![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
–Induced Proliferation and Matrix Metalloproteinase Expression in Human Vascular Smooth Muscle Cells
Department of Anatomy, College of Medicine, Konkuk University, Chungju City, Chungbuk 380–701, South Korea and * Department of Food and Biotechnology, Chungju National University, Chungju, Chungbuk 380–702, South Korea
1To whom correspondence should be addressed. E-mail: sumoon66{at}dreamwiz.com.
ABSTRACT
Resveratrol (RV), a polyphenolic substance found in grape skin, was suggested to play a role in preventing the development of atherosclerotic disease. Although RV has antiatherogenic effects on vascular smooth muscle cells (VSMC), the molecular mechanisms associated with tumor necrosis factor (TNF)-
–induced VSMC are unclear. The goal of this study was to determine the effect of RV on the modulation of cell proliferation, cell-cycle regulation, and matrix metalloproteinase (MMP)-9 expression in TNF-–induced human VSMC. RV treatment inhibited DNA synthesis in cultured VSMC in the presence of TNF-. These inhibitory effects were associated with reduced levels of extracellular signal-regulated kinase (ERK) 1/2 activity and G1 cell-cycle arrest. Treatment with RV, which blocks the cell cycle in the G1 phase, downregulated the expression of cyclins and cyclin-dependent kinases (CDKs) and upregulated the expression of p21/WAF1, a CDK inhibitor. RV did not upregulate p27. Moreover, RV increased the promoter activity of the p21/WAF1 gene. Immunoblot and deletion analysis of the p21/WAF1 promoter showed that RV induced the expression of p21/WAF1 and that this expression was independent of the p53 pathway. Furthermore, zymographic and immunoblot analyses showed that RV dose dependently suppressed the TNF-–induced expression of MMP-9. This inhibition was characterized by the downregulation of MMP-9, which was transcriptionally regulated at the activator protein-1 (AP-1) and nuclear factor-
B (NF-B) sites in the MMP-9 promoter. Collectively, these results suggest that RV inhibits cell proliferation, G1 to S phase cell-cycle progress, and MMP-9 expression through the transcription factors NF-B and AP-1 in TNF-–induced VSMC.
KEY WORDS: • resveratrol • vascular smooth muscle cell • matrix metalloproteinase-9 • G1 cell-cycle arrest
The proliferation of vascular smooth muscle cells (VSMC)2 is an important event during the development of atherosclerosis and contributes to the failure of clinical interventions used to treat patients with coronary heart disease such as in-stent restenosis and late vein graft failure. VSMC is the principal cell type in both atherosclerotic and restenotic lesions (1). Vascular lesions form as the result of a number of pathological processes involving the accumulation of inflammatory cells and the release of cytokines (2). The cytokine, tumor necrosis factor (TNF)-, is secreted by VSMC in the neointima after balloon-injury as well as by macrophages in atherosclerotic lesions (3–5). It was shown previously that TNF- induces an increase in DNA synthesis and the activation of extracellular signal-regulated kinases (ERK) 1/2, in VSMC (6,7). A recent study reported that TNF- may also play an important role in cell-cycle regulation in VSMC (8).
During the development of atherosclerosis or in response to vessel injury, VSMC migrate into the intimal layer of the arterial wall, where they exit from their quiescent state (G0/G1 phase of the cell cycle) and reenter the cell cycle (1). Progression through the G1 phase of the cell cycle is regulated by the cyclins (D, E), which associate with and activate their catalytic partners, the cyclin-dependent kinases (CDK2 and CDK4) (9–12). In many cells, transit through the G1 phase of the cell cycle and entry into the S phase require the binding and activation of cyclin/CDKs complexes, a process in which cyclin D1/CDK4 and cyclin E/CDK2 play major roles (9–12). The kinase activities of the cyclin/CDK complexes are positively regulated by a cyclin subunit and are negatively regulated by the CDK inhibitors p21/WAF1 and p27 (13,14). It is clear that p21/WAF1 also plays an essential and positive role in the assembly of certain cyclin/CDK complexes (15–19).
