Cignarella). 0939-4753/$ - see front matter ª 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.numecd.2005.05.010
Lack of vascular anti-oxidant activity by resveratrol
Introduction The inflammatory response in vascular tissue and plasma is an important mediator of cardiovascular disease [1]. Vascular dysfunction is typically found in diabetes and is thought to result primarily from sustained hyperglycaemia and increased oxidant burden [2,3]. As a result, marked alterations of vascular NO pathways have been described during diabetes and inflammation. Inducible nitric oxide synthase (iNOS) is expressed in blood vessels in response to inflammation and its expression is critically involved in vascular dysfunction during diabetes [4,5]. Another important inflammatory enzyme in vascular tissue is cyclooxygenase 2 (COX-2). This enzyme can be constitutively expressed in some tissues and generates proinflammatory substances from arachidonic acid, particularly prostaglandin D2 (PGD2) and prostaglandin E2 (PGE2) (reviewed in ref. [6]). Resveratrol, a plant polyphenolic compound, has revealed anti-oxidant, immunomodulating and chemopreventive effects in several in vitro biological systems, thereby showing potential as antitumour and cardioprotective agent [7,8]. It has been reported that resveratrol enhances expression and activity of endothelial NOS in human endothelial cells [9]. This naturally occurring bioflavonoid may exert anti-inflammatory effects by inhibiting COX-2 and iNOS expression in macrophages [10e12]. In addition, the generation of reactive oxygen species and the proliferation of bovine aortic smooth muscle cells (SMC) induced by oxidized low-density lipoprotein are inhibited by resveratrol and related compounds [13]. Since 17b-estradiol blocks iNOS expression in vascular SMC from control [14] but not from diabetic rats [15], and resveratrol can interact with estrogen receptors (ER) as a mixed agonistantagonist [16,17], we set out to assess the antiinflammatory properties of resveratrol in SMC from both non-diabetic and diabetic rats. Quite unexpectedly, it turned out that resveratrol enhanced cytokine-induced iNOS functional expression, at least in part, via an ER-dependent mechanism and did not affect COX-2 expression, while reducing COX-2 activity in SMC from control but not diabetic rats.
Methods
323 182,270 was purchased from Tocris (Bristol, UK). The anti-iNOS polyclonal antibody was purchased from Transduction Laboratories, whereas the antiCOX-2 polyclonal antibody was from Santa Cruz. Peroxidase-coupled secondary antibodies were obtained from Vector. Resveratrol and genistein were freshly dissolved in DMSO.
Diabetes induction Diabetes was induced by injecting a single dose of STZ (65 mg/kg) freshly dissolved in citrate buffer (pH 4.5) to male SpragueeDawley rats weighing 200e225 g (Charles River, Calco, Italy). Control animals were injected with vehicle. At sacrifice animals had plasma glucose levels above 25 mM. The Principles of laboratory care as issued by the US National Institutes of Health (publication 85 23, revised 1985) were followed in this study.
Cell culture SMC were obtained from aortic intimal-medial layers of non-diabetic and 28-day diabetic rats according to Ross [18]. Cells were grown in medium 199 (M199) as previously described [14]. Experiments were carried out in phenol red-free M199. SMC were incubated for the indicated time with a cytokine mixture [14] comprising 10 ng/mL interleukin-1b (IL-1b), 10 ng/mL interferon-g (IFNg), 25 ng/mL tumour necrosis factor (TNF)-a and 1 mg/mL lipopolysaccharide (LPS). Such a mixture, which is likely to occur in vivo in a setting of vascular inflammation [1e3], consistently stimulated iNOS protein synthesis in SMC. Test compounds were added along with cytokines. Vehicle concentration was 0.1% in each well.
Western blot analysis At the end of incubations, cells were harvested in lysis buffer as described elsewhere [19]. At least 30 mg cell protein were loaded onto 10% SDSacrylamide gels. At the end of the run, proteins were transferred to nitrocellulose membrane and incubated with the primary antibodies (all 1:1000) overnight, then with the peroxidase-conjugated secondary antibodies for 1 h. Proteins were detected by chemiluminescence (Amersham Biosciences). Loading control was performed using tubulin immunodetection.
