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Effect of red wine polyphenols on vascular smooth muscle cell function—molecular mechanism of the ‘French paradox’
Mechanisms of Ageing and Development 123 (2002) 1033– 1039 www.elsevier.com/locate/mechagedev
Effect of red wine polyphenols on vascular smooth muscle cell function—molecular mechanism of the ‘French paradox’ Katsuya Iijima, Masao Yoshizumi, Yasuyoshi Ouchi * Department of Geriatric Medicine, Graduate School of Medicine, The Uni6ersity of Tokyo, 7 -3 -1 Hongo, Bunkyo-ku, Tokyo 113 -8655, Japan
Abstract Red wine polyphenols (RWP) have been shown to have an anti-atherogenic activity mainly through anti-oxidative effects on low-density lipoprotein (LDL) oxidation. Though proliferation of vascular smooth muscle cells (VSMC) is critical to atherosclerosis formation, the effect of RWP on VSMC proliferation has not been elucidated. In this study, we investigated whether RWP, which extracted from red wine using column chromatography, could affect the 10% serum-stimulated VSMC proliferation. Treatment with RWP showed a potent inhibitory effect on the proliferation and DNA syntheses is in cultured rat VSMC. In contrast, the inhibitory effect of RWP on the proliferation of bovine vascular endothelial cells (EC) was only observed at much higher doses. Moreover, RWP significantly inhibited the proliferation and DNA synthesis of human VSMC but no human vascular EC in a dose-dependent manner. To elucidate the molecular mechanisms of these anti-proliferative effects of RWP on VSMC, but not on vascular EC, we investigated the effects of RWP on the cell cycle regulation. RWP downregulated the expression and promoter activity of cyclin A gene, one of cell cycle regulators. In addition, RWP inhibited the binding of nuclear proteins to the activating transcription factor (ATF) site in the cyclin A promoter, and downregulated the expression of transcription factors, cyclic AMP-responsive element binding protein (CREB) and ATF-1. In conclusion, these results demonstrate one possible finding that the anti-proliferative effect of RWP on VSMC may be associated with the downregulation of cyclin A gene expression through the inhibition of transcription factor expression. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Red wine polyphenols; French paradox; Vascular smooth muscle cells
1. Introduction Polyphenols are found in grapes and other fruits and vegetables. Although polyphenols are * Corresponding author. Tel.: +81-3-5800-8830; fax: + 813-5800-6529. E-mail address: [email protected] (Y. Ouchi).
widely distributed in plants, they are found in high concentrations in only a few foods and beverages, such as red wine and tea. Grapes are one of the most widely consumed fruits in the world, and are rich in polyphenols and 90–95% of grape polyphenols exist in grape seeds and grape skin. Most of the grape seed polyphenols are quite different from tea polyphenols. Grape seed
0047-6374/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 4 7 - 6 3 7 4 ( 0 1 ) 0 0 3 8 6 - 4
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K. Iijima et al. / Mechanisms of Ageing and De6elopment 123 (2002) 1033–1039
polyphenols are mainly rich in polymers, whereas most tea polyphenols are monomers, such as catechins. Epidemiological studies have demonstrated that the French population has lower mortality from coronary heart disease compared with other countries, in spite of high-cholesterol diet (Renaud and Lorgeril, 1992). Recently, it has been reported that this paradoxical finding, the ‘French paradox’, may be attributed to regular consumption of red wine, and the unique antiatherogenic effects of red wine reside in the action of polyphenols (Renaud and Lorgeril, 1992). Development of atherosclerotic plaque is characterized by dysfunction of endothelial cells (EC), oxidation of low-density lipoprotein (LDL) and foam cell formation from macrophage, migration of vascular smooth muscle cells (VSMC) from the arterial media into intima, excessive proliferation of VSMC in the neointima, and increased extracellular matrix deposition (Ross, 1993). It has been previously reported that red wine polyphenols (RWP) inhibit the oxidation of LDL in vitro and in vivo (Anonymous, 1993), and this anti-oxidative effects of RWP may be involved in a part of the mechanism of the French paradox (Anonymous, 1993). Moreover, it has been recently reported that RWP exert some beneficial effects, such as decreasing activity of lipoprotein (a) and increasing levels of HDL-cholesterol (Stephan et al., 1999). Additionally, it has been also reported that RWP have not only anti-oxidation of LDL cholesterol but also other various antiatherogenic effects, such as inhibition of adhesion molecule expression through downregulation of NF-kB activation in vascular EC (Murase et al., 1999), induction of nitric oxidedependent vaso-relaxation (Flesch et al., 1998), downregulation of tissue factor expression (Pendurthi et al., 1999), and inhibition of platelet aggregation (Ruf, 1999). The VSMC proliferation plays a key role in progressive intimal thickening. In this study, we demonstrate the effect of RWP on proliferation of vascular cells in relation to cell cycle regulation.
2. Methods and results
2.1. Preparation and characterization of red wine polyphenolic compound Polyphenolic substances (dry weight 24 g in total) were extracted from ten-bottles (7600 ml in total) of red wine (Suntory Co., Osaka, Japan) by adsorption column chromatography using a Diaion HP-20 column (40× 40 cm; Mitsubishi Chemical Industries, Japan). The column was eluted with de-ionized water (150 l), and the eluent, which did not contain polyphenolic substances, was discarded. Total polyphenolic fraction, named RWP (24 g in total; dry weight), was obtained by eluting the column with 100% ethanol (150 l). RWP compound contained a wide variety of polyphenols including monomers and polymers, which have different molecular weights. To measure the content of total polyphenols, all fractions were systematically analyzed by ultra-violet spectrophotometry according to the Folin–Denis method. Just before the addition of these compounds to cells, we dissolved each compound in 50% ethanol using ultrasonication in all experiments. The total polyphenolic fraction (RWP) extracted from red wine contained wide varieties of polyphenols, which have different molecular weights. RWP was separated into six fractions and broadly divided into two groups. The first group (fraction 1–4) contained various polyphenolic monomer components, such as anthocyanidins, catechins, and flavonoids, and was characterized by lower average molecular weight (approximately 200–400). Although the percentage of each monomer may be quite different among the four fractions, we have not found any specific polyphenolic substance that is dominant in a specific fraction. Another group (fraction 5 and 6) contained a significant amount of specific polyphenolic compounds, proanthocyanidins, which are polymerized anthocyanidins, and was characterized by higher average molecular weight (approximately 1600– 2000).
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2.2. Effects of red wine polyphenols on cell proliferation
2.3. Effect of red wine polyphenols on cyclin A gene expression
The effects of RWP on proliferation of cultured rat aortic smooth muscle cells (RASMC) or bovine carotid endothelial cells (BCEC) stimulated by 10% serum were investigated. In all experiments, we confirmed that ethanol had no effects on cell growth and expression of cell cycle regulator as follow. RWP treatment (1– 100 mg/ml for 72 h) significantly inhibited cell proliferation of RASMC in a dose-dependent manner (Fig. 1A). In contrast, RWP inhibited the proliferation of BCEC at much higher concentrations, especially at 30 and 100 mg/ml (Fig. 1B). Moreover, in the experiment of DNA synthesis of two cell types, RWP inhibited thymidine incorporation into RASMC in a dose-dependent manner (Fig. 1C). In BCEC, however, RWP did not inhibit thymidine incorporation except at much higher concentrations (Fig. 1D). These findings suggest that the inhibitory effect of RWP is specific on cell proliferation and DNA synthesis in VSMC but not in vascular EC. To rule out the possibility that the anti-proliferative effect of RWP on VSMC but not vascular EC is dependent on the difference of species, the effect of RWP on human VSMC or vascular EC was examined in the same way. RWP significantly inhibited the DNA synthesis of human VSMC but not human vascular EC in a dose-dependent manner (data not shown). To confirm the difference of these inhibitory effects on VSMC growth in each polyphenolic fraction separated from RWP, we investigated the effects of each fraction on DNA synthesis in serum-stimulated VSMC. Six polyphenolic fractions (Fraction 1– 6) decreased thymidine incorporation into RASMC to 41, 37, 30, 26, 30, and 22% of control containing 0.15% ethanol as vehicle. Interestingly, the inhibitory effect of each fraction was in proportion to its respective total polyphenolic content. It is noteworthy that polyphenolic fractions which have different molecular weight showed similar potent antiproliferative effects on VSMC (data not shown).
To elucidate molecular mechanisms of these anti-proliferative effects of RWP on VSMC, the effect of RWP on expression of cell cycle regulators, especially cyclin A gene, was examined. Cyclin A, one of these cyclins, is essential in the cell cycle, and has a critical role in DNA replication during the S phase (Girard et al., 1991; Sobczak et al., 1993). It has been previously reported that cyclin A is one of the key regulators of VSMC proliferation caused by mechanical balloon injury in vivo (Wei et al., 1997), and by chemical stimulation, such as homocysteine or serum, in vitro (Tsai et al., 1996). In time-course experiment, the expression of cyclin A mRNA was slightly decreased at 4 h, and completely suppressed at 48–72 h in RASMC treated with RWP at a final concentration of 30 mg/ml (data not shown). RWP significantly inhibited the levels of cyclin A mRNA of 10% serum-stimulated RASMC in a dose-dependent manner (Fig. 1E). However, RWP did not inhibit the cyclin A expression in BCEC except at much higher concentrations (Fig. 1F). In case of human cells, RWP dose-dependently inhibited the cyclin A mRNA expression in human VSMC but not human vascular EC (data not shown).
2.4. Effect of red wine polyphenols on transcription of cyclin A gene 2.4.1. Cyclin A promoter acti6ity To further investigate the mechanisms of the downregulation of cyclin A gene expression, the effect of RWP on the cyclin A promoter activity was examined (Fig. 2A). Reporter constructs containing a fragment of the human cyclin A 5%-flanking region (bp − 266 to + 205) were inserted into the promoterless firefly luciferase reporter plasmid. The cyclin A promoter contains the activating transcription factor (ATF) site as a potential regulatory element. The wild-typed ATF consensus sequence (TGACGTCA) in the plasmid − 266/ + 205 was mutated to TGCCCCCA by PCR to generate the plasmid mut −266/ + 205. Twenty-four hours after transfection with this
Fig. 1. Effects of RWP on proliferation and cyclin A expression. After 48 h incubation, 10% serum-stimulated RASMC or BCEC were treated with RWP (1 –100 mg/ml) for 72 h. Effects of RWP on number of RASMC (A) and thymidine incorporation into RASMC (C) after 72 h treatment is expressed as percentage of the control containing 0.5% ethanol as vehicle. Similarly, the effects of RWP on number of BCEC (B) and thymidine incorporation into BCEC (D) are shown. Data are expressed as mean 9S.E.M. (n= 6; **, PB 0.05, **, P B0.01 vs. vehicle). To examine the effect of RWP on expression of cyclin A mRNA, total RNA was extracted from 10% serum-stimulated RASMC (E) or BCEC (F) treated with RWP for 72 h. Northern analysis with cyclin A and 18S probes was performed.
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Fig. 2. Effects of RWP on transcription of cyclin A gene. (A) RASMC were transfected with luciferase reporter plasmids containing the human cyclin A promoter containing a normal ATF site (ATF) or a mutated ATF site (mut ATF). After transfection for 24 h, the cells were treated with RWP (1 –100 mg/ml) for 72 h. Luciferase activity was compared with that of control (defined as 100%) containing 0.5% ethanol as vehicle. Data are expressed as mean 9S.E.M. (n= 4; **, P B 0.01 vs. vehicle). (B) Nuclear extracts from RASMC treated with RWP for 72 h were incubated with 32P-labeled ATF probe and analyzed by 5% native polyacrylamide gel electrophoresis. A 100-fold molar excess of unlabeled oligonucleotides encoding the ATF site (lane 3) or mutated ATF sequence (mut ATF, lane 4) were added to the nuclear extracts as competitors to determine the specificity of DNA-protein complexes. (C) Effect of RWP on expression of CREB mRNA and ATF-1 mRNA was examined.
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plasmid in RASMC or BCEC, the cells were incubated with each concentration of RWP, and then the luciferase activity, as the cyclin A promoter activity, was measured. Induction of the cyclin A promoter activity was based on ATF site but not mutated ATF site, and RWP decreased the transcriptional activity in a dose-dependent manner. In contrast, RWP did not inhibit the transcriptional activity of the cyclin A promoter in BCEC (data not shown).
2.4.2. Binding of nuclear proteins The binding of nuclear proteins from RASMC to the ATF site was examined using gel mobility shift assay (Fig. 2B). Twenty two base pair oligonucleotide probe was synthesized according to the cyclin A 5%-flanking sequence containing a typical ATF site. Specific ATF-binding complexes, which be shown by the arrow, were competed by addition of excessive cold probes encoding wild-type ATF sequence (lane 3), however, was not competed by mutated ATF sequence (lane 4). The specific binding complexes were downregulated by treatment with RWP in a dose-dependent manner (lane 5– 10). 2.4.3. Transcription factor expression These binding nuclear proteins are important in the induction of cyclin A gene, and these proteins contain several transcription factors, such as cyclic AMP-responsive element binding protein (CREB) and activating transcription factor-1 (ATF-1) (Yoshizumi et al., 1997). The effect of RWP on the expression of these transcription factors in 10% serum-stimulated RASMC is shown in Fig. 2C. The time-course of the downregulation of CREB and ATF-1 mRNA was very similar to that of cyclin A mRNA. Moreover, the dose-dependency of the downregulation of these transcription factors was also very similar to that of cyclin A. 2.5. Effect of red wine polyphenols on cell morphological changes To rule out the possibility that RWP induces apoptosis in RASMC, morphological change of RSMC was examined under fluorescent mi-
croscopy following nucleic acid staining (Hoechst 33258 dye). RWP even at the highest concentration (100 mg/ml) did not cause any morphological changes (data not shown). These findings suggest that an apoptotic process does not mediate the inhibitory effects of RWP on VSMC.
3. Conclusion RWP have potent inhibitory effects on the VSMC proliferation in the context of atherogenesis and the mechanism of the inhibitory effects on VSMC is associated with the downregulation of cyclin A gene through the inhibition of transcription factor expression. Our findings suggest that the antiproliferative effect of RWP may be one possible mechanism for the antiatherogenic effects of red wine, other than the antioxidative effects of RWP on LDL. When detachment of the endothelial monolayer is induced by several injuries, including mechanical intervention, chemical substances, and inflammatory processes, the proliferation and migration of vascular EC is mainly responsible for the wound repair of damaged vessels. In this study, we demonstrated that RWP had no inhibitory effect on proliferation, DNA synthesis, and cyclin A gene expression in serum-stimulated vascular EC. Although RWP had no significant effects on proliferation of vascular EC, the mechanism of these phenomena is unclear. However, we found that RWP inhibited expression of vascular cell adhesion molecule-1 in TNF-a-stimulated vascular EC in a concentration-dependent manner (data not shown). Moreover, it has been recently reported that gallates, a content of RWP, inhibited cytokine-induced adhesion molecule expression in vascular EC through a reduction of NF-kB activity (Murase et al., 1999). These beneficial effects of RWP on vascular EC suggest that RWP may play a role in not only maintenance of vascular structural integrity, but also in the restoration of function of damaged vascular EC. In the future, the effects of RWP on vascular endothelial cell function, including membrane sensitivity and intracellular signaling, need to be elucidated.
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According to some reports that have demonstrated absorption efficiency of RWP (Duthie et al., 1998), it may be reasonable to suppose that the physiological concentration of RWP in blood after a normal intake of red wine may be 1– 10 mg/ml. It can be assumed that the long-term (for years) effect of even lower concentrations of RWP may have the inhibitory effect on VSMC proliferation in vivo. Further investigation for cell cycle regulation modulating the VSMC proliferation may clarify the mechanism of the ‘French paradox’, and may enable us to innovate a therapeutic approach for the prevention of atherosclerotic progression.
References Anonymous, 1993. Inhibition of LDL oxidation by phenolic substances in red wine: a clue to the French paradox. Nutr. Rev. 51, 185 – 187 Review. Duthie, G.G., Pedersen, M.W., Gardner, P.T., Morrice, P.C., Jenkinson, A.M., McPhail, D.B., Steele, G.M., 1998. The effect of whisky and wine consumption on total phenol content and antioxidant capacity of plasma from healthy volunteers. Eur. J. Clin. Nutr. 52, 733 –736. Flesch, M., Schwarz, A., Bohm, M., 1998. Effects of red and white wine on endothelium-dependent vasorelaxation of rat aorta and human coronary arteries. Am. J. Physiol. 275, H1183 – H1190 (4 Pt. 2). Girard, F., Strausfeld, U., Fernandez, A., Lamb, N.J., 1991. Cyclin A is required for the onset of DNA replication in mammalian fibroblasts. Cell 67, 1169 –1179. Murase, T., Kume, N., Hase, T., Shibuya, Y., Nishizawa, Y., Tokimitsu, I., Kita, T., 1999. Gallates inhibit cytokine-in-
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duced nuclear translocation of NF-kB and expression of leukocyte adhesion molecules in vascular endothelial cells. Arterioscler. Thromb. Vasc. Biol. 19 (6), 1412 – 1420. Pendurthi, U.R., Williams, J.T., Rao, L.V., 1999. Resveratrol, a polyphenolic compound found in wine, inhibits tissue factor expression in vascular cells: a possible mechanism for the cardiovascular benefits associated with moderate consumption of wine. Arterioscler. Thromb. Vasc. Biol. 19 (2), 419 – 426. Renaud, S., Lorgeril, M., 1992. Wine, alcohol, platelets, and the French paradox for coronary heart disease. Lancet 339, 1523 – 1526. Ross, R., 1993. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 362, 801 – 809. Ruf, J.C., 1999. Wine and polyphenols related to platelet aggregation and atherothrombosis. Drugs. Exp. Clin. Res. 25 (2 – 3), 125 – 131. Sobczak, T.J., Harper, F., Florentin, Y., Zindy, F., Brechot, C., Puvion, E., 1993. Localization of cyclin A at the sites of cellular DNA replication. Exp. Cell. Res. 206, 43 – 48. Stephan, E.M., Schneider, S.H., Khachadurian, A.K., 1999. Red wine inhibits the cell-mediated oxidation of LDL and HDL. J. Am. Coll. Nutr. 18 (2), 137 – 143. Tsai, J.C., Wang, H., Perrella, M.A., Yoshizumi, M., Sibinga, N.E., Tan, L.C., Haber, E., Chang, T.H., Schlegel, R., Lee, M.E., 1996. Induction of cyclin A gene expression by homocysteine in vascular smooth muscle cells. J. Clin. Invest. 97, 146 – 153. Wei, G.L., Krasinski, K., Kearney, M., Isner, J.M., Walsh, K., Andres, V., 1997. Temporally and spatially coordinated expression of cell cycle regulatory factors after angioplasty. Circ. Res. 80, 418 – 426. Yoshizumi, M., Wang, H., Hsieh, C.M., Sibinga, N.E., Perrella, M.A., Lee, M.E., 1997. Down-regulation of the cyclin A promoter by transforming growth factor-beta1 is associated with a reduction in phosphorylated activating transcription factor-1 and cyclic AMP-responsive elementbinding protein. J. Biol. Chem. 272, 22259 – 22264.
Effect of red wine polyphenols on vascular smooth muscle cell function—molecular mechanism of the ‘French paradox’ Katsuya Iijima, Masao Yoshizumi, Yasuyoshi Ouchi * Department of Geriatric Medicine, Graduate School of Medicine, The Uni6ersity of Tokyo, 7 -3 -1 Hongo, Bunkyo-ku, Tokyo 113 -8655, Japan
Abstract Red wine polyphenols (RWP) have been shown to have an anti-atherogenic activity mainly through anti-oxidative effects on low-density lipoprotein (LDL) oxidation. Though proliferation of vascular smooth muscle cells (VSMC) is critical to atherosclerosis formation, the effect of RWP on VSMC proliferation has not been elucidated. In this study, we investigated whether RWP, which extracted from red wine using column chromatography, could affect the 10% serum-stimulated VSMC proliferation. Treatment with RWP showed a potent inhibitory effect on the proliferation and DNA syntheses is in cultured rat VSMC. In contrast, the inhibitory effect of RWP on the proliferation of bovine vascular endothelial cells (EC) was only observed at much higher doses. Moreover, RWP significantly inhibited the proliferation and DNA synthesis of human VSMC but no human vascular EC in a dose-dependent manner. To elucidate the molecular mechanisms of these anti-proliferative effects of RWP on VSMC, but not on vascular EC, we investigated the effects of RWP on the cell cycle regulation. RWP downregulated the expression and promoter activity of cyclin A gene, one of cell cycle regulators. In addition, RWP inhibited the binding of nuclear proteins to the activating transcription factor (ATF) site in the cyclin A promoter, and downregulated the expression of transcription factors, cyclic AMP-responsive element binding protein (CREB) and ATF-1. In conclusion, these results demonstrate one possible finding that the anti-proliferative effect of RWP on VSMC may be associated with the downregulation of cyclin A gene expression through the inhibition of transcription factor expression. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Red wine polyphenols; French paradox; Vascular smooth muscle cells
1. Introduction Polyphenols are found in grapes and other fruits and vegetables. Although polyphenols are * Corresponding author. Tel.: +81-3-5800-8830; fax: + 813-5800-6529. E-mail address: [email protected] (Y. Ouchi).
widely distributed in plants, they are found in high concentrations in only a few foods and beverages, such as red wine and tea. Grapes are one of the most widely consumed fruits in the world, and are rich in polyphenols and 90–95% of grape polyphenols exist in grape seeds and grape skin. Most of the grape seed polyphenols are quite different from tea polyphenols. Grape seed
0047-6374/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 4 7 - 6 3 7 4 ( 0 1 ) 0 0 3 8 6 - 4
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K. Iijima et al. / Mechanisms of Ageing and De6elopment 123 (2002) 1033–1039
polyphenols are mainly rich in polymers, whereas most tea polyphenols are monomers, such as catechins. Epidemiological studies have demonstrated that the French population has lower mortality from coronary heart disease compared with other countries, in spite of high-cholesterol diet (Renaud and Lorgeril, 1992). Recently, it has been reported that this paradoxical finding, the ‘French paradox’, may be attributed to regular consumption of red wine, and the unique antiatherogenic effects of red wine reside in the action of polyphenols (Renaud and Lorgeril, 1992). Development of atherosclerotic plaque is characterized by dysfunction of endothelial cells (EC), oxidation of low-density lipoprotein (LDL) and foam cell formation from macrophage, migration of vascular smooth muscle cells (VSMC) from the arterial media into intima, excessive proliferation of VSMC in the neointima, and increased extracellular matrix deposition (Ross, 1993). It has been previously reported that red wine polyphenols (RWP) inhibit the oxidation of LDL in vitro and in vivo (Anonymous, 1993), and this anti-oxidative effects of RWP may be involved in a part of the mechanism of the French paradox (Anonymous, 1993). Moreover, it has been recently reported that RWP exert some beneficial effects, such as decreasing activity of lipoprotein (a) and increasing levels of HDL-cholesterol (Stephan et al., 1999). Additionally, it has been also reported that RWP have not only anti-oxidation of LDL cholesterol but also other various antiatherogenic effects, such as inhibition of adhesion molecule expression through downregulation of NF-kB activation in vascular EC (Murase et al., 1999), induction of nitric oxidedependent vaso-relaxation (Flesch et al., 1998), downregulation of tissue factor expression (Pendurthi et al., 1999), and inhibition of platelet aggregation (Ruf, 1999). The VSMC proliferation plays a key role in progressive intimal thickening. In this study, we demonstrate the effect of RWP on proliferation of vascular cells in relation to cell cycle regulation.
2. Methods and results
2.1. Preparation and characterization of red wine polyphenolic compound Polyphenolic substances (dry weight 24 g in total) were extracted from ten-bottles (7600 ml in total) of red wine (Suntory Co., Osaka, Japan) by adsorption column chromatography using a Diaion HP-20 column (40× 40 cm; Mitsubishi Chemical Industries, Japan). The column was eluted with de-ionized water (150 l), and the eluent, which did not contain polyphenolic substances, was discarded. Total polyphenolic fraction, named RWP (24 g in total; dry weight), was obtained by eluting the column with 100% ethanol (150 l). RWP compound contained a wide variety of polyphenols including monomers and polymers, which have different molecular weights. To measure the content of total polyphenols, all fractions were systematically analyzed by ultra-violet spectrophotometry according to the Folin–Denis method. Just before the addition of these compounds to cells, we dissolved each compound in 50% ethanol using ultrasonication in all experiments. The total polyphenolic fraction (RWP) extracted from red wine contained wide varieties of polyphenols, which have different molecular weights. RWP was separated into six fractions and broadly divided into two groups. The first group (fraction 1–4) contained various polyphenolic monomer components, such as anthocyanidins, catechins, and flavonoids, and was characterized by lower average molecular weight (approximately 200–400). Although the percentage of each monomer may be quite different among the four fractions, we have not found any specific polyphenolic substance that is dominant in a specific fraction. Another group (fraction 5 and 6) contained a significant amount of specific polyphenolic compounds, proanthocyanidins, which are polymerized anthocyanidins, and was characterized by higher average molecular weight (approximately 1600– 2000).
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2.2. Effects of red wine polyphenols on cell proliferation
2.3. Effect of red wine polyphenols on cyclin A gene expression
The effects of RWP on proliferation of cultured rat aortic smooth muscle cells (RASMC) or bovine carotid endothelial cells (BCEC) stimulated by 10% serum were investigated. In all experiments, we confirmed that ethanol had no effects on cell growth and expression of cell cycle regulator as follow. RWP treatment (1– 100 mg/ml for 72 h) significantly inhibited cell proliferation of RASMC in a dose-dependent manner (Fig. 1A). In contrast, RWP inhibited the proliferation of BCEC at much higher concentrations, especially at 30 and 100 mg/ml (Fig. 1B). Moreover, in the experiment of DNA synthesis of two cell types, RWP inhibited thymidine incorporation into RASMC in a dose-dependent manner (Fig. 1C). In BCEC, however, RWP did not inhibit thymidine incorporation except at much higher concentrations (Fig. 1D). These findings suggest that the inhibitory effect of RWP is specific on cell proliferation and DNA synthesis in VSMC but not in vascular EC. To rule out the possibility that the anti-proliferative effect of RWP on VSMC but not vascular EC is dependent on the difference of species, the effect of RWP on human VSMC or vascular EC was examined in the same way. RWP significantly inhibited the DNA synthesis of human VSMC but not human vascular EC in a dose-dependent manner (data not shown). To confirm the difference of these inhibitory effects on VSMC growth in each polyphenolic fraction separated from RWP, we investigated the effects of each fraction on DNA synthesis in serum-stimulated VSMC. Six polyphenolic fractions (Fraction 1– 6) decreased thymidine incorporation into RASMC to 41, 37, 30, 26, 30, and 22% of control containing 0.15% ethanol as vehicle. Interestingly, the inhibitory effect of each fraction was in proportion to its respective total polyphenolic content. It is noteworthy that polyphenolic fractions which have different molecular weight showed similar potent antiproliferative effects on VSMC (data not shown).
To elucidate molecular mechanisms of these anti-proliferative effects of RWP on VSMC, the effect of RWP on expression of cell cycle regulators, especially cyclin A gene, was examined. Cyclin A, one of these cyclins, is essential in the cell cycle, and has a critical role in DNA replication during the S phase (Girard et al., 1991; Sobczak et al., 1993). It has been previously reported that cyclin A is one of the key regulators of VSMC proliferation caused by mechanical balloon injury in vivo (Wei et al., 1997), and by chemical stimulation, such as homocysteine or serum, in vitro (Tsai et al., 1996). In time-course experiment, the expression of cyclin A mRNA was slightly decreased at 4 h, and completely suppressed at 48–72 h in RASMC treated with RWP at a final concentration of 30 mg/ml (data not shown). RWP significantly inhibited the levels of cyclin A mRNA of 10% serum-stimulated RASMC in a dose-dependent manner (Fig. 1E). However, RWP did not inhibit the cyclin A expression in BCEC except at much higher concentrations (Fig. 1F). In case of human cells, RWP dose-dependently inhibited the cyclin A mRNA expression in human VSMC but not human vascular EC (data not shown).
2.4. Effect of red wine polyphenols on transcription of cyclin A gene 2.4.1. Cyclin A promoter acti6ity To further investigate the mechanisms of the downregulation of cyclin A gene expression, the effect of RWP on the cyclin A promoter activity was examined (Fig. 2A). Reporter constructs containing a fragment of the human cyclin A 5%-flanking region (bp − 266 to + 205) were inserted into the promoterless firefly luciferase reporter plasmid. The cyclin A promoter contains the activating transcription factor (ATF) site as a potential regulatory element. The wild-typed ATF consensus sequence (TGACGTCA) in the plasmid − 266/ + 205 was mutated to TGCCCCCA by PCR to generate the plasmid mut −266/ + 205. Twenty-four hours after transfection with this
Fig. 1. Effects of RWP on proliferation and cyclin A expression. After 48 h incubation, 10% serum-stimulated RASMC or BCEC were treated with RWP (1 –100 mg/ml) for 72 h. Effects of RWP on number of RASMC (A) and thymidine incorporation into RASMC (C) after 72 h treatment is expressed as percentage of the control containing 0.5% ethanol as vehicle. Similarly, the effects of RWP on number of BCEC (B) and thymidine incorporation into BCEC (D) are shown. Data are expressed as mean 9S.E.M. (n= 6; **, PB 0.05, **, P B0.01 vs. vehicle). To examine the effect of RWP on expression of cyclin A mRNA, total RNA was extracted from 10% serum-stimulated RASMC (E) or BCEC (F) treated with RWP for 72 h. Northern analysis with cyclin A and 18S probes was performed.
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Fig. 2. Effects of RWP on transcription of cyclin A gene. (A) RASMC were transfected with luciferase reporter plasmids containing the human cyclin A promoter containing a normal ATF site (ATF) or a mutated ATF site (mut ATF). After transfection for 24 h, the cells were treated with RWP (1 –100 mg/ml) for 72 h. Luciferase activity was compared with that of control (defined as 100%) containing 0.5% ethanol as vehicle. Data are expressed as mean 9S.E.M. (n= 4; **, P B 0.01 vs. vehicle). (B) Nuclear extracts from RASMC treated with RWP for 72 h were incubated with 32P-labeled ATF probe and analyzed by 5% native polyacrylamide gel electrophoresis. A 100-fold molar excess of unlabeled oligonucleotides encoding the ATF site (lane 3) or mutated ATF sequence (mut ATF, lane 4) were added to the nuclear extracts as competitors to determine the specificity of DNA-protein complexes. (C) Effect of RWP on expression of CREB mRNA and ATF-1 mRNA was examined.
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plasmid in RASMC or BCEC, the cells were incubated with each concentration of RWP, and then the luciferase activity, as the cyclin A promoter activity, was measured. Induction of the cyclin A promoter activity was based on ATF site but not mutated ATF site, and RWP decreased the transcriptional activity in a dose-dependent manner. In contrast, RWP did not inhibit the transcriptional activity of the cyclin A promoter in BCEC (data not shown).
2.4.2. Binding of nuclear proteins The binding of nuclear proteins from RASMC to the ATF site was examined using gel mobility shift assay (Fig. 2B). Twenty two base pair oligonucleotide probe was synthesized according to the cyclin A 5%-flanking sequence containing a typical ATF site. Specific ATF-binding complexes, which be shown by the arrow, were competed by addition of excessive cold probes encoding wild-type ATF sequence (lane 3), however, was not competed by mutated ATF sequence (lane 4). The specific binding complexes were downregulated by treatment with RWP in a dose-dependent manner (lane 5– 10). 2.4.3. Transcription factor expression These binding nuclear proteins are important in the induction of cyclin A gene, and these proteins contain several transcription factors, such as cyclic AMP-responsive element binding protein (CREB) and activating transcription factor-1 (ATF-1) (Yoshizumi et al., 1997). The effect of RWP on the expression of these transcription factors in 10% serum-stimulated RASMC is shown in Fig. 2C. The time-course of the downregulation of CREB and ATF-1 mRNA was very similar to that of cyclin A mRNA. Moreover, the dose-dependency of the downregulation of these transcription factors was also very similar to that of cyclin A. 2.5. Effect of red wine polyphenols on cell morphological changes To rule out the possibility that RWP induces apoptosis in RASMC, morphological change of RSMC was examined under fluorescent mi-
croscopy following nucleic acid staining (Hoechst 33258 dye). RWP even at the highest concentration (100 mg/ml) did not cause any morphological changes (data not shown). These findings suggest that an apoptotic process does not mediate the inhibitory effects of RWP on VSMC.
3. Conclusion RWP have potent inhibitory effects on the VSMC proliferation in the context of atherogenesis and the mechanism of the inhibitory effects on VSMC is associated with the downregulation of cyclin A gene through the inhibition of transcription factor expression. Our findings suggest that the antiproliferative effect of RWP may be one possible mechanism for the antiatherogenic effects of red wine, other than the antioxidative effects of RWP on LDL. When detachment of the endothelial monolayer is induced by several injuries, including mechanical intervention, chemical substances, and inflammatory processes, the proliferation and migration of vascular EC is mainly responsible for the wound repair of damaged vessels. In this study, we demonstrated that RWP had no inhibitory effect on proliferation, DNA synthesis, and cyclin A gene expression in serum-stimulated vascular EC. Although RWP had no significant effects on proliferation of vascular EC, the mechanism of these phenomena is unclear. However, we found that RWP inhibited expression of vascular cell adhesion molecule-1 in TNF-a-stimulated vascular EC in a concentration-dependent manner (data not shown). Moreover, it has been recently reported that gallates, a content of RWP, inhibited cytokine-induced adhesion molecule expression in vascular EC through a reduction of NF-kB activity (Murase et al., 1999). These beneficial effects of RWP on vascular EC suggest that RWP may play a role in not only maintenance of vascular structural integrity, but also in the restoration of function of damaged vascular EC. In the future, the effects of RWP on vascular endothelial cell function, including membrane sensitivity and intracellular signaling, need to be elucidated.
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According to some reports that have demonstrated absorption efficiency of RWP (Duthie et al., 1998), it may be reasonable to suppose that the physiological concentration of RWP in blood after a normal intake of red wine may be 1– 10 mg/ml. It can be assumed that the long-term (for years) effect of even lower concentrations of RWP may have the inhibitory effect on VSMC proliferation in vivo. Further investigation for cell cycle regulation modulating the VSMC proliferation may clarify the mechanism of the ‘French paradox’, and may enable us to innovate a therapeutic approach for the prevention of atherosclerotic progression.
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