Matrix metalloproteinases (MMPs), specifically gelatinases MMP-2 and MMP-9, have been implicated in VSMC migration, contributing to the intimal thickening that is characteristic of in vivo vascular lesions. In most cases, MMP-2 is constitutively present in tissues in the form of a 72-kDa proenzyme. In contrast, MMP-9, like most other MMPs, is expressed and secreted only on demand, and regulation occurs at the gene transcription level. The synthesis and secretion of MMP-9 can be stimulated by a variety of stimuli including growth factors and cytokines (20–23,48). The expression of MMP-9 was implicated in the progression of atherosclerotic lesions (24,25). Recent reports, based on in vivo studies, concluded that MMP-9 is critical for the development of arterial lesions via its ability to regulate both migration and proliferation (26,27). On the basis of in-depth reports from several different laboratories, it has generally been concluded that basal levels of MMP-9 are usually low, and that its expression can be induced by treatment of vascular smooth muscle cells with TNF- (8,20,22,23). In addition, recent results demonstrated that TNF-–induced MMP-9 expression in VSMC is mediated by increased activities of nuclear factor (NF)-B and activator protein-1 (AP-1) and involves the activation of the Ras/ERK1/2 pathway (8,23).
Resveratrol (RV), a polyphenolic phytoalexin, is produced by grapes and other plants in response to an infection or injury (28). As a major constituent of red wine, RV was proposed to account, at least in part, for the beneficial effects attributed to this beverage in cardiovascular diseases (29,30). RV was reported to inhibit a wide variety of biological events associated with cell proliferation and tumor progression (31,32). It was also reported to have a variety of anti-inflammatory (33), antiplatelet (34), and antimutagenic (35) effects. A recent study reported that RV can inhibit VSMC proliferation in vitro (36–38). An in vivo study suggested that RV can protect against intimal hyperplasia after endothelial denudation in an experimental rabbit model (39). A more recent study demonstrated that RV reversibly inhibits cell-cycle inhibition in the early S phase in calf serum–treated VSMC (40). In addition, Haider et al. (41) showed previously that RV suppresses angiotensin II-induced VSMC hypertrophy, most likely by interfering with Akt-governed pathways. However, information is lacking on the effect of RV on VSMC responses via ERK1/2 phosphorylation, cell-cycle regulation, and MMP-9 expression in TNF--induced VSMC.
We previously reported findings relative to signaling pathways, cell-cycle regulation, and MMP-9 expression in TNF-–induced VSMC proliferation (8,23,42). In the present study, we report on the effect of RV on the ERK1/2 signaling pathway, cell-cycle regulation, and MMP-9 expression in TNF-–treated VSMC.
MATERIALS AND METHODS
Materials.
RV and dimethyl sulfoxide (DMSO) were purchased from Sigma. RV was dissolved in DMSO. TNF- was obtained from R&D systems. Polyclonal antibodies to cyclin D1, cyclin E, CDK2, CDK4, p21/WAF1, p53 and p27 were obtained from New England Biolabs. The polyclonal MMP-9 antibody was obtained from Chemicon.
Cell cultures. Human aortic smooth muscle cells (VSMC) were purchased from Bio-Whittaker and cultured in smooth muscle cell growth medium-2 containing 10% fetal bovine serum, 2 µg/L human basic fibroblast growth factor, 0.5 µg/L human epidermal growth factor, 50 mg/L gentamicin, 50 mg/L amphotericin-B, and 5 mg/L bovine insulin.
[3H]thymidine incorporation. VSMC, grown to near confluence in 24-well tissue culture plates, were made quiescent and treated with RV, as indicated. The [3H]thymidine incorporation experiment was performed as described previously (8).
Cell-cycle analysis. Cells were harvested and fixed in 70% ethanol and stored at –20°C. They were then washed twice with ice-cold PBS and incubated with RNase and the DNA intercalating dye, propidium iodide, and a cell-cycle phase analysis was performed by flow cytometry using a Becton Dickinson Facstar flow cytometer and the Becton Dickinson cell fit software.
Immunoprecipitation, Western blotting, and immune complex kinase assays.