Materials Nitrite assay 17b-estradiol, resveratrol, genistein and streptozotocin (STZ) were obtained from Sigma (Milan, Italy). The estrogen receptor antagonist ICI
For nitrite measurement, 250 ml of medium samples were treated with 20 ml of 6.5 M HCl and 20 ml
324
A. Cignarella et al.
of 37.5 mM sulfanilic acid. After 10-min incubation, 20 ml of 12.5 mM N-(1-naphthyl)-ethylendiamine was added. Optical density was read at 550 nm after 15 min. Values were expressed as mmol nitrite/mg cell protein.
Determination of prostaglandin E2 After incubation, the culture medium was collected and centrifuged at 12,000 rpm for 5 minutes. PGE2 was measured with an ELISA kit (Cayman Chemical) according to the manufacturer’s instructions. Results were expressed as pg PGE2 /mg cell protein.
Statistical analysis Data were obtained from at least 3 independent experiments, each value representing meanGSEM of duplicate or triplicate determinations. Comparison between groups was performed by unpaired Student’s t-test. For multiple comparisons, oneway ANOVA with Bonferroni’s post-hoc test was used. Values of P!0.05 were considered statistically significant.
Results Cytokine stimulation of cultured aortic SMC for 24 h resulted in the production of detectable levels of iNOS, which was virtually absent in unstimulated SMC (Fig. 1). Treatment with resver-
Control
atrol (0.1e100 mM) during cytokine stimulation enhanced iNOS expression in a concentrationdependent manner (up to 10 mM) in vascular SMC from normoglycemic rats (Fig. 1) but had no effect at 100 mM, showing a bell-shaped activity profile. In SMC from diabetic rats, iNOS up-regulation by resveratrol was greatest at 100 mM (Fig. 1), showing a lower sensitivity than in SMC from control rats. For comparison, we tested the effects of a structurally related compound, genistein (1 nMe 1 mM), on the same system. Like resveratrol, genistein increased cytokine-induced iNOS production by up to 70% in control and 40% in diabetic SMC (Fig. 2). Neither resveratrol nor genistein induced iNOS expression in the absence of the cytomix nor affected cell viability (data not shown). In previous studies, we demonstrated that 17b-estradiol reduces the functional expression of iNOS in cultured aortic SMC exposed to a cytokine mixture [14,15]. This finding was reproduced in SMC from control and, to a lesser extent, from diabetic rats under the experimental conditions selected for the present study (data not shown). Cytokine stimulation increased significantly nitrite levels in the medium of both SMC groups (Fig. 3). In partial agreement with protein results, resveratrol increased cytokine-induced nitrite levels, reaching a peak at 1 mM in control SMC (Fig. 3), while no effect was seen at 100 mM. In diabetic SMC, at concentrations of 1 mM and above resveratrol enhanced the effect of cytokines to a comparable extent (Fig. 3). By contrast, genistein failed to affect nitrite release into the medium after
Diabetic iNOS 2.50
iNOS protein (arbitrary units)
iNOS protein (arbitrary units)
2.50
* 2.00
* *
1.50 1.00 0.50 0
Cytomix Resveratrol
-
+ -
+ 10-7
+ 10-6
+ 10-5
+ 10-4 M
* 2.00
*
1.50 1.00 0.50
0 Cytomix Resveratrol
-
+ -
+ 10-7
+ 10-6
+ 10-5
+ 10-4 M
Figure 1 Effects of resveratrol on cytokine-induced iNOS protein synthesis in aortic SMC from control (panel A) and diabetic (panel B) rats. SMC were grown in phenol red-free M199 medium for two days, synchronized in 0.4% FCS for 24 h and stimulated with a cytokine cocktail (10 ng/ml IL-1b, 10 ng/ml IFN-g, 25 ng/ml TNF-a and 1 mg/ml LPS) for 24 h. Increasing concentrations of resveratrol (0.1e100 mM) were added at the same time as cytokines where indicated. The amount of iNOS in cell lysates was quantitated by scanning densitometry, whereby the intensity of the cytomix band was set arbitrarily to 1. Representative Western blots are shown. Data are expressed as meanGSEM of four to six independent experiments. )P!0.05 compared with cytokines alone.
