Migration of vascular smooth muscle cells (VSMCs) from the media to the intima is thought to be a key event in the development of atherosclerosis and post-angioplasty vascular remodeling.1,2 In the intima, these SMCs assume a synt hetic phenotype, resulting in the deposition of collagen within the neointimal tissue.3 The mechanisms leading to the migration, proliferation, and collagen synthesis by VSMCs are not fully understood. Growth factors and reactive oxygen species (ROS) produced during vascular injury are thought to play a major role in this process.
The importance of the growth factors such as the platelet-derived growth factor (PDGF) in the development of neointima has been established in arterial injury models.4 PDGF produced by platelets, VSMCs, and endothelial cells during vascular injury activates a variety of intracellular signal transduction pathways leading to VSMC proliferation, migration, and collagen synthesis.5 ROS produced during vascular injury can also act as second messengers as a part of signaling mediated by growth factors.6 Consequently, inhibition of growth factor or ROS-mediated signaling may represent a potential therapeutic strategy for interference with the progression of atherosclerosis and restenosis.
We have recently reported that curcumin [Figure 1; diferuloyl methane; 1,7-bis(4-hydroxy-3-methoxy-phenyl)-1,6-heptadiene-3-5-dione)], the major yellow pigment extracted from spice turmeric (Curcuma longa) is a potent inhibitor of PDGF-stimulated migration, proliferation, and collagen synthesis in VSMCs.7 Perivascular delivery of curcumin significantly attenuated carotid artery neointima formation after balloon catheter injury,7 indicating that curcumin may protect against vascular injury by inhibiting key PDGF-stimulated VSMC functions.
Dehydrozingerone, 4-(4-hydroxy-3-methoxyphenyl)-3-buten-2-one isolated from Ginger (Zingiber officinale), is a biosynthetic intermediate of curcumin. Structurally, dehydrozingerone is a half-analog of curcumin - curcumin comprises two molecules of dehydrozingerone linked together with a methylene bridge (Figure 1). Dehydrozingerone has been found to possess antioxidant and antimutagenic properties.8,9 However, the role of dehydrozingerone on growth factor signaling in VSMC has not been reported. Thus, the objective of this study was to investigate the effect of dehydrozingerone on PDGF-stimulated VSMC migration, proliferation, and collagen synthesis. Further, in an attempt to understand the mechanism, we studied the effect of dehydrozingerone on hydrogen peroxide (H2O2)-stimulated PDGF receptor signaling. Growth factor-mediated cell proliferation is negatively regulated by protein tyrosine phosphatases (PTPs); therefore, we also assessed the effect of dehydrozingerone on PTP activity in cells treated with H2O2. Finally, to understand the structural requirements for activity, we compared dehydrozingerone with curcumin and isoeugenol; the latter is an active component of clove with similar structural features to dehydrozingerone (Figure 1).
MATERIALS AND METHODS
Commercial curcumin contains amounts of the isomers demethoxycurcumin and bisdemethoxycurcumin thus rendering it impure; hence, pure curcumin (Figure 1) was synthesized by condensing vanillin with acetyl acetone as a boron complex as previously reported.10 The purity and the chemical structure were confirmed by melting point, elemental analysis, and spectral studies. Dehydrozingerone, isoeugenol, and PDGF-BB were obtained from Sigma Chemical Co. Phospho-PDGF receptor β (Tyr751), PDGF receptor β (28E1), phospho-Akt (Ser473), and pan Akt were obtained from Cell Signaling Technology (Boston, MA).
Smooth Muscle Cell Isolation and Culture
Thoracic aorta SMCs were obtained from male Sprague-Dawley rats weighing between 100 and 150 g as previously described using collagenase and elastase.11 This protocol was approved by the Institutional Animal Care and Use Committee of the University of Wyoming in accordance with the Guide for the Care and Use of Laboratory Animals (Department of Health and Human Services, publication No. NIH 86-23). Each isolate consisted of cells pooled from 2 to 4 individual aortas. All experiments were performed using early cell passages (less than 5) of VSMCs. All cultures were maintained in a humidified atmosphere of 5% CO2-95% air. Isolated cells were seeded on tissue culture plates in DMEM/F-12 media supplemented with penicillin (50 U/ml), streptomycin (50 mg/ml), insulin (5 mg/ml), transferrin (5 mg/ml), and selenous acid (5 ng/ml) at a density of 2-3 × 104 cells/cm2 and were grown to confluence in 10% fetal bovine serum. Cultures were treated with serum-free medium for 24 hours before experiments were initiated. Subcultured cells were derived from primary cultures via detachment of cells with trypsin, replating at lower densities, and growth to confluence as described.