Growth-arrested VSMC were treated with TNF- in the presence or absence of RV for specified time periods at 37°C. Cell lysates were prepared, and immunoprecipitation, Western blotting, and immune complex kinase assays were performed as described previously (8,19,23,42).
Zymography. The conditioned medium was electrophoresed in a polyacrylamide gel containing 1 g/L gelatin. The gel was then washed at room temperature for 2 h with 2.5% Triton X-100 and then at 37°C overnight in a buffer containing 10 mmol/L CaCl2, 150 mmol/L NaCl, and 50 mmol/L Tris-HCl, pH 7.5. The gel was stained with 0.2% Coomassie blue and photographed on a light box. Proteolysis was detected as a white zone in a dark blue field.
Creation of p21/WAF1WAF1 promoter reporter constructs.
The human p21/WAF1 promoter construct, WWW-luc (p21/WAF1P), was a gift from Dr. Bert Vogelstein (43). p21/WAF1P 
2.3 was described by Datto et al. (44).
Creation of MMP-9 promoter reporter construct and transient transfection. A 0.7-kb segment (pGL2-MMP-9WT) at the 5'-flanking region of the human MMP-9 gene was amplified by PCR using specific primers from the human MMP-9 gene (Accession No. D10051): 5'-ACATTTGCCCGAGCTCCTGAAG (forward/SacI) and 5'-AGGGGCTGCCAGAAGCTTATGGT (reverse/Hind III) (8,23).
Each plasmid was transfected into VSMC using the Superfect reagent (Qiagen) according to the manufacturer’s instructions (8,23). The luciferase activity was tested using a luciferase assay system (Promega) according to the manufacturer’s instructions. Firefly luciferase activities were standardized for ß-galactosidase activity.
Nuclear extracts and electrophoretic mobility shift assay. Nuclear extracts were prepared essentially as described elsewhere (8,19,23,42). The electrophoretic mobility shift assay was performed as described previously (8,19,23,42).
Statistical analysis. Data are expressed as means ± SE unless stated otherwise. Data were analyzed by factorial ANOVA and Fisher’s least significant difference test when appropriate. Differences were considered significant at P < 0.05.
RESULTS
RV inhibits VSMC proliferation.
TNF- stimulated the proliferation of VSMC, as measured by DNA synthesis, and the proliferation was significantly inhibited by RV in a dose-dependent manner (Fig. 1A). The complete inhibition of thymidine incorporation occurred at a dose of 50 µmol/L. RV alone or vehicle (DMSO) did not affect basal levels of thymidine incorporation (Fig. 1A). TNF- stimulation of vehicle did not affect thymidine incorporation. Moreover, the rate of thymidine incorporation into VSMC was inhibited in a time-dependent manner (Fig. 1B). After 24 h, RV (50 µmol/L) greatly reduced the rate of thymidine incorporation. TNF-–induced ERK1/2 activation was inhibited by RV in a concentration-dependent manner (10–50 µmol/L) (Fig. 1C).
|
|
|
increased CDK2 and CDK4 activities. RV inhibited the kinase activities of both the CDK2- and CDK4- immunoprecipitates in a dose-dependent manner at 24 h (Fig. 3B).
RV-induced cell-cycle arrest is associated with the upregulation of the CKI, p21/WAF1.
We next assessed the effect of RV on the induction of p21/WAF1, which regulates the entry of cells at the G1-S phase transition checkpoint (13,14). An immunoblot analysis revealed that RV treatment of the TNF-–induced VSMC resulted in a significant dose-dependent induction in p21/WAF1 compared with the basal levels (Fig. 4A). RV did not affect the inhibition of p27 induced by TNF-. However, under similar experimental conditions, the levels of expression of p53 tumor suppressor protein were unaffected, suggesting that it is unlikely that p27 and p53 are involved in the cell-cycle arrest induced by RV (Fig. 4A).
|
stimulation (Fig. 4B). Luciferase activity was increased as the result of TNF- treatment (Fig. 4C). Collectively, these results demonstrate that TNF- induces the expression of p21/WAF1 protein and activates its promoter activity.
p21/WAF1, one of the direct downstream targets of p53, activates p21/WAF1 via its interaction with 2 p53 binding sites residing on the p21/WAF1P promoter (Fig. 4C) (45,46). The deletion mutant p21/WAF1P2.3, which is devoid of p53 binding sites (Fig. 4C), exhibited a modestly reduced TNF- response but had no effect on the extent of TNF- induction relative to basal or TNF-–induced activity (Fig. 4C). This indicates that RV is capable of inducing p21/WAF1, independently of the p53 pathway. This TNF-–stimulated p21/WAF1 promoter activity was increased after the treatment of VSMC with RV (Fig. 4C). In addition, the luciferase activity of the deletion mutant p21/WAF1P2.3 remained unchanged, compared with p21/WAF1P (Fig. 4C), suggesting that RV induces p21/WAF1 induction in VSMC via a p53-independent pathway.
RV inhibits TNF--induced MMP-9 expression.
Media from control smooth muscle cells did not demonstrate any proteolytic activity at 92 kDa, corresponding to MMP-9. In contrast, treatment with 100 µg/L of TNF- induced the expression of proteolytic MMP-9 activity, as evidenced by the presence of a band. This induction of MMP-9 activity by TNF- was inhibited in the presence of RV in a dose-dependent manner. Similar results were found for the lysates and immunoblot results (Fig. 5). These data indicate that RV inhibited the TNF-–stimulated increase in MMP-9 activity. In addition, under similar zymographic experimental conditions, the level of expression of proteolytic MMP-2 activity was also significantly reduced (Fig. 5).
|
B and AP-1 binding activities.
To better understand the mechanism that is operative in the decrease in MMP-9 expression, we examined MMP-9 promoter activity before and after TNF- stimulation in the absence or presence of RV. In a previous study, we found that the minimal response elements, NF-B and AP-1, for TNF- stimulation are located in the region –710 bp upstream of the transcription start site in VSMC (8,23). Therefore, we used a plasmid containing a luciferase reporter gene driven by a 710-bp segment from the 5'-promoter region of the human MMP-9 gene to examine TNF--mediated MMP-9 promoter activation in the absence or presence of RV. VSMC were transiently transfected with the pGL2-MMP-9WT plasmid and subsequently treated with TNF- for 24 h. TNF- strongly increased the reporter activity, which can be attributed to the MMP-9 promoter sequence in VSMC (Fig. 6A). In addition, this TNF--stimulated MMP-9 promoter activity was reduced by >90% after the treatment of VSMC with RV, suggesting that the repressive effect of the RV is due, at least in part, to the reduced transcription of the MMP-9 gene. To determine whether the repressive effect of RV on MMP-9 expression was mediated through these 2 types of motifs, NF-B and AP-1 cis-elements, an electrophoretic mobility shift assay (EMSA) was performed on nuclear extracts of VSMC after treatment with TNF- in the absence or presence of RV. Nuclear extracts from VSMC treated with TNF- had increased binding to the NF-B and AP-1 motifs (Fig. 6B). Thus, RV effectively suppressed the increased NF-B and AP-1 binding activities (Fig. 6B). These data suggest that RV blocks MMP-9 expression, at least in part, by decreasing the binding of transcription factors NF-B and AP-1 to DNA.
|
DISCUSSION
The uptake of thymidine, as an index of DNA synthesis in TNF-–stimulated VSMC after RV treatment, indicated that a cessation in DNA synthesis occurred. Recent studies clarified the ability of the cytokine TNF- to activate the ERK1/2 signaling pathway (6,7,8,42). We then examined the effect of RV on the early signal transduction pathway (ERK 1/2) by TNF- stimulation. Consistent with the cell proliferation study (Fig. 1A, B), RV treatment inhibited the ERK signaling pathway in TNF-–stimulated VSMC. These results suggest that RV may have an antiproliferative effect on VSMC by inhibiting TNF-–induced ERK 1/2 activation. Although our observations in this experiment differed from those of an earlier report, indicating that serum-induced ERK1/2 activation was not inhibited after RV treatment (40), other findings showed that RV inhibits angiotensin II–induced ERK1/2 activity (41). Thus, these findings may explain the stimulant-dependent differences in the inhibition of ERK1/2 activity on VSMC after RV.
Our data also show that RV led to G1 cell-cycle arrest, compared with TNF-–stimulated VSMC. This is consistent with other studies showing that RV induces G1 cell-cycle arrest in several cell lines (38,47). To further elucidate the cell-cycle regulation underlying the action of RV on VSMC, we investigated the involvement of the CKI-cyclin-CDK machinery during the induction of cell-cycle arrest by RV in VSMC. Because our studies demonstrated that RV treatment of VSMC resulted in the G1-phase arrest of the cell cycle (Fig 2C), we examined the effect of RV on cell-cycle regulatory molecules that are operative in the G1 phase of the cell cycle. The G1 to S cell-cycle progression is controlled by several CDK complexes, including cyclinD1/CDK4 and cyclinE/CDK2; the activities of these complexes are dependent on the balance of cyclins and CDK inhibitors (CKIs), such as p27 and p21. To determine whether RV-induced cell growth inhibition is due to the downregulation of cyclins and CDKs or the upregulation of CKIs, we then analyzed the expression of these cell-cycle regulators in VSMC after RV treatment. Our experiment indicated that the treatment of VSMC with RV significantly downmodulated cyclinD1/CDK4 and cyclinE/CDK2, although to different extents. RV treatment dose dependently inhibited kinase activities associated with all of the CDKs examined.
Our data demonstrated a significant upregulation in the p21waf, CKI, during G1-phase arrest of VSMC by RV. Interestingly, however, treatment with RV did not affect the inhibition of p27 after TNF-. Many studies showed that the regulation of G1 cell-cycle arrest can be attributed to a number of cellular proteins, including p53 (45). However, RV had no effect on p53 protein levels in VSMC, as determined by immunoblot analysis and a promoter assay, suggesting that the RV-induced accumulation of p21 could also be responsible for G1-phase arrest.
The action of MMP has emerged recently as an important component of the natural history of atherosclerosis (24,25,48), and of the vascular response to injury (24,25). Of considerable interest in this study was the marked decrease in the secretion of MMP-9 activity from TNF-–stimulated VSMC in response to RV. Consistent with the zymography and immunoblot analyses, our data showed that MMP-9 promoter activity is effectively suppressed by RV. Finally, using consensus AP-1 and NF-B probes, there was a marked decrease in both AP-1 and NF-B binding activities in response to TNF- in VSMC after treatment with RV (Fig. 6). A few studies of the inhibition of cell invasion and MMP expression in cancer cell lines have appeared (49,50). However, the molecular and cellular mechanisms underlying the inhibition of MMP expression by RV in VSMC have not been examined. To our knowledge, this is the first systematic study demonstrating the inhibition of AP-1 and NF-B binding activities by RV in TNF-–induced MMP-9 expression. Our study clearly revealed that the ability of RV to reduce MMP-9 expression in VSMC is achieved via a reduction in NF-B and AP-1 binding as well as diminished transactivation of the MMP-9 promoter. The antiatherogenic effects of RV appear to be mediated through the transcriptional downregulation of MMP-9; this effector molecule was implicated in regulating the progression of plaque rupture (25). However, a limitation in our study is that the mechanism of MMP-2 on RV-treated VSMC has not been clarified. The exact mechanism requires elucidation.
The present study provides some important new insights into the molecular mechanisms of action of RV in VSMC (Fig. 7). Our results clearly show that RV influences important TNF-–activated pathways in VSMC to a substantial extent. First, they suggest that RV acts via the ERK1/2 pathway to reduce TNF-–mediated cell proliferation. In addition, RV arrests the cell cycle at the G1 phase in VSMC and the RV-induced VSMC arrest can be attributed to the inhibition of cyclin D1/CDK4 and cyclin E/CDK2 complexes by the increased expression of p21/WAF1 without altering p53 protein levels. Moreover, RV stimulates p21/WAF1 gene promoter activity, suggesting that p21/WAF1 gene induction is mediated through the activation of its promoter in a p53-independent manner. Furthermore, RV potently inhibits TNF-–induced MMP-9 expression by suppressing NF-B and AP-1 binding activities. The findings of the present study may explain in part the therapeutic benefits of red wine on cardiovascular diseases that were observed in several clinical studies.
|
ACKNOWLEDGMENTS
We thank Sook-Nyeo Han and Se-Jeong Lee for their kind assistance in the above experiments.
FOOTNOTES
2 Abbreviations used: AP-1, activator protein-1; CDK, cyclin-dependent kinase; DMSO, dimethyl sulfoxide; EMSA, electrophoretic mobility shift assay; ERK, extracellular signal-regulated kinase; MMP, matrix metalloproteinase; NF-B, nuclear factor-B; RV, resveratrol; TNF, tumor necrosis factor; VSMC, vascular smooth muscle cell. 
Manuscript received 13 July 2005. Initial review completed 4 August 2005. Revision accepted 19 September 2005.
LITERATURE CITED
1. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993;362:801-809.[Medline]
2. Abedi H, Zachary I. Signalling mechanisms in the regulation of vascular cell migration. Cardiovasc Res. 1995;30:544-556.[Medline]
3. Jovinge S, Hultgardh-Nilsson A, Regnstrom J, Nilsson J. TNF-alpha activates smooth muscle cell migration in culture and is expressed in the balloon-injured rat aorta. Arterioscler Thromb Vasc Biol. 1997;17:490-497.
4. Tipping PG, Hancock WW. Production of tumor necrosis factor and interleukin-1 by macrophages from human atherosclerotic plaques. Am J Pathol. 1993;142:1721-1728.[Abstract]
5. Clausell N, Lima VC, Molossi S. Expression of tumor necrosis factor alpha and accumulation of fibronectin in coronary artery restenotic lesions retrieved by atherectomy. Br Heart J. 1995;73:534-539.
6. Goetze S, Xi XP, Kawano Y. TNF--induced migration of vascular smooth muscle cells is MAPK-dependent. Hypertension. 1999;33:183-189.
7. Goetze S, Kintscher U, Kaneshiro K, Meehan WP, Collins A, Fleck E, Hsueh WA, Law RE. TNF- induces expression of transcription factors c-fos, Egr-1, and Ets-1 in vascular lesions through extracellular signal-regulated kinases 1/2. Atherosclerosis. 2001;159:93-101.[Medline]
8. Moon SK, Cho GO, Jung SY, Gal SW, Kwon TK, Lee YC, Madamanchi NR, Kim CH. Quercetin exerts multiple inhibitory effects on vascular smooth muscle cells: role of ERK1/2, cell-cycle regulation, and matrix metalloproteinase-9. Biochem Biophys Res Commun. 2003;301:1069-1078.[Medline]
9. Sherr CJ. G1 phase progression: cycling on cue. Cell. 1994;79:551-555.[Medline]
10. Sherr CJ. Cancer cell cycles. Science. 1996;274:1672-1677.
11. Collins K, Jacks T, Pavletich NP. The cell cycle and cancer. Proc Natl Acad Sci U S A. 1997;94:2776-2778.
12. Jacks T, Weinberg RA. Cell-cycle control and its watchman. Nature. 1996;381:643-644.[Medline]
13. Xiong Y, Hannon GJ, Zhang H, Casso D, Kobayashi R, Beach D. p21 is a universal inhibitor of cyclin kinases. Nature. 1993;366:701-704.[Medline]
14. Toyoshima H, Hunter T. p27, a novel inhibitor of G1 cyclin-cdk protein kinase activity, is related to p21. Cell. 1994;78:67-74.[Medline]
15. Weiss RH, Joo A, Randour C. p21(Waf1/Cip1) is an assembly factor required for platelet-derived growth factor-induced vascular smooth muscle cell proliferation. J Biol Chem. 2000;275:10285-10290.
16. LaBaer J, Garrett MD, Stevenson LF, Slingerland JM, Sandhu C, Chou HS, Fattaey A, Harlow E. New functional activities for the p21 family of CDK inhibitors. Genes Dev. 1997;11:847-862.
17. Zhang H, Hannon GJ, Beach D. p21-containing cyclin kinases exist in both active and inactive states. Genes Dev. 1994;8:1750-1758.
18. Zhan Y, Kim S, Izumi Y, Izumiya Y, Nakao T, Miyazaki H, Iwao H. Role of JNK, p38, and ERK in platelet-derived growth factor-induced vascular proliferation, migration, and gene expression. Arterioscler Thromb Vasc Biol. 2003;23:795-801.
19. Moon SK, Jung SY, Choi YH, Lee YC, Kim CH. Platelet-derived growth factor induces p21/WAF1 promoter in vascular smooth muscle cells via activation of an Sp1 site. FEBS Lett. 2003;552:130-134.[Medline]
20. Galis ZS, Muszynski M, Sukhova GK, Simon-Morrissey E, Unemori EN, Lark WW, Amento E, Libby P. Cytokine-stimulated human vascular smooth muscle cells synthesize a complement of enzymes required for extracellular matrix digestion. Circ Res. 1994;75:181-189.
21. Fabunmi RP, Baker AH, Murray EJ, Booth RFG, Newby AC. Divergent regulation by growth factors and cytokines of 95 kDa and 72 kDa gelatinases and tissue inhibitors or metalloproteinases-1, -2, and -3 in rabbit aortic smooth muscle cells. Biochem J. 1996;315:335-342.[Medline]
22. Cho A, Graves J, Reidy MA. Mitogen-activated protein kinases mediate matrix metalloproteinase-9 expression in vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 2000;20:2527-2532.
23. Moon SK, Cha BY, Kim CH. ERK1/2 mediates TNF-alpha-induced matrix metalloproteinase-9 expression in human vascular smooth muscle cells via the regulation of NF-kappaB and AP-1: involvement of the ras dependent pathway. J Cell Physiol. 2004;198:417-427.[Medline]
24. Ross R. Atherosclerosis—an inflammatory disease. N Engl J Med. 1999;340:115-126.
25. Newby AC, Zaltsman AB. Molecular mechanisms in intimal hyperplasia. J Pathol. 2000;190:300-309.[Medline]
26. Cho A, Reidy MA. Matrix metalloproteinase-9 is necessary for the regulation of smooth muscle cell replication and migration after arterial injury. Circ Res. 2002;91:845-851.
27. Galis ZS, Johnson C, Godin D, Magid R, Shipley JM, Senior RM, Ivan E. Targeted disruption of the matrix metalloproteinase-9 gene impairs smooth muscle cell migration and geometrical arterial remodeling. Circ Res. 2002;91:852-859.
28. Schneider Y, Duranton B, Gosse F, Schleiffer R, Seiler N, Raul F. Resveratrol inhibits intestinal tumorigenesis and modulates host-defense-related gene expression in an animal model of human familial adenomatous polyposis. Nutr Cancer. 2001;39:102-107.[Medline]
29. Soleas GJ, Diamandis EP, Goldberg DM. The world of resveratrol. Adv Exp Med Biol. 2001;492:159-182.[Medline]
30. 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;8:3-17.[Medline]
31. Gusman J, Malonne H, Atassi G. A reappraisal of the potential chemopreventive and chemotherapeutic properties of resveratrol. Carcinogenesis. 2001;22:1111-1117.
32. Bhat KP, Kosmeder JW, Pezzuto JM. Biological effects of resveratrol. Antioxid Redox Signal. 2001;3:1041-1064.[Medline]
33. Jang M, Cai L, Udeani GO, Slowing KV, Thomas CF, Beecher CWW, Fong HHS, Farnsworth NR, Kinghorn AD, Mehta RG, Moon RC, Pezutto JM. Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Science. 1997;275:218-220.
34. Bertilli AA, Giovannini L, Gianessi D, Migliori M, Bernini W, Fregoni M, Bertilli A. Antiplatelet activity of synthetic and natural resveratrol in red wine. Int J Tissue React. 1995;17:1-3.[Medline]
35. Uenobe F, Nakamura S, Miyazama M. Antimutagenic effect of resveratrol against Trp-P-1. Mutat Res. 1997;373:197-200.[Medline]
36. Mnjoyan ZH, Fujise K. Profound negative regulatory effects by resveratrol on vascular smooth muscle cells: a role of p53–p21(WAF1/CIP1) pathway. Biochem Biophys Res Commun. 2003;311(2):546-552.[Medline]
37. Araim O, Ballantyne J, Waterhouse AL, Sumpio BE. Inhibition of vascular smooth muscle cell proliferation with red wine and red wine polyphenols. J Vasc Surg. 2002;35(6):1226-1232.[Medline]
38. Zou J, Huang Y, Chen Q, Wang N, Cao K, Hsieh TC, Wu JM. Suppression of mitogenesis and regulation of cell cycle traverse by resveratrol in cultured smooth muscle cells. Int J Oncol. 1999;15(4):647-651.[Medline]
39. Zou J, Huang Y, Cao K, Yang G, Yin H, Len J, Hsieh TC, Wu JM. Effect of resveratrol on intimal hyperplasia after endothelial denudation in an experimental rabbit model. Life Sci. 2000;68:153-163.[Medline]
40. 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(4):925-932.
41. 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(4):772-777.
42. Moon SK, Kim HM, Lee YC, Kim CH. Disialoganglioside (GD3) synthase gene expression suppresses vascular smooth muscle cell responses via the inhibition of ERK1/2 phosphorylation, cell cycle progression, and matrix metalloproteinase-9 expression. J Biol Chem. 2004;279(32):33063-33070.
43. El-Deiry WS, Harper JW, O’Connor PM, Velculescu VE, Canman CE, Jackman J, Pietenpol JA, Burrell M, Hill DE, Wang Y, Wiman KG, Mercer WE, Kastan MB, Kohn KW, Elledge SJ, Kinzler KW, Vogelstein B. WAF1/CIP1 is induced in p53-mediated G1 arrest and apoptosis. Cancer Res. 1994;54:1169-1174.
44. Datto MB, Yu Y, Wang XF. Functional analysis of the transforming growth factor beta responsive elements in the WAF1/Cip1/p21 promoter. J Biol Chem. 1995;270:28623-28628.
45. Macleod K, Sherry N, Hannon G, Beach D, Tokino T, Kinzler K, Vogelstein B, Jacks T. p53-dependent and independent expression of p21 during cell growth, differentiation, and DNA damage. Genes Dev. 1995;9:935-944.
46. Hu PP, Shen X, Huang D, Liu Y, Counter C, Wang XF. The MEK pathway is required for stimulation of p21(WAF1/CIP1) by transforming growth factor-beta. J Biol Chem. 1999;274:35381-35387.
47. Hsieh T, Halicka D, Lu X, Kunicki J, Guo J, Darzynkiewicz Z, Wu J. Effects of resveratrol on the G(0)-G(1) transition and cell cycle progression of mitogenically stimulated human lymphocytes. Biochem Biophys Res Commun. 2002;297(5):1311-1317.[Medline]
48. Bond M, Fabunmi RP, Baker AH, Newby AC. Synergistic upregulation of metalloproteinase-9 by growth factors and inflammatory cytokines: an absolute requirement for transcription factor NF-kappa B. FEBS Lett. 1998;435:29-34.[Medline]
49. Woo JH, Lim JH, Kim YH, Suh SI, Min do S, Chang JS, Lee YH, Park JW, Kwon TK. Resveratrol inhibits phorbol myristate acetate-induced matrix metalloproteinase-9 expression by inhibiting JNK and PKC delta signal transduction. Oncogene. 2004;23(10):1845-1853.[Medline]
50. Li YT, Shen F, Liu BH, Cheng GF. Resveratrol inhibits matrix metalloproteinase-9 transcription in U937 cells. Acta Pharmacol Sin. 2003;24(11):1167-1171.[Medline]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
, increased activity; 
, decreased activity. Arrows outside targets indicate positive inputs.