Lack of vascular anti-oxidant activity by resveratrol
Control
325
Diabetic iNOS 2.50
*
2.00
* 1.50 1.00 0.50
iNOS protein (arbitrary units)
iNOS protein (arbitrary units)
2.50
2.00
*
1.50 1.00 0.50
0
Cytomix Genistein
* *
0 -
+ -
+
+
+
10-9
10-8
10-7
+ 10-6 M
Cytomix Genistein
-
+ -
+
+
+
+
10-9
10-8
10-7
10-6 M
Figure 2 Effects of genistein on cytokine-induced iNOS protein synthesis in aortic SMC from control (panel A) and diabetic (panel B) rats. SMC were grown and stimulated as described in the legend to Fig. 1. Increasing concentrations of genistein (1 nMe1 mM) were added at the same time as cytokines where indicated. The amount of iNOS in cell lysates was quantitated as described in the legend to Fig. 1. Representative Western blots are shown. Data are expressed as meanGSEM of five to seven independent experiments. )P!0.05 compared with cytokines alone.
cytokine stimulation in both control and diabetic SMC (data not shown), suggesting that iNOS function was affected by genistein also at a post-translational level. Neither resveratrol nor genistein induced any significant change in nitrite levels in the absence of cytomix (data not shown). The effects of resveratrol were also tested at an earlier time point of inflammatory challenge, when iNOS functional expression started to be consistently detectable. As shown in Fig. 4, there was no evidence of iNOS down-regulation in response to resveratrol when control SMC were stimulated with cytokines for 4 h. Conversely, the relative increase in iNOS synthesis following treatment with 10e100 mM resveratrol as compared with cytokines alone was significant after stimulation
Control
Diabetic 0.03
** Nitrite (µmol/mg protein)
Nitrite (µmol/mg protein)
0.03
** **
0.02
* 0.01
**
0.02
**
**
* 0.01
0
0
Cytomix Resveratrol
for 4 h, showing a linear rather than bell-shaped activity profile as was the case after stimulation for 24 h (see Fig. 1). In fact, no change in iNOS levels was observed with 0.1 and 1 mM resveratrol. The amount of iNOS after 4-h stimulation was increased only by 100 mM resveratrol treatment in diabetic SMC (Fig. 4). There was a general decrease in SMC sensitivity to resveratrol after 4-h with respect to 24-h stimulation. To assess whether the effects of resveratrol on iNOS expression were mediated by interaction with ER, SMC were preincubated for 30 min with the pure estrogen antagonist ICI 182,780 (1 mM). Preliminary experiments indicated that the ICI compound alone induced no change in iNOS protein synthesis (data not shown). Treatment with 0.1 mM resveratrol with
-
+ -
+
+
+
10-7
10-6
10-5
+ 10-4 M
Cytomix Resveratrol
-
+ -
+
+
+
10-7
10-6
10-5
+ 10-4 M
Figure 3 Effects of resveratrol on cytokine-induced nitrite accumulation in the culture medium of aortic SMC from control (panel A) and diabetic (panel B) rats. SMC were grown and stimulated as described in the legend to Fig. 1. Results are meanGSEM of four to six independent experiments performed in duplicate. )P!0.01 compared with untreated; ))P!0.05 compared with cytokines alone.
326
A. Cignarella et al.
Control
Diabetic iNOS
*
2.00
*
1.50 1.00 0.50
2.50
iNOS protein (arbitrary units)
iNOS protein (arbitrary units)
2.50
* 1.50 1.00 0.50 0
0
Cytomix Resveratrol
2.00
-
+ -
+
+
+
10-7
10-6
10-5
+ 10-4 M
Cytomix Resveratrol
-
+ -
+
+
+
10-7
10-6
10-5
+ 10-4 M
Figure 4 Effects of resveratrol on cytokine-induced iNOS protein synthesis in SMC from control (panel A) and diabetic (panel B) rats after short cytokine stimulation. SMC were grown and stimulated for 4 h as described in the legend to Fig. 1. The amount of iNOS in cell lysates was quantitated as described in the legend to Fig. 1. Representative Western blots are shown. Data are expressed as meanGSEM of three independent experiments. ) P!0.01 vs cytokines alone.