Cells were seeded in 24-well tissue culture plates and were allowed to adhere for 24 hours. Cells were treated for 48 hours with ethanol (control) or with 10, 25, and 50 μM dehydrozingerone. Triplicate samples were used for each treatment. Viable cells were counted by trypan blue exclusion assay.
Cell Migration Assay
Two types of cell migration assays were used. In the first, modified Boyden transwell chambers were used for monitoring cell migration as described by us previously.12 Cells (35,000 cells/well) were seeded onto the apical (upper) chamber of the transwell, and the lower chamber contained the experimental reagents. Cells were allowed to migrate for 6 hours, after which the inserts were removed. Nonmigrating cells in the upper chamber were removed, and cells in the bottom membrane were fixed with 3.7% formaldehyde for 10 minutes and stained with 0.4% hematoxylin for 5 minutes. The number of migrated cells was measured by counting the number of stained nuclei from 4 randomly chosen high-power (400×) fields.
In the second assay, migration was measured using a monolayer-wounding protocol in which cells migrated from a confluent area into an area that was mechanically denuded of cells.11 For monolayer-wounding cell migration assay, confluent cells were treated with serum-free medium containing hydroxyurea (5 mmol/L) for 24 hours before the start of the experiments. Hydroxyurea was used to prevent proliferation of cells13 to eliminate potential effects dehydrozingerone on cell proliferation. Hydroxyurea by itself did not have any effect on cell migration (data not shown). Area of migration was photographed with a video camera system using Scion Image software at the intersection of the previously marked line and wound edge at 0 hours (WW0) and at 24 hours (WW24) after treatment with PDGF (10 ng/mL) in the presence or absence of dehydrozingerone. Cell migration was calculated as wound width covered at time t(WW0 - WW24) and expressed as percentage.
Cell Proliferation Assay
Cell proliferation was assessed by [3H]-thymidine incorporation in mitogenically quiescent VSMCs.12 Cells were incubated for 18 hours with or without PDGF (10 ng/mL) and various concentrations of dehydrozingerone and then pulse-labeled with 1μCi/mL of [3H] thymidine for 6 hours. Cells were washed 3 times with phosphate-buffered saline, precipitated with 10% (wt/vol) ice-cold trichloroacetic acid, and rinsed with absolute ethanol and air dried. For analysis, the monolayer was dissolved in 250 mL of 0.5 M sodium hydroxide per well at room temperature overnight. Duplicate samples of 100 mL were counted in scintillation fluid in a liquid scintillation counter (Beckmann LC 6000IC). A second aliquot was used for the determination of protein content via the Bradford assay (BioRad Laboratories Inc., Hercules, CA) per the manufacturer's specifications.
Collagen synthesis was determined by measuring [3H]L-proline incorporation.7 Collagen synthesis was initiated by treating quiescent VSMCs with [3H]-L-proline (5 μCi/mL) in the presence or absence of PDGF and dehydrozingerone. Cells were processed in a similar manner as that described for the proliferation assay for determining the radioactivity.
SDS-PAGE and Immunoblotting
Western blotting for protein was performed as described previously.7 Cells were lysed in RIPA buffer containing 1 mM sodium vanadate, 1 mM phenyl-methyl-sulfonyl fluoride, 5 mg/ml aprotinin, and 5 mg/ml leupeptin. Protein concentration was determined by the bicinchoninic acid method (Pierce Biotechnology Inc., Rockford, IL). Lysates corresponding to equal amounts of proteins were boiled in Laemmli sample buffer, and the supernatants were loaded onto gels for SDS-PAGE. Proteins were transferred onto nitrocellulose membranes and probed with the following primary antibodies: anti-phospho PDGF receptor and anti-phospho Akt (Thr308) (at dilutions of 1:1000). Appropriate horseradish peroxidase-coupled secondary antibodies (HRPO-conjugated) were used at 1:5000. Immunoreactive bands were visualized using Renaissance chemiluminescence reagents. Films were scanned, and the intensity of immunoblot bands was detected with a BioRad Calibrated Densitometer (Model: GS-800).
Subconfluent VSMCs were serum-starved for 24 hours and stimulated with H2O2 (0.1 mM) for 5 minutes in the presence or absence of dehydrozingerone. Cells were washed twice with ice-cold phosphate buffered saline (without calcium or magnesium) and lysed in RIPA buffer (without phosphatase inhibitor orthovanadate). After adjustment for protein concentration, tissue lysates were subjected to phosphatase activity measurement using a phosphate assay kit from Upstate Biotech per the manufacturers' specifications.
Analysis of variance (ANOVA) followed by Fischer post-hoc test were performed for statistical analysis. Data were expressed as mean ± standard error of the mean (SEM). P < 0.05 was considered to be statistically significant for all comparisons.
Dehydrozingerone Inhibits PDGF-stimulated VSMC Migration
To assess the role of dehydrozingerone in VSMC migration, we used two types of cell migration assays: a transwell migration assay and the monolayer-wounding assay. In the transwell migration assay (Figure 2A) that represents chemotaxis, PDGF (10 ng/mL) enhanced basal migration of VSMCs by about 5-fold (basal migration 3.1 ± 0.4 cells/mm2 versus 14.8 ± 2.0 cells/mm2 after PDGF stimulation). Treatment with dehydrozingerone (1 to 50 μM) resulted in a concentration-dependent inhibition of cell migration with statistical significance achieved at 10 mM (9.0 ± 1.2 cells/mm2, P < 0.05) concentration of dehydrozingerone. At 50 μM concentration of dehydrozingerone, an almost complete inhibition of PDGF-stimulated VSMC migration was achieved.
Similar results were obtained in the cell monolayer-wounding assay that represents chemokinesis (Figure 2B). Treatment with PDGF (10 ng/mL) for 24 hours caused the cells to migrate to cover about 90% of the wound width. This robust cell migration induced by PDGF was inhibited by dehydrozingerone in a concentration-dependent manner with almost complete inhibition observed at a concentration of 50 μM dehydrozingerone.
Cell viability analyzed with or without dehydrozingerone demonstrated that dehydrozingerone did not affect cell viability at these concentrations (Figure 3).
Dehydrozingerone Inhibits PDGF-stimulated VSMC Proliferation and Collagen Synthesis
We next determined the ability of dehydrozingerone to inhibit PDGF-stimulated VSMC proliferation and collagen synthesis. In the [3H]-thymidine incorporation assay (Figure 4A), stimulation with PDGF (10 ng/mL) increased cell proliferation by about 5-fold. Dehydrozingerone (1 to 50 μM) inhibited PDGF-stimulated cell proliferation in concentration-dependent manner, with 73% inhibition observed at 50 μM dehydrozingerone. When quiescent cells were treated with dehydrozingerone (1 to 50 μM) for 24 hours in the absence of PDGF, no significant difference (from control) was observed in the extent of 3[H]-thymidine incorporation, suggesting that dehydrozingerone did not significantly cause cytotoxicity to the cells.
VSMCs are thought to assume a synthetic phenotype (as against the normal contractile phenotype) after vascular injury3; we therefore assessed the effect of dehydrozingerone on PDGF-stimulated [3H]L-proline incorporation in VSMCs. As seen in Figure 4B, treatment with dehydrozingerone (1 to 50 μM) inhibited PDGF-stimulated [3H]L-proline incorporation in a concentration-dependent manner; at 50 μM concentration, dehydrozingerone exhibited 81% inhibition of collagen synthesis in VSMCs.
Dehydrozingerone Attenuates PDGF-stimulated Tyrosine Phosphorylation of PDGFR β and Akt
Given the inhibition of VSMC function by dehydrozingerone, we next analyzed the effect of dehydrozingerone on the signal transduction pathways mediated by PDGF. As anticipated, stimulation of VSMCs with PDGF (10 ng/mL) resulted in robust phosphorylation of PDGFR β (Figure 5A). Treatment with dehydrozingerone significantly reduced the PDGF-stimulated receptor phosphorylation in a concentration-dependent manner. Neither treatment with PDGF nor dehydrozingerone altered the protein levels of the PDGFR β. In a similar manner, dehydrozingerone blocked the capacity of PDGF to induce phosphorylation (Ser473) of its downstream effector Akt without altering the protein levels of Akt (Figure 5B).
Dehydrozingerone Attenuates H2O2-stimulated Tyrosine Phosphorylation of PDGFR β and Akt
In an attempt to understand the potential mechanisms of inhibitory effects of dehydrozingerone, we next assessed its ability to inhibit ligand-independent transactivation of PDGFR mediated by H2O2 stimulation. ROS such as H2O2 are known to phosphorylate PDGFR β and Akt, thus functioning as a regulator of growth factor signaling.14 Stimulation of VSMCs by H2O2 induced a concentration-dependent phosphorylation of PDGFR β, with a maximum phosphorylation seen with 1 mM concentration of H2O2 (Figure 6). Previous studies have shown that achievement of the intracellular H2O2 concentrations seen after PDGF stimulation requires extracellular concentrations of H2O2 ranging from 1 to 10 mM.14-16 Accordingly, a 1 mM concentration of H2O2 was used to test the effects of dehydrozingerone on H2O2-stimulated phosphorylation of PDGFR β and Akt. Dehydrozingerone inhibited H2O2-stimulated tyrosine phosphorylation of PDGFR β (Figure 7A) and Ser (473) phosphorylation of Akt (Figure 7B) in a concentration-dependent manner without altering the levels of these proteins.
Dehydrozingerone Inhibits H2O2-induced Inactivation of Phosphotyrosine Phosphatase (PTP)
Enhancement of PDGF signaling by H2O2 is attributed in part to the oxidative inactivation of PTPs that negatively regulate growth factor signaling.6 We therefore analyzed whether treatment with dehydrozingerone prevents H2O2-induced inactivation of PTP. As reported previously,16,17 treatment of VSMCs with H2O2 (100 μM) was associated with a significant reduction in PTP activity as seen in Figure 8. In contrast, treatment with dehydrozingerone inhibited H2O2-mediated inactivation of PTP. Dehydrozingerone by itself did not have any effect on PTP activity.
Effect of Dehydrozingerone, Curcumin, and Isoeugenol on PDGF Signaling
We next compared the abilities of molecules structurally similar to dehydrozingerone for their ability to inhibit PDGF signaling in VSMCS. At 50 μM concentrations, both curcumin and dehydrozingerone completely inhibited PDGF-stimulated tyrosine phosphorylation of PDGFR β (Figure 9). In contrast, isoeugenol, a structural analog of dehydrozingerone lacking the carbonyl side-chain (Figure 1) failed to show a statistically significant inhibition in the PDGF-stimulated phosphorylation of PDGFR β at equimolar concentrations. Neither did isoeugenol inhibit H2O2-stimulated phosphorylation of PDGFR β (figure not shown).
In this study, we demonstrate that dehydrozingerone, an active principle of ginger, has potent and dose-dependent inhibitory effects on PDGF-stimulated VSMC proliferation, migration, and collagen synthesis. These effects of dehydrozingerone may be mediated via its inhibition of PDGF-stimulated phosphorylation of PDGFR β and the phosphorylation of downstream effector Akt. Besides agonist-induced phosphorylation of PDGFR, dehydrozingerone also inhibits ROS-stimulated phosphorylation of PDGFR as evidenced by the inhibition of H2O2-stimulated phosphorylation of PDGFR. Blunting of PDGF signaling by dehydrozingerone may be at least partially attributable to the attenuation of H2O2-mediated oxidation of phosphatases in the cells. Inhibition of PDGF signaling by dehydrozingerone is comparable to that of curcumin and is completely lost when the carbonyl group in the side chain is removed, as is the case with isoeugenol, suggesting the importance of the carbonyl group for activity.
PDGF is thought to play a vital role in a large number of diseases involving remodeling of the vascular wall.4 PDGF receptors are expressed at low levels in the arteries of healthy adults. In contrast, its expression in the arteries is upregulated by several folds during endothelial injury after angioplasty or early stage of atherosclerosis.18 Targeting PDGF with anti-PDGF antibody,19 or inhibiting the receptor expression with antisense oligonucleotides 20 have been shown to attenuate neointima development. We have previously shown that curcumin inhibits balloon catheter-induced neointima formation in rat carotid arteries via attenuation of PDGF-mediated signaling.7 These effects of curcumin were in part mediated by its inhibition of PDGF-stimulated migration, proliferation, and collagen synthesis in VSMCs. Here we demonstrate that dehydrozingerone, half the structure of curcumin, is sufficient to cause inhibition of PDGF signaling in VSMCs.
Binding of PDGF to its receptor causes tyrosine phosphorylation of the receptor, which functions as a kinase, inducing tyrosine-phosphorylation of different substrate proteins, including phospholipase C-γ1 (PLC-γ1), phosphatidylinositol 3′-kinase (PI 3′-K), Akt, and extracellular response kinases 1/2 (ERK1/2). Here we show that dehydrozingerone attenuates PDGF-mediated phosphorylation of Akt, which is thought to be a critical downstream mediator of PDGF signal transduction cascade. In a similar manner, dehydrozingerone also inhibited PDGF-stimulated phosphorylation of ERK 1/2 (figure not shown), completely abrogating downstream PDGF signaling.
In our previous studies, we had compared the ability of curcumin to inhibit signals transduced by different growth factors and found that curcumin showed more potent inhibition of PDGF receptor signaling compared to fibroblast growth factor receptor or insulin receptor signaling mediated by the respective ligands.7 Part of this specificity may be attributed to the ability of curcumin to compete with PDGF for its receptors.21 However, competition for the receptor alone could not completely explain the potent inhibitory activity of curcumin on PDGF signal transduction, suggesting that more than one mechanism may be involved in this process.7
Excessive production of reactive oxygen species (ROS) within the arterial wall has been implicated in the pathophysiology of a variety of cardiovascular diseases.22 ROS is also generated in vascular cells in response to growth factor stimulation and are thought to be important regulators of growth factor signal transduction.14 Several studies have demonstrated that H2O2 produced within the vasculature mediate diverse physiological functions such as cell proliferation, differentiation, and migration.23 Phosphorylation of tyrosine residues in proteins in response to growth factor stimulation is governed by reciprocal activities of protein tyrosine phosphatases and protein tyrosine kinases.6 ROS generated during growth factor-mediated stimulation of cells can oxidize a critical cysteine on PTPs, rendering them inactive.24 By inactivating phosphatases, H2O2 appears to promote tyrosine phosphorylation and propagation of signals that mediate cell proliferation and migration. This hypothesis is further supported by the studies that demonstrate enhanced PDGF signaling in transgenic animals lacking phosphatases.25 The present study shows that dehydrozingerone is a potent inhibitor of H2O2-stimulated activation of the PDGF signaling cascade as assessed by estimating the phosphorylation levels of PDGFR β and Akt. Furthermore, treatment of VSMCs with dehydrozingerone inhibited H2O2-mediated inactivation of PTP in these cells. These results suggest that inhibition of the oxidation of the negative regulatory protein PTP may play a role in the inhibitory actions of dehydrozingerone on PDGF-mediated signaling.
Curcumin has interesting structural features (Figure 1), including a diketone system (occurs as a keto-enol tautomer) and the phenoxy and methoxy groups on the benzene rings. Dehydrozingerone is half the analog of curcumin and its biosynthetic intermediate and shares many of the structural features of curcumin. Like curcumin, dehydrozingerone has phenoxy and methoxy groups on the benzene ring and a carbonyl group on the side chain. Isoeugenol, a component of clove is structurally similar to curcumin and dehydrozingerone in that it has the phenoxy and methoxy groups but is devoid of the carbonyl group on the side chain. In an attempt to understand the structural features required for inhibiting PDGF signaling, we compared the effects of curcumin, dehydrozingerone, and isoeugenol on H2O2-stimulated phosphorylation of PDGFR β. At 50 μM concentration, both curcumin and dehydrozingerone completely blocked the H2O2-stimulated phosphorylation of PDGF receptor, whereas isoeugenol failed to have any effect. This suggests that the carbonyl group on the side chain is essential for the inhibition of PDGF signaling by curcumin and derivatives.
Inhibitors of growth factor signaling via varied mechanisms have been shown to successfully combat pathological processes associated with proliferative diseases such as atherosclerosis and cancer. Drugs such as imatinib mesylate, now used clinically as an antitumor agent, were developed with the primary goal of preventing restenosis after coronary angioplasty through inhibition of abnormal PDGF-receptor activation.26 Clinical trials have demonstrated that trapidil, a nonspecific PDGFR-inhibitor can inhibit stenosis caused due to vascular injury.27 The data presented here demonstrate that dehydrozingerone may prove to be a potential therapeutic agent for prevention and possibly treatment of stenotic vascular remodeling and/or contributory mechanisms of atherosclerosis.
Curcumin has been used as a food additive in curry and in indigenous medicine for centuries. Nutritional supplementation with curcumin has been credited with a variety of beneficial effects in chronic diseases.28 Recent studies have shown that curcumin may have beneficial effects in the treatment of cancer,29 Alzheimer's disease,30 and cystic fibrosis.31 However, in vivo bioavailability of curcumin is poor owing to its rapid metabolism and degradation.32 There are no reports available on the bioavailability of dehydrozingerone. Although dehydrozingerone was not as potent as curcumin in inhibiting PDGF-stimulated signaling and functional changes in smooth muscle cells, the lack of the diketone system could render dehydrozingerone more stable, which may be an advantage over curcumin in terms of susceptibility to hