It is generally appreciated that atherosclerosis is initiated by damage to the vascular endothelium, resulting in endothelial release of mitogenic factors that promote monocyte recruitment to the vessel wall, smooth muscle cell migration from the media to the intima, and smooth muscle cell proliferation.1 However, elevated levels of the amino acid homocysteine can initiate atherosclerosis by inducing vascular smooth muscle cell (VSMC) proliferation,2 and reduction of VSMC proliferation using pharmacological agents can attenuate atherosclerosis.3,4 Thus, VSMC proliferation is an important event in the etiology of atherosclerosis.
The reported cardioprotective effects of red wine consumption was prompted by epidemiological studies documenting the “French Paradox,” a term coined to describe the reduced incidence of death due to coronary heart disease (CHD) in areas of southwest France.5 Inhabitants of this area exhibit increased serum cholesterol and blood pressure and eat more lard and butter than do Americans, yet suffer 40% fewer deaths due to CHD than other western societies.5 This paradoxical effect is attributed to their daily consumption of red wine. Although epidemiological studies suggest a decreased risk of CHD in populations regularly consuming alcohol, considerable data indicate that wine provides greater protection as compared to other alcoholic beverages.6,7
Resveratrol is a phytoalexin polyphenol found in foods such as grapes, mulberries, peanuts, and grapevine.7 Within the grape itself, resveratrol is most abundant in the skin (∼50-100 μg/g).7 One fluid ounce of red wine provides ∼160 μg resveratrol,7 but its concentration in red wine can vary from 0.1-15 mg/L.8 Immediately after consumption, resveratrol can be measured in the plasma, heart, liver, and kidney.9 Chronic consumption further increases levels of resveratrol in tissues such as the heart and liver.9 Importantly, even modest doses can produce biologic activity in vivo.10,11
Prior reports demonstrate resveratrol's efficacy in inhibiting the proliferation of VSMCs.12,13 In VMSC isolated from spontaneously hypertensive rats, resveratrol inhibited proliferation induced by exposure to advanced glycation end-products, and this effect was diminished by cotreatment with the estrogen receptor (ER) antagonist ICI 182,780.14 In another related epidemiological report, the cardiovascular benefit of red wine consumption was shown more pronounced in women as compared to men.15,16 Taken together, these studies implicate estrogen receptor modulation in the cardioprotection mediated by resveratrol or other red wine polyphenols. The objective of this study was thus to determine whether ER modulation is required for resveratrol-mediated decrease in VSMC proliferation. ER activation is known to promote the activity and/or expression of isoforms of nitric oxide synthase (NOS) and thus the production of nitric oxide (NO).17-20 Although NO is known to exert antimitogenic effects on VSMC growth,21,22 VSMCs express at least two isoforms of NOS, inducible NOS (iNOS) and neuronal NOS (nNOS). Thus, our hypothesis was the resveratrol mediates VSMC proliferation through an ER-dependent modulation of NOS activity and/or expression. Experiments in this study were aimed at determining the specific roles of the ER and its regulation of NO production in resveratrol-mediated inhibition of vascular smooth muscle cell proliferation.
MATERIALS AND METHODS
Chemicals
Resveratrol (trans-3,4,5-trihydroxystilbene) was purchased from Alexis Biochemicals (Lausen, Switzerland). ICI 182,780 was obtained from Tocris (Ellisville, MO). N5-(1-Iminomethyl)-L-ornithine, dihydrochloride (L-NIO) and S,S′-1,4-phenylene-bis (1,2-ethanediyl)bis-isothiourea, dihydrobromide (PBIT) were purchased from Cayman Chemical Co (Ann Arbor, MI). 7-Nitroindazole (7-NI) was obtained from Calbiochem (San Diego, CA). Stock solutions of resveratrol or ICI 182,780 were prepared in ethanol and were diluted 1:2000 in Dulbecco Modified Eagle's Minimal Essential Medium (DMEM) growth medium (low-glucose, phenol red-free DMEM containing 10% fetal bovine serum). In some experiments, DMEM growth medium contained 10% charcoal-treated fetal bovine serum. Stock solutions of NOS inhibitors were prepared in phosphate-buffered saline (PBS) prior to dilution in DMEM growth medium.
Vascular Smooth Muscle Cell Culture and Treatment With Resveratrol
VSMC were isolated from the aortas (ascending to descending) of 6- to 8-week old male and female Sprague-Dawley rats using a method described previously.23 All animal procedures were approved by an institutional animal care and use committee and were in compliance with the guidelines set forth by the Guide for the Care and Use of Laboratory Animals. The VSMC were grown in DMEM growth medium. Upon confluence, cell cycle was synchronized by replacing the growth medium with phenol red-free DMEM containing 0.1% fetal bovine serum (FBS) and incubating for 72 hours. Mitogenesis was then stimulated by incubation for 24 to 72 hours in DMEM containing 10% serum and either vehicle (0.05% ethanol) or resveratrol. For comparison, in some experiments, DMEM used for culturing and treating cells contained charcoal-treated FBS. To determine whether resveratrol's effects on VSMC were dependent upon estrogen receptor (ER) modulation, following growth arrest, VSMC were cotreated with 10 nM ICI 182,780 (half maximal inhibitory concentration [IC50] = 0.29 nM24).
Assessing the Effects of Resveratrol on Rates of Cellular Proliferation
To assess cell proliferation, DNA synthesis was measured using the 5-bromo-2′-deoxyuridine (BrDU) incorporation assay (BrDU Labeling and Detection Kit III, Boehringer-Mannheim, Indianapolis, IN).25 To assess VSMC growth, the number of cells in each sample was determined by removing the cells from the plates using trysinization and then counting them using a hemocytometer.
Determining the Effects of Resveratrol on VSMC Apoptosis
VSMC were grown to confluence in eight-chambered microscope slides and were synchronized by serum deprivation (described above). The cells were treated for 0 to 72 hours with DMEM growth medium containing vehicle (0.05% ethanol) or resveratrol, were washed with PBS, and were fixed for 15 minutes in 2% cold paraformaldehyde in PBS, pH 7.4, and then for 1 hour in 70% ice-cold ethanol. The cells were again washed with PBS, were stained using the fluorescent probe 4′,6-diamidine-2′phenylindole dihydrochloride (DAPI) at 1 μg/mL, and were incubated for 30 minutes in the dark at room temperature. The fixed cells were then washed twice with PBS, were mounted with coverslips using DAKO fluorescent mounting fluid, and were viewed using an Olympus Bx50 microscope. The morphology of DAPI-stained cell nuclei was examined for the identification of apoptosis.26 Numbers of apoptotic cells were expressed as a percent of total cells.
As an additional test for apoptosis, DNA fragmentation was also assessed. To accomplish this, the cells were lysed, treated with RNase A and then proteinase K, and the DNA was isolated using the Apoptotic DNA Ladder Kit from Roche (Indianapolis, IN). The DNA samples were electrophoresed using 1% agarose gels containing ethidium bromide. As a positive control, DNA from lysates of U937 cells treated with 4 μg/mL camptothecin for 3 hours was also isolated and electrophoresed.
Competitive Estrogen Receptor Binding Assays
A whole cell ligand binding assay was conducted, as described by Stoessel and Leclercq.27 Confluent cultures of VSMC grown in six-well plates were treated with 5 nM [3H]-17β-estradiol and increasing concentrations of resveratrol for 2 hours at 37°C. The [3H]-estradiol-containing medium was aspirated, the cells were washed with PBS 2 to 3 times, and the VSMC were incubated with 300 μL/well ethanol for 30 minutes. Aliquots of the ethanol extracts were added to scintillation fluid and counted using a Wallac 1409 scintillation counter. An additional aliquot was used for measuring cell protein by the bicinchoninic (BCA) assay (Pierce, Rockford, IL). To determine nonspecific binding, some cell samples were incubated with a 200-fold excess of nonradiolabeled estradiol. Finally, samples were normalized to cell protein prior to calculating total specific binding.
Measuring Nitric Oxide Production
After 24 to 72 hours of incubation with resveratrol, VSMC plus spent medium were removed from the plate by scraping, were homogenized together, and were assayed for nitrite plus nitrate using the Oxford Nitric Oxide assay kit (catalog no. NB 88). Finally, absorbance was read at 540 nM, and the concentration of nitrite was calculated by comparison to sodium nitrite used as a standard. To confirm the specificity of the nitrite measurement for NO production, in some samples, 2 μM L-NIO, a general NOS inhibitor,28 was also included.
Determining the Effects of Resveratrol on iNOS Activity
VSMC were treated for 24 to 48 hours with resveratrol. To test the role of the ER in resveratrol-mediated modulation of iNOS activity, some cell samples were cotreated with 5 nM ICI 182,780. Additionally, to test whether modulation of tetrahydrobiopterin (BH4) levels altered iNOS activity, in some wells, BH4 synthesis was inhibited by cotreatment with 10 nM methotrexate. The cells were then washed with PBS, were harvested in PBS containing 1 mM ethylenediamine tetraacetic acid (EDTA), were transferred to Microfuge tubes, and were centrifuged. Finally, the pellet was isolated, and the calcium-independent conversion of [3H]-L-arginine to [3H]-L-citrulline was measured using the Cayman NOS Activity Assay Kit (Ann Arbor, MI).29 Note that in calculating conversion of L-arginine to L-citrulline, all measured activities were corrected for blank levels of activity observed in the presence of excess L-NIO.
To further understand the role of NO in modulating resveratrol-mediated effects on VSMC proliferation, VSMC were cotreated with resveratrol plus either 2 μM L-NIO (a general NOS inhibitor; IC50 = 3.9 μM),30 20 nM PBIT (an iNOS-selective inhibitor; IC50 = 47 nM),31 or 750 nM 7-NI (an nNOS-selective inhibitor; IC50 = 900 nM),32 and BrDU incorporation was assessed at 48 hours after treatment. Note that a low concentration of each of the NOS inhibitors was required in these experiments, such that VSMC proliferation was only minimally altered by treatment with the NOS inhibitor alone.
Determining Cellular Levels of Tetrahydrobiopterin
To evaluate the role of the ER in resveratrol-induced effects on BH4, VSMC were treated for 24 to 48 hours with resveratrol ± 5 nM ICI 182,780. BH4 was determined using a modification of the method described by Howells et al.33 First, the cells were lysed by scraping in 50 mM Tris-HCl, pH 8, containing 300 mM NaCl and 0.5% Triton X-100, and were diluted 1:1 with 3.8% perchloric acid containing 1 mg/mL dithioerythritol (DTE) and 1 mg/mL diethylenetriaminepentaacetic acid (DTPA). The lysates were centrifuged and the supernatant was stored at −80°C. BH4 levels were determined using a Waters 2695 high-performance liquid chromatography (HPLC; Milford, MA), interfaced to an ESA Coularray four-channel electrochemical detector (Chelmsford, MA). The separation was accomplished using a 250 mm × 4.6 mm I.D. Ultrasphere reversed-phase C18 column (Beckman Coulter, Fullerton, CA) and isocratic elution of 10% methanol/90% 83 mM sodium acetate, containing 5.5 mM citric acid, 54 μM EDTA, and 160 μM DTE, at a flow rate of 0.4 mL/min. The four channels of the electrochemical detector were set at 150, 350, 700, and 850 mV, and BH4 was quantitated on the latter three channels, combined. Solutions of ultrapure BH4 (Alexis Biochemicals, San Diego, CA) were freshly prepared in 3.8% perchloric acid containing 1 mg/mL DTPA and 1 mg/mL DTE and were used to construct a standard curve. BH4 eluted at ∼9 min, and the BH4 peak was confirmed by spiking the experimental samples with authentic standard.
Assessing Levels of iNOS and Guanosine Triphosphate (GTP) Cyclohydrolase-I Protein
VSMC were treated with resveratrol with and without ICI 182,780, were washed with PBS, and were lysed using a 30-minute incubation at 4°C in 50 mM Tris-HCl buffer containing 300 mM NaCl, 0.5% Triton X-100, 1 mM phenylmethylsulphonylfluoride (PMSF), and a protease inhibitor cocktail (Roche, Indianapolis, IN; 1:20 dilution). The lysate protein concentrations were determined using the BCA protein assay (Pierce, Rockford, IL). Approximately 10 to 15 μg were loaded in each lane and electrophoresed on Novex 4% to 20% Tris-glycine gradient gels (Invitrogen, Carlsbad, CA). Note, however, that equivalent amounts of protein were loaded in each well of a single gel. As a positive control for iNOS expression, 1 μg mouse macrophage cell lysate was loaded. The protein was transferred to polyvinylidene difluoride (PVDF) membranes, and the blots were blocked by incubation at 4°C overnight with PBS containing 0.1% Tween 20 and 5% nonfat dry milk. The blots were then incubated for 2 hours at room temperature with 1:1000 rabbit ∝ mouse polyclonal iNOS antibody (Cayman Chemicals, Ann Arbor, MI), 1:20,000 rabbit ∝ mouse polyclonal GTP cyclohydrolase I (GTPCH) antibody (generously provided by Dr. Ron Mandel, University of Florida), or 1:10,000 GAPDH mouse monoclonal antibody (Calbiochem), diluted in PBS-0.1% Tween 20 containing 5% nonfat dry milk. In some experiments, blots comparing lysates from male versus female VSMC were incubated with 1:200 rabbit ∝ mouse ER-α polyclonal antibody (Santa Cruz Biotechnology). After washing 3 times in PBS-0.1% Tween, the blots were incubated for 1 hour with a 1:20,000 dilution of horseradish peroxidase-conjugated donkey ∝ rabbit immunoglobulin (Ig; Amersham Biosciences Corp., Piscataway, NJ), for measurement of GTP cyclohydrolase-I (GTPCH) and iNOS, or 1:20,000 sheep ∝ mouse IgG (Amersham), for determining GAPDH. The blots were again washed in PBS-0.1% Tween 20 containing 5% nonfat dry milk and then in PBS-0.1% Tween 20. The chemiluminescent signal was detected on X-ray film using the ECL Plus Detection System (Amersham). The films were imaged using a BioRad VersaDoc 3000 (Hercules, CA), and densitometric data was collected. Finally, levels of iNOS and GTPCH were normalized using densitometric data for GAPDH.
Statistics
Statistical analyses were performed using SPSS, version 10.0 (SPSS, Inc., Chicago, IL). Effects of treatment and time were assessed using analysis of the variance (ANOVA). When significant differences between data sets were observed, post-hoc t tests (eg, Tukey or Bonferroni) were performed to determine differences between data points. In all cases, P < 0.05 was accepted as statistical significance.
RESULTS
Effects of Resveratrol on VSMC Proliferation
In our initial studies, we measured the effects of resveratrol on proliferation of VSMC isolated from male as compared to female rats. In these studies, rates of DNA synthesis were used as an indication of VSMC proliferation. Though resveratrol inhibited VSMC proliferation in cells isolated from male rats, resveratrol-induced effects were comparatively less dramatic than for VSMC isolated from females. As shown in Figure 1A, a moderate dose of resveratrol (25 μM) reduced VSMC proliferation in female VSMC by 70% at 24 hours, and this effect was sustained through 72 hours of treatment. However, in VSMC isolated from male rats, resveratrol reduced DNA synthesis by 50% at 24 hours, but this effect was diminished by 72 hours. Because resveratrol is a known phytoestrogen, we measured the protein levels of ER-α in male versus female VSMC using Western blot analysis, and found that female VSMC express 2.0 ± 0.2 times greater ER-α protein compared to male VSMC (data not shown). Because these experiments demonstrated a greater efficacy of resveratrol in female as compared to male VSMC, in all further experiments, only female cells were utilized.
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FIGURE 1: Effects of resveratrol on DNA synthesis in vascular smooth muscle cells (VSMC). DNA synthesis, determined by measuring the incorporation of BrDU, was assessed in VSMC isolated from male versus female rats treated with 25 μM resveratrol (A) and VSMC isolated from female rats and treated for 24 hours with 0.5 to 100 μM resveratrol (B). Data are means ± SE for four experiments. For both sexes, two-way ANOVA revealed a significant effect of treatment but no effect of time. *Significant differences as compared to vehicle for the same time point. #Significant differences between the genders.
We next determined the dose-response for resveratrol-mediated effects and found that in VSMC isolated from females, treatment with resveratrol induced a dose-dependent, 20% to 80% decrease in DNA synthesis at 24 hours after treatment (Fig. 1B). In this experiment, resveratrol-mediated decrease in proliferation exhibited an IC50 of 3.73 ± 0.57 μM, with doses as low as 0.5 μM significantly decreasing proliferation (Fig. 1B).
Because our hypothesis was that resveratrol mediates ER activation, we made every effort to utilize conditions minimizing interference from other estrogenic compounds. Utilization of phenol red-free medium aided in the reduction of variability in our data. However, utilization of charcoal-treated serum, to remove any interfering serum hormones, produced data nearly identical reductions in BrDU incorporation compared to that obtained using ordinary heat-inactivated serum (data not shown). Thus, due to cost limitations, heat-inactivated serum was used for all further experimentation.
To ascertain whether effects on DNA synthesis were translated into decreases in cell growth, we treated VSMC isolated from female rats with micromolar concentrations of resveratrol and determined cell number at 0 to 72 hours. In agreement with our observation of resveratrol-mediated inhibition of DNA synthesis, resveratrol treatment induced a dose-dependent decrease in cell number by 72 hours after treatment (Fig. 2). This decrease ranged from 20% at the lowest dose (0.5 μM) to 80% at the highest dose (100 μM; Fig. 2).
FIGURE 2: Effects of resveratrol on VSMC growth. VSMC were treated with vehicle or resveratrol for 0 to 72 hours, and cell growth was determined by quantifying cell number. Data represent means ± SE for three experiments. Two-way ANOVA revealed significant effects of time and treatment. *Significant differences as compared to vehicle for the same time point.
Effects of Resveratrol on VSMC Apoptosis
To determine whether decreases in VSMC growth were due to an induction of apoptosis, we treated VSMC with micromolar amounts of resveratrol and quantitated apoptotic cells by morphological assessment of cell nuclei following DAPI fluorescence staining.26 Although there was a trend toward increased numbers of apoptotic cells at 72 hours and at higher micromolar concentrations of resveratrol (ie, 40 μM), no significant effect of treatment was noted (Fig. 3A). In addition, no visible signs of stress, including cells floating in the medium or cells with unusual morphology, were observed up to 100 μM resveratrol.
FIGURE 3: Effects of resveratrol on VSMC apoptosis, determined using morphological assessment of nuclei fluorescently stained using DAPI (A) and the DNA fragmentation assay (B). For DAPI fluorescence analysis (A), data are means ± SE for three experiments; two-way ANOVA revealed a significant effect of time but not treatment. For the DNA fragmentation assay (B), VSMC apoptosis was assessed at 24, 48, and 72 hours (left, middle, and right panels, respectively) after treatment. In each panel, lane 1 is the Lambda Hind III DNA marker, lane 2 contains DNA from VSMC treated with vehicle, and lanes 3, 4, and 5 contain DNA from VSMC treated with 20, 40, and 100 μM resveratrol, respectively. Lane 6 in the right panel contains the positive control, (DNA isolated from U937 cells treated for 3 hours with 4 μg/mL camptothecin).
As an additional test for apoptosis, we measured DNA fragmentation following resveratrol treatment. Once more, no significant amount of DNA fragmentation was observed for cells treated with either vehicle or resveratrol (Fig. 3B). However, DNA fragmentation was clearly visible for the positive control (U937 cells treated with 4 μg/mL camptothecin for 3 hours). Thus, based on results from these two assays, resveratrol treatment did not significantly increase the level of apoptosis in VSMC.
Effects of an Estrogen Receptor Antagonist on Resveratrol-Mediated Decrease in Proliferation
The role of the ER in the antimitogenic effects of resveratrol was determined pharmacologically by treating VSMC + resveratrol, with and without the ER antagonist ICI 182,780, and then measuring DNA synthesis. Resveratrol treatment again induced a dose-dependent decrease in DNA synthesis 24 to 48 hours after treatment (Fig. 4A). However, because ANOVA detected no significant effect of time, to simplify the data presentation, only the 48-hour time point is shown. In VSMC treated with resveratrol plus ICI 182,780, only the highest doses of resveratrol (50 and 100 μM) decreased DNA synthesis (Fig. 4A), and VSMC treated with ICI plus 0.5-50 μM resveratrol exhibited significantly higher rates of DNA synthesis as compared to resveratrol treatment alone. Importantly, treatment with ICI alone produced no significant effect on cell proliferation. ANOVA revealed a significant difference between cells treated with resveratrol plus ICI and cells treated with only resveratrol. In support of these data, whole-cell competitive binding assays conducted in VSMC showed that relatively low doses of resveratrol effectively competed with [3H]-estradiol for ER binding, exhibiting an IC50 of 8.92 ± 0.14 μM (Fig. 4B).
FIGURE 4: Effect of estrogen receptor (ER) modulation on resveratrol-mediated inhibition of VSMC proliferation. A) VSMC from female rats were treated for 0 to 72 hours with vehicle, resveratrol, or resveratrol plus 5 nM ICI 182,780, an ER antagonist. DNA synthesis was determined using the BrDU incorporation assay. Because two-way ANOVA revealed no significant effect of time, only data from the 48-hour time point are shown. Analysis of the variance indicated a significant effect of dose of resveratrol and a significant effect of treatment with ICI. Data are means ± SE for four experiments. *Significant differences compared to vehicle. #Significant differences between treatment with resveratrol + ICI as compared to treatment with resveratrol alone. B) Competitive ER binding determined in VSMC treated for 2 hours with 5 nM [3H]-17β-estradiol plus resveratrol.
Effects of Resveratrol on Cellular Nitric Oxide
To address whether resveratrol modulates VSMC nitric oxide, we measured total nitrite (ie, nitrite plus nitrate) in VSMC treated with 0 to 100 μM resveratrol. Note that in these studies we measured nitrite in cell homogenates and spent medium, combined, to maximize the sensitivity of the assay. Treatment of VSMC with resveratrol induced dose-dependent 50% to 200% increases in nitrite at 24 hours, 12% to 100% increases at 48 hours (Fig. 5), and 33% to 118% increases at 72 hours (data not shown). Again, because two-way ANOVA revealed no significant effect of time for these analyses, only the 48-hour time point is shown (Fig. 5).
FIGURE 5: Resveratrol-induced modulation of VSMC nitric oxide and its inhibition by the NOS inhibitor L-NIO (A) and the ER antagonist ICI 182,780 (B). NO production by VSMC was assessed by measuring nitrite plus nitrate using a modified Griess assay. Analysis of the variance revealed a significant effect of dose of resveratrol and of treatment with L-NIO or ICI. No effect of time was detected; therefore, only the 48-hour time point is shown. Data are means ± SE for three experiments. *Significant increases as compared to vehicle. #Significance compared to resveratrol alone.
To confirm the specificity of the nitrite assay and to ensure that the measured increase in nitrite was indicative of an increase in NO production, we treated VSMC with resveratrol plus the NOS inhibitor L-NIO. In cells treated with only L-NIO, nitrite levels were comparable to that observed for vehicle alone (Fig. 5A). An important note is that the concentration of L-NIO used in these experiments, 2 μM, is relatively low in comparison to its IC50 (3.9 μM).24,30 Thus, dramatic inhibition of baseline NO levels was not expected. However, the concentration was maintained at this low level such that L-NIO treatment alone did not alter VSMC proliferation, thus complicating later experiments exploring the role of NO in resveratrol-mediated effects on VSMC proliferation (discussed below). In cells treated with resveratrol plus L-NIO, no significant increase in nitrite was observed (Fig. 5A). These data thus confirm that that the resveratrol-mediated increases in cellular nitrite are dependent upon resveratrol-induced effects on NO production.
The role of the ER in resveratrol-mediated increase in NO was then determined by treating VSMC with resveratrol ± ICI 182,780 and measuring cellular nitrite. Though treatment with 0.5 to 100 μM resveratrol induced a 20% to 60% increase in nitrite at 48 hours, no significant increase in nitrite was observed in cells treated with resveratrol plus ICI (Fig. 5B). Although the baseline levels of NO were also decreased by ∼20%, this effect cannot account for the inability of resveratrol to increase NO in the presence of ICI.
Source of Nitric Oxide in Resveratrol-Mediated Effects
To mechanistically link ER-mediated increases in NO with resveratrol-induced decrease in VSMC proliferation, we measured DNA synthesis in cells treated with resveratrol plus or minus L-NIO (a general NOS inhibitor), PBIT (an iNOS-selective inhibitor), or 7-NI (an nNOS-selective inhibitor). L-NIO and PBIT cotreatments partially blunted the effects of low doses of resveratrol (0.5 to 25 μM); however, neither could reverse the effects of resveratrol at higher doses (Fig. 6). Note that relatively low doses of L-NIO and PBIT were chosen such that treatment with NOS inhibitor alone had only marginal effects on VSMC proliferation. In contrast, 7-NI cotreatment did not reverse resveratrol-mediated effects. As an additional note, treatment with the indicated doses of L-NIO, PBIT, and 7-NI exhibited trends toward increased proliferation, as one might expect. However, these effects were not significantly different from treatment with vehicle alone. Overall, these data suggest that even relatively low doses of L-NIO and PBIT can blunt resveratrol-mediated decrease in VSMC proliferation.
FIGURE 6: Effect of selective NOS inhibition on resveratrol-mediated modulation of VSMC proliferation. VSMC were treated for 0 to 72 hours with vehicle (0.05% ethanol), resveratrol, or resveratrol plus 2 μM L-NIO, a nonselective NOS inhibitor (A), 20 nM PBIT, an iNOS-selective inhibitor (B), or 750 nM 7-NI, an nNOS-selective inhibitor (C). DNA synthesis was assessed using the BrDU incorporation assay. Data are means ± SE for three to five experiments. Analysis of the variance revealed a significant effect of dose of resveratrol and of treatment with L-NIO and PBIT. *Denotes significant increases in BrDU incorporation as compared to vehicle. #Indicates significant differences compared to resveratrol alone.
Because an iNOS-selective inhibitor effectively blunted resveratrol-mediated effects, we next measured the activity of iNOS by determining the calcium-independent conversion of L-arginine to L-citrulline. Resveratrol dose-dependently increased iNOS activity three- to fivefold at both 24 and 48 hours after treatment, although there was no additional increase in iNOS activity between 24 and 48 hours (Fig. 7A). Calcium-dependent NOS activity (likely derived from nNOS) was very small by comparison to calcium-independent activity and was unaffected by cotreatment with resveratrol (data not shown). Resveratrol-mediated increase in iNOS activity was inhibited by cotreatment with ICI (Fig. 7B). Treatment with methotrexate, an inhibitor of the biosynthesis of the iNOS cofactor BH4, conferred a near complete (ie, ∼90%) inhibition of iNOS activity (Fig. 7B). On the other hand, Western blot analysis revealed no significant effect of 0.5 to 100 μM resveratrol on iNOS protein levels at 8 to 24 hours after treatment (Fig. 8). For brevity, only the 8-hour timepoint is shown.
FIGURE 7: Resveratrol-induced modulation of iNOS activity. VSMC were treated with resveratrol for 24 and 48 hours (A) and resveratrol ± 5 nM ICI 182,780 or 10 nM methotrexate for 24 hours (B). The activity of iNOS was determined as the calcium-independent conversion of [3H]-L-arginine to [3H]-L-citrulline. Data are expressed as means ± SE for three experiments. Analysis of the variance revealed a significant effect of treatment with resveratrol, but no effect of time. *Significant increases in activity as compared to vehicle. #Significance compared to treatment with resveratrol alone.
FIGURE 8: Effects of resveratrol on protein levels of iNOS in VSMC. VSMC isolated from female rats were treated for 8 to 24 hours with resveratrol, and protein levels of iNOS were determined by Western blot analysis (top). The densitometric data were collected from digitized images, were normalized to that obtained for GAPDH, and were expressed as a percent of vehicle (bottom). ANOVA revealed no significant effect of resveratrol or time on iNOS protein levels. Thus, for brevity, only the 8-hour timepoint is shown.
Effects of Resveratrol on Levels of BH4 and Protein Levels of GTP Cyclohydrolase-I
Absolute levels of BH4 were increased as much as 30% by 24 to 48 hours after treatment with resveratrol, and this effect was abolished by cotreatment with ICI 182,780 (Fig. 9A). However, ICI alone reduced levels of BH4 by ∼35%. We thus expressed the data as a percent of the respective vehicle in each case, such as a percent of 0.05% ethanol or as a percent of 0.05% ethanol plus ICI. Expressed in this way, ICI significantly reduced resveratrol-mediated increases in BH4 levels (Fig. 9B).
FIGURE 9: Resveratrol-induced modulation of tetrahydrobioterin (BH4) levels. VSMC were treated with resveratrol or resveratrol plus 5 nM ICI 182,780 for 24 hours and BH4 levels were measured by HPLC with electrochemical detection. Levels of BH4 are expressed as either fmol/mg protein (A) or as a percent of the vehicles (B). Data are means ± SE for four experiments. *Significance compared to vehicle alone. ΨSignificance compared to resveratrol alone. #Significance compared to ICI alone.
Finally, protein levels of GTPCH, the rate limiting enzyme in BH4 biosynthesis, were measured by Western blot analysis following treatment with resveratrol or resveratrol plus ICI. As shown in Figure 10, resveratrol treatment increased GTPCH 10% to 40% by 8 hours, and this effect was abolished by cotreatment with ICI 182,780.
FIGURE 10: Effects of resveratrol on protein levels of GTP cyclohydrolase I. VSMC were treated with resveratrol (A) or resveratrol plus 5 nM ICI 182,780 (B) for 8 to 24 hours. GTPCH protein levels were determined by Western blot analysis (top). The densitometric data were collected from digitized images, were normalized to that obtained for GAPDH, and were expressed as a percent of vehicle (bottom). ANOVA revealed significant effects of resveratrol and significant differences between resveratrol and resveratrol + ICI. For brevity, only the 8-hour time point is shown. *Significance compared to vehicle. #Significance compared to resveratrol alone.
DISCUSSION
Resveratrol Induces Cell Cycle Arrest but not Apoptosis
Resveratrol dose-dependently suppressed proliferation of VSMC, a biologically important process in the progression of atherosclerosis. Supporting earlier reports of resveratrol-mediated inhibition of VSMC proliferation, data presented here demonstrate that resveratrol inhibits VSMC DNA synthesis (Fig. 1) and cell growth (Fig. 2), with the effects of resveratrol apparent at doses as low as 0.5 μM (Fig. 4A). In addition, the IC50 for resveratrol-induced decrease in proliferation was calculated to be ∼4 μM (Fig. 1B). Although one report suggests that 50 to 100 μM resveratrol induces cell cycle arrest,13 another reports that resveratrol decreases cell proliferation at low micromolar concentrations (ie, <25 μM) and induces apoptosis at higher doses (doses >25 μM).34 To address this discrepancy, we evaluated both cell proliferation, using measurements of cell number and rates of DNA synthesis, and apoptosis, using DAPI fluorescence staining and assessments of DNA fragmentation. Resveratrol induced a significant decrease in DNA synthesis (Fig. 1) and inhibited VSMC growth (Fig. 2). In contrast, neither apoptosis assay employed here demonstrated a significant increase in apoptosis up to 72 hours after resveratrol treatment (Fig. 3). Thus, these studies support the assertion that resveratrol-induced decrease in VSMC growth is dependent upon effects on cell cycle and not on an induction of apoptosis.
Of interest was the more pronounced effect of resveratrol on VSMC isolated from female as compared to male rats (Fig. 1A). We hypothesized that sex differences in resveratrol-mediated effects were related to ER binding. Tissue expression of ER is reportedly higher in tissue isolated from females as compared to males,35 although males certainly express some level of ER protein. Frank evidence of this is the fact that men, like women, are susceptible to ER-dependent breast cancer,36 albeit with a lower risk factor. Supporting these prior reports, we measured a 2.0 ± 0.2 times higher level of ER-α protein in VSMC isolated from females as compared to males (data not shown). To examine the ER dependence of resveratrol-mediated inhibition of VSMC proliferation, we measured VSMC DNA synthesis in cells isolated from female rats treated with resveratrol ± the ER antagonist ICI 182,780. Though resveratrol treatment dramatically reduced VSMC proliferation, this effect was attenuated by cotreatment with ER antagonist ICI 182,780 (Fig. 4A), indicating a role for the ER in resveratrol-mediated effects on VSMC. Because ICI was incapable of reversing the effects of resveratrol at the higher doses (ie, 40 to 100 μM) and because the concentration of ICI utilized was well above its reported IC50, it is possible that, at these doses, resveratrol acts through alternate pathways. However, these doses of resveratrol are also much larger than what might be achieved after oral supplementation.9,10 Thus, our data suggest that at pharmacologically relevant concentrations, resveratrol acts through an ER-dependent mechanism.
Although resveratrol is known to bind and stimulate the ER in cancer cells,37 our competitive binding studies in VSMC confirmed these findings (Fig. 4B). It is furthermore important to note that the IC50 for binding in VSMC (∼9 μM) was comparable to the IC50 for resveratrol-mediated reduction in VSMC proliferation (∼4 μM). In support of these in vitro findings, epidemiological studies now demonstrate that the cardiovascular benefit of red wine consumption is more pronounced in women as compared to men.15,16 Presumably, both sexes may benefit from red wine consumption and a resveratrol-induced stimulation of ER, but perhaps with a larger response in females as compared to males.
Estrogen Receptor Stimulation, Nitric Oxide, and VSMC Proliferation
NO modulates vascular tone and VSMC growth by elevating cGMP levels, in turn activating cGMP-dependent protein kinases.38 Chronic inhibition of NO production leads to medial hyperplasia of carotid arteries and hypertension,39 and treatment of VSMC with NO donors decreases VSMC proliferation39 and replication in the S-phase.22
Estradiol has been shown to modulate the production of NO, and this relationship has been deemed the primary protective effect of estradiol against vascular disease.40 Generally speaking, it is well-appreciated that treatment of endothelial cells with estradiol increases the expression41 and, in some cases, the activity42 of eNOS. Estradiol has likewise been shown to modulate iNOS expression. In prior reports, estradiol treatment reduced iNOS expression in aortic endothelial cells stimulated with interleukin-1β43 and inhibited interferon-γ and lipopolysaccharide-induced iNOS in murine macrophages.17 However, in cardiomyocytes20 as well as coronary artery,19 estradiol treatment increased the protein expression of both eNOS and iNOS, and in smooth muscle cells, estradiol inhibited cytokine-stimulated expression of iNOS.18 Thus, although the specific effects of estradiol in reducing or stimulating iNOS protein expression appear dependent upon the type of cell and/or cell stimulus, estradiol clearly modulates iNOS-derived NO.
Resveratrol Modulates VSMC Nitric Oxide Through Estrogen Receptor Stimulation
Effects of resveratrol on NO production and NOS expression in endothelial cells and macrophages are well documented. In studies utilizing pulmonary artery endothelial cells, resveratrol decreased cell proliferation and increased eNOS expression.44 Like estradiol, resveratrol was shown to inhibit LPS-stimulated induction of iNOS in macrophages.45 In addition, Zou et al46 demonstrated that resveratrol-mediated improvement in endothelial function in hypercholesterolemic rabbits was correlated with increased plasma NO levels. Although the effects of resveratrol on endothelial cell NOS activity/expression has been addressed at length, no prior reports have addressed whether resveratrol acts directly on smooth muscle cells to modulate NO production. However, resveratrol-mediated increase in VSMC nitric oxide likely involves modulation of NOS isoforms other than eNOS. VSMC of various tissue sources can contain any of the three isoforms of NOS, including neuronal, inducible, and endothelial.38,47 However, in the more elastic arteries such as the aorta, nNOS and iNOS isoforms predominate.38
In these studies, resveratrol clearly induced ER-dependent increase in NO, increasing cellular nitrite 50% to 200% through 48 hours (Fig. 5) in a manner inhibitable by the ER antagonist ICI 182,780 (Fig. 5B). Further, resveratrol-mediated increase in nitrite was clearly inhibited by coincubation with the NOS inhibitor L-NIO (Fig. 5A), thus confirming that the observed increases in nitrite were specific to NO production, and not alternate cellular sources of nitrite/nitrate. It is important to point out that using the assay employed in these studies, the Griess assay, time courses of 24 hours or greater were required for measuring increases in nitrite. However, we suspect that this was due to the need to accumulate sufficient levels of nitrite in the medium, such that it was measurable by this relatively insensitive assay, and presumably increases in nitrite might actually have occurred as early as a few hours after resveratrol treatment. In support of this, NOS activity was indeed at a maximal level by 24 hours (Fig. 7A), with additional incubation achieving no greater levels of activity.
To evaluate whether resveratrol's ER-dependent stimulation of NO was translated into effects on VSMC proliferation, we measured DNA synthesis in VSMC treated with resveratrol ± L-NIO. While resveratrol again decreased VSMC proliferation in a dose-dependent manner, this effect was blunted by L-NIO (Fig. 6A). Experiments utilizing selective inhibitors of iNOS and nNOS (Fig. 6B, C) suggested that the observed increase in NO is likely derived from iNOS and not nNOS. Thus, the totality of the data generated in these studies suggests that at least part of the antimitogenic effects of resveratrol is mediated by its stimulation of ER and its concomitant increase in NO production.
Resveratrol Modulates iNOS Activity Through Regulation of Tetrahydrobiopterin Levels
To determine whether the observed increases in NO were due to an increase in iNOS activity or its protein expression, we assayed iNOS activity directly by determining the Ca-independent conversion of L-arginine to L-citrulline, and iNOS protein by Western blot analysis. In these studies, we present the novel finding that in VSMC, resveratrol increases iNOS activity (Fig. 7A) in an ER-dependent manner (Fig. 7B) without concomitantly inducing iNOS expression (Figure 8). Although conventional wisdom would suggest that iNOS is modulated at only a transcriptional level, both its translation and activity are now known to be regulated through modulation of two of its cofactors, arginine48 and BH4,49 as well as through modulation of its interactions with other biomolecules. For example, iNOS activity can be diminished through pathways involving disruption of the iNOS-nitrosyl50 or iNOS-α-actinin-4 complexes.51
BH4 is an important cofactor for iNOS activity, likely providing five electrons for the oxidation of arginine to produce NO plus citrulline.52 Induction of pathways involved in BH4 biosynthesis (for example, the inducible expression of GTPCH, the rate-limiting enzyme in de novo BH4 biosynthesis) is imperative for supporting iNOS activity following immunoactivation.53
To understand how BH4 modulates iNOS activity, several molecular aspects must be considered. First, unlike eNOS or nNOS, iNOS activation is not dependent upon calcium/calmodulin because it constitutively possesses bound calmodulin.54 However, on the amino terminus of the iNOS protein is an oxygenase domain to which heme, L-arginine, and BH4 bind.55 The carboxy terminus possesses a reductase domain that binds NADPH, FAD, and FMN,56 and between the two domains lies a calmodulin binding sequence.57 The two domains can be expressed separately and can fold and function independent of one another.58 For the oxygenase domain to assume a stable and active form apparently requires the binding of both arginine and BH4.56 The oxygenase monomer can possess at least loosely-bound heme, but no BH4.59 Binding to BH4 and arginine causes a conformational change in the protein that culminates in closing of the heme pocket to stabilize heme binding.60 BH4 thus stabilizes the dimeric form of the protein, promoting its enzymatic activity.60
To further support our findings of resveratrol-mediated effects on ER-dependent stimulation of iNOS activity, prior reports suggest that estradiol treatment can increase both the concentration and biosynthesis of BH4,61 and BH4 levels were shown diminished in ovariectomized animals.62 Because arginine levels in the VSMC culture system employed here were already quite high (ie, in the millimolar range), we hypothesized that resveratrol regulated iNOS activity by modulating levels of BH4. Cellular levels of BH4 were indeed dose-dependently increased by resveratrol, and this effect was attenuated in the presence of the ER antagonist ICI 182,780 (Fig. 9).
To demonstrate the importance of BH4 in VSMC iNOS activity, we measured iNOS activity in the presence of a low dose of methotrexate (MTX), a selective inhibitor of dihydrofolate reductase, an enzyme catalyzing the terminal step in the synthesis of BH4 from sepiapterin.63 Surprisingly, even low nanomolar concentrations of MTX reduced iNOS activity by nearly 90% (Fig. 7B).
Finally, to elucidate how resveratrol and its ER stimulation modulate BH4 levels, we measured GTPCH protein levels by Western blot analysis. GTPCH is an inducible enzyme that catalyzes the rate-limiting step in BH4 biosynthesis.53 Resveratrol increased GTPCH protein levels in a manner dependent upon ER modulation, since cotreatment with ICI 182,780 abolished the increase in GTPCH protein levels (Fig. 10). Thus, resveratrol-mediated increase in iNOS activity appears to be linked to its regulation of GTPCH expression, and an important mechanism underlying resveratrol-mediated decrease in VSMC proliferation appears tightly linked with its regulation of VSMC iNOS and its concomitant production of NO.
CONCLUSIONS
In these studies, we present the novel finding that resveratrol reduces VSMC proliferation via ER-dependent increases in iNOS activity but not iNOS protein. We furthermore demonstrate that increases in iNOS activity are associated with ER-dependent increases in levels of the iNOS cofactor BH4, and in protein levels of GTP cyclohydrolase-I. In support of these findings with resveratrol, one recent report indicates that 17β-estradiol acts via ER activation to increase gene expression of GTP cyclohydrolase-I.64 Clearly, while the signal transduction pathways linking the ER with regulation of GTP cyclohydrolase-I and by extension, BH4 levels, have yet to be elucidated, additional molecular studies examining the mechanism for resveratrol-mediated modulation of BH4 appear in order.
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