or without ICI 182,780 did not affect iNOS immunoreactivity (Fig. 5). By contrast, ICI 182,780 prevented the rise in cytokine-induced iNOS protein levels elicited by 10 mM resveratrol in control as well as diabetic SMC (Fig. 5), consistent with an action mediated through ER pathways. Therefore, resveratrol controlled iNOS expression in vascular SMC, at least in part, via ER-dependent mechanisms. The pro-inflammatory potential of resveratrol was tested on another inflammatory enzyme expressed by vascular SMC, COX-2. Under the present
Control
experimental conditions, COX-2 turned out to be constitutively expressed in both control and diabetic SMC, as shown in Fig. 6. Stimulation with the cytokine mixture did not lead to significant changes in COX-2 protein levels in either group. Resveratrol was ineffective on COX-2 levels in control SMC but increased them by w25% at the highest concentration tested (100 mM) in diabetic SMC. For comparison, 17b-estradiol elicited no effect whatsoever on COX-2 protein levels (data not shown). Despite detectable levels of COX-2 protein, PGE2 release into the medium was close to
Diabetic iNOS
*
1.50
#
1.00
0.50
0
Cytomix Resveratrol ICI 182,780
iNOS protein (arbitrary units)
iNOS protein (arbitrary units)
1.50
* # 1.00
0.50
0 -
+ -
+ 10-7 -
+ 10-7
+ 10-5
+ 10-5 M
10-6
-
10-6 M
Cytomix Resveratrol ICI 182,780
-
+ -
+ 10-7 -
+ 10-7
+ 10-5
+ 10-5 M
10-6
-
10-6 M
Figure 5 Effects of resveratrol and the estrogen receptor antagonist ICI 182,780 on cytokine-induced iNOS protein synthesis in aortic SMC from control (panel A) and diabetic (panel B) rats. SMC were grown and stimulated as described in the legend to Fig. 1. Preincubation with 1 mM ICI 182,780 for 30 min was performed before adding resveratrol (0.1 and 10 mM) at the same time as cytokines. The amount of iNOS in cell lysates was quantitated as described in the legend to Fig. 1. Representative Western blots are shown. Data are expressed as meanGSEM of three independent experiments. )P!0.05 compared with cytomix, #P!0.05 compared with 10 mM resveratrol alone.
Lack of vascular anti-oxidant activity by resveratrol
Control
327
Diabetic COX-2 protein (arbitrary units)
COX-2 protein (arbitrary units)
COX-2 1.50
1.00
0.50
0
Cytomix Resveratrol
-
+ -
+
+
+
10-7
10-6
10-5
+ 10-4 M
1.50
*
1.00
0.50
0
Cytomix Resveratrol
-
+ -
+
+
+
10-7
10-6
10-5
+ 10-4 M
Figure 6 Effects of resveratrol on COX-2 protein synthesis in aortic SMC from control (panel A) and diabetic (panel B) rats. SMC were grown and stimulated as described in the legend to Fig. 1. The amount of COX-2 in cell lysates was determined by scanning densitometry, whereby the intensity of the cytomix band was set arbitrarily to 1. Representative Western blots are shown. Data are expressed as meanGSEM of three to five independent experiments. )P!0.05 compared with cytokines alone.
detection limit in the basal state and was enhanced by cytokine stimulation in both SMC groups (Table 1). Treatment with resveratrol reduced PGE2 levels in the medium of SMC from normoglycaemic rats only at 100 mM but had no effect in SMC from diabetic rats as compared with cytokines alone (Table 1). For comparison, 17b-estradiol treatment did not affect significantly PGE2 levels in both SMC groups (data not shown).
Table 1 Effect of increasing concentrations of resveratrol on prostaglandin E2 (PGE2) levels released into the incubation medium by SMC after inflammatory challenge with cytokines for 24 h PGE2 (pg/mg cell protein) Basal Cytokines CytokinesCresveratrol 0.1 mM CytokinesCresveratrol 1 mM CytokinesCresveratrol 10 mM CytokinesCresveratrol 100 mM
Control
Diabetes
197G71 3715G1301a 3622G1262
16G1 4197G1476a 5130G1834
3395G1197
5885G1112
2470G847
6274G1251
186G62b
5299G1149
Data are expressed as meanGSEM of three independent experiments each performed in triplicate. a P!0.01 vs. basal. b P!0.01 vs. cytokines (ANOVA and Bonferroni’s post-hoc test).
Discussion The naturally occurring polyphenol resveratrol has recently attracted considerable interest because of the appreciation of its antioxidant and antiinflammatory effects, which could possibly lead to cardioprotective and chemiopreventive actions [7,8]. This compound, abundant in grapes, may contribute to the reduced risk of coronary heart disease in humans who have consumed moderate amounts of alcohol from red wine regularly [20]. With this background, it was expected that resveratrol would reduce the functional expression of inflammatory enzymes such as iNOS in vascular SMC from non-diabetic and also from diabetic rats, which feature a vascular dysfunction of inflammatory nature [2,3]. By contrast, resveratrol enhanced cytokine-mediated iNOS activation in both SMC groups at concentrations that could be achieved in vivo, at least in part via an ERdependent mechanism. In SMC from normoglycaemic rats, however, resveratrol had no effect on iNOS functional expression at pharmacological concentrations (100 mM), possibly due to interactions with additional and rather aspecific molecular targets such as protein kinase C [21,22] that apparently were not activated in SMC from diabetic rats. Furthermore, the duration of inflammatory challenge affected iNOS regulation by resveratrol, suggesting that multiple molecular targets ultimately control its biological effects. Thus, two major questions arise. First, why did resveratrol fail to exert an anti-inflammatory effect in our experimental system? Second, what
328 kind of interaction with ER could account for the observed pro-inflammatory effects? Inhibition of iNOS function by resveratrol has been reported to occur particularly in macrophage cells [10,12,24e26]. In vascular SMC, however, the molecular pathways mediating the anti-proliferative properties of resveratrol [13,27e29], but not its potential anti-inflammatory activity, have been reported in detail. Most of the anti-inflammatory and growth inhibitory properties of resveratrol in different cell types have been ascribed to impaired activation of NF-kB [23]. It is conceivable that the lack of anti-inflammatory action of resveratrol in SMC from both non-diabetic and diabetic rats was accounted for by the use of a cytokine mix, which more closely resembles inflammatory settings in vivo than a single mediator. Even if we assume that resveratrol effectively down-regulated NF-kB, other transcription factors might have fostered iNOS induction. It is important to note, though, that our findings do not provide information concerning endothelial function, which can be stimulated by resveratrol via ER resulting in MAPK and eNOS activation [30]. The estrogenic component of resveratrol action was involved in the findings herein reported. ERa and ERb are expressed in vascular SMC of rats [14,15] and humans [31]. Resveratrol exhibits variable degrees of ER agonism in different test systems [17] and is known to act as a mixed agonist/antagonist for ERa and ERb according to cell type, response element sequence and coactivator/corepressor protein expression [16]. Our working hypothesis is that resveratrol, as much as the phytoestrogen genistein, enhanced iNOS activation in vascular SMC by virtue of preferential ERb agonist activity. Several lines of evidence support this notion. First, it is well established that genistein has remarkable affinity for ERb [32]; second, the synthetic selective ERb agonist 2, 3-bis(4-hydroxyphenyl)-propionitrile, unlike 17bestradiol, up-regulated iNOS function in aortic SMC from both control and diabetic rats (unpublished observations); third, the anti-inflammatory activity of 17b-estradiol in the brain has been shown to be mediated by ERa [33]. Post-translational modulation of iNOS activity by genistein may occur via different mechanisms including interaction with PPARs [34], while tyrosine kinase inhibition is unlikely to occur below mM concentrations. Under the present experimental conditions COX-2 protein was found to be constitutively expressed in vascular SMC. There have been reports of constitutive COX-2 expression in vascular cells [35], yet it cannot be ruled out that COX-2 expression as observed in the present study was
A. Cignarella et al. triggered by serum [36]. However, COX-2 protein in the basal state was virtually inactive, possibly due to impaired activity of the 85-kD phospholipase A2 in the absence of inflammatory stimuli [37]. Resveratrol at high concentration (100 mM) reduced COX-2 activity but not protein levels, in contrast to previous reports in other cell types [11,12,38]. Conversely, the apparent increase in PGE2 levels induced particularly by 10-mM resveratrol treatment in SMC from diabetic rats, to the best of our knowledge, was not reported previously. The different response to resveratrol in terms of COX-2 protein levels and activity suggests that COX-1 is a major source of inflammatory PGE2 in the rat [39]. In conclusion, using iNOS and COX-2 as surrogate endpoints, resveratrol showed no significant antiinflammatory properties in vascular SMC from normoglycaemic and diabetic rats at concentrations approaching putative peak plasma levels in vivo [40,41]. Conversely, a potential pro-inflammatory activity might be associated with this compound.
References [1] Blake GJ, Ridker PM. Novel clinical markers of vascular wall inflammation. Circ Res 2001;89:763e7. [2] De Vriese AS, Verbeuren TJ, Van de Voorde J, Lameire NH, Vanhoutte PM. Endothelial dysfunction in diabetes. Br J Pharmacol 2000;130:963e74. [3] Creager MA, Luscher TF, Cosentino F, Beckman JA. Diabetes and vascular disease: pathophysiology, clinical consequences, and medical therapy e part I. Circulation 2003;108:1527e32. [4] Bardell AL, MacLeod KM. Evidence for inducible nitricoxide synthase expression and activity in vascular smooth muscle of streptozotocin-diabetic rats. J Pharmacol Exp Ther 2001;296:252e9. [5] Gunnett CA, Heistad DD, Faraci FM. Gene-targeted mice reveal a critical role for inducible nitric oxide synthase in vascular dysfunction during diabetes. Stroke 2003;34: 2970e4. [6] Warner TD, Mitchell JA. Cyclooxygenases: new forms, new inhibitors, and lessons from the clinic. FASEB J 2004;18: 790e804. [7] Fre ´mont L. Biological effects of resveratrol. Life Sci 2000; 66:663e73. [8] Pervaiz S. Resveratrol: from grapevines to mammalian biology. FASEB J 2003;17:1975e85. [9] Wallerath T, Deckert G, Ternes T, et al. Resveratrol, a polyphenolic phytoalexin present in red wine, enhances expression and activity of endothelial nitric oxide synthase. Circulation 2002;106:1652e8. [10] Chan MM, Mattiacci JA, Hwang HS, Shah A, Fong D. Synergy between ethanol and grape polyphenols, quercetin, and resveratrol in the inhibition of the inducible nitric oxide synthase pathway. Biochem Pharmacol 2000;60:1539e48. [11] Martinez J, Moreno JJ. Effect of resveratrol, a natural polyphenolic compound, on reactive oxygen species and prostaglandin production. Biochem Pharmacol 2000;59: 865e70.
Lack of vascular anti-oxidant activity by resveratrol [12] Tsai SH, Lin-Shiau SY, Lin JK. Suppression of nitric oxide synthase and the down-regulation of the activation of NFkB in macrophages by resveratrol. Br J Pharmacol 1999; 126:673e80. [13] Liu Y, Liu G. Isorhapontigenin and resveratrol suppress oxLDL-induced proliferation and activation of ERK1/2 mitogen-activated protein kinases of bovine aortic smooth muscle cells. Biochem Pharmacol 2004;67:777e85. [14] Zancan V, Santagati S, Bolego C, Vegeto E, Maggi A, Puglisi L. 17b-estradiol decreases nitric oxide synthase II synthesis in vascular smooth muscle cells. Endocrinology 1999;140:2004e9. [15] Maggi A, Cignarella A, Brusadelli A, Bolego C, Pinna C, Puglisi L. Diabetes undermines estrogen control of inducible nitric oxide synthase function in rat aortic smooth muscle cells through overexpression of estrogen receptor-b. Circulation 2003;108:211e7. [16] Bowers JL, Tyulmenkov VV, Jernigan SC, Klinge CM. Resveratrol acts as a mixed agonist/antagonist for estrogen receptors a and b. Endocrinology 2000;141:3657e67. [17] Gehm BD, McAndrews JM, Chien PY, Jameson JL. Resveratrol, a polyphenolic compound found in grapes and wine, is an agonist for the estrogen receptor. Proc Natl Acad Sci USA 1997;94:14138e43. [18] Ross R. The smooth muscle cell II - growth of smooth muscle in culture and formation of elastic fibers. J Cell Biol 1971;103:2787e96. [19] Idel S, Ellinghaus P, Wolfrum C, et al. Branched chain fatty acids induce nitric oxide-dependent apoptosis in vascular smooth muscle cells. J Biol Chem 2002;277:49319e25. [20] Wu JM, Wang ZR, Hsieh TC, Bruder JL, Zou JG, Huang YZ. Mechanism of cardioprotection by resveratrol, a phenolic antioxidant present in red wine. Int J Mol Med 2001;8:3e17. [21] Ferriola PC, Cody V, Middleton Jr E. Protein kinase C interaction by plant flavonoids: kinetic mechanism and structure activity relationships. Biochem Pharmacol 1989; 38:1617e24. [22] Slater SJ, Seiz JL, Cook AC, Stagliano BA, Buzas CJ. Inhibition of protein kinase C by resveratrol. Biochim Biophys Acta 2003;1637:59e69. [23] Manna SK, Mukhopadhyay A, Aggarwal BB. Resveratrol suppresses TNF-induced activation of nuclear transcription factors NF-kB, activator protein-1, and apoptosis: potential role of reactive oxygen intermediates and lipid peroxidation. J Immunol 2000;164:6509e19. [24] Cho DI, Koo NY, Chung WJ, et al. Effects of resveratrolrelated hydroxystilbenes on the nitric oxide production in macrophage cells: structural requirements and mechanism of action. Life Sci 2002;71:2071e82. [25] Wadsworth TL, Koop DR. Effects of the wine polyphenolics quercetin and resveratrol on pro-inflammatory cytokine expression in RAW 2647 macrophages. Biochem Pharmacol 1999;57:941e9. [26] Bi XL, Yang JY, Dong YX, et al. Resveratrol inhibits nitric oxide and TNF-a production by lipopolysaccharide-activated microglia. Int Immunopharmacol 2005;5:185e93.
329 [27] Haider UG, Sorescu D, Griendling KK, Vollmar AM, Dirsch VM. Resveratrol suppresses angiotensin II-induced Akt/protein kinase B and p70 S6 kinase phosphorylation and subsequent hypertrophy in rat aortic smooth muscle cells. Mol Pharmacol 2002;62:772e7. [28] Haider UG, Sorescu D, Griendling KK, Vollmar AM, Dirsch VM. Resveratrol increases serine15-phosphorylated but transcriptionally impaired p53 and induces a reversible DNA replication block in serum-activated vascular smooth muscle cells. Mol Pharmacol 2003;63:925e32. [29] Zou J, Huang Y, Cao K, et al. Effect of resveratrol on intimal hyperplasia after endothelial denudation in an experimental rabbit model. Life Sci 2000;68:153e63. [30] Klinge CM, Blankenship KA, Risinger KE, et al. Resveratrol and estradiol rapidly activate MAPK signaling through estrogen receptors a and b in endothelial cells. J Biol Chem 2005;280:7460e8. [31] Hodges YK, Tung L, Yan XD, Graham JD, Horwitz KB, Horwitz LD. Estrogen receptors a and b: prevalence of estrogen receptor b mRNA in human vascular smooth muscle and transcriptional effects. Circulation 2000;101: 1792e8. [32] Kuiper GG, Lemmen JG, Carlsson B, et al. Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor b. Endocrinology 1998;139:4252e63. [33] Vegeto E, Belcredito S, Etteri S, et al. Estrogen receptora mediates the brain antiinflammatory activity of estradiol. Proc Natl Acad Sci USA 2003;100:9614e9. [34] Dang ZC, Audinot V, Papapoulos SE, Boutin JA, Lowik CW. Peroxisome proliferator-activated receptor g (PPARg) as a molecular target for the soy phytoestrogen genistein. J Biol Chem 2003;278:962e7. [35] Parfenova H, Parfenov VN, Shlopov BV, et al. Dynamics of nuclear localization sites for COX-2 in vascular endothelial cells. Am J Physiol 2001;281:C166e78. [36] Rimarachin JA, Jacobson JA, Szabo P, Maclouf J, Creminon C, Weksler BB. Regulation of cyclooxygenase-2 expression in aortic smooth muscle cells. Arterioscler Thromb 1994;14:1021e31. [37] Mitchell JA, Warner TD. Cyclo-oxygenase-2: pharmacology, physiology, biochemistry and relevance to NSAID therapy. Br J Pharmacol 1999;128:1121e32. [38] Subbaramaiah K, Chung WJ, Michaluart P, et al. Resveratrol inhibits cyclooxygenase-2 transcription and activity in phorbol ester-treated human mammary epithelial cells. J Biol Chem 1998;273:21875e82. [39] Giuliano F, Warner TD. Origins of prostaglandin E2: involvements of cyclooxygenase (COX)-1 and COX-2 in human and rat systems. J Pharmacol Exp Ther 2002;303: 1001e6. [40] Gescher AJ, Steward WP. Relationship between mechanisms, bioavailibility, and preclinical chemopreventive efficacy of resveratrol: a conundrum. Cancer Epidemiol Biomarkers Prev 2003;12:953e7. [41] Walle T, Hsieh F, Delegge MH, Oatis Jr JE, Walle UK. High absorption but very low bioavailability of oral resveratrol in humans. Drug Metab Dispos 2004;32:1377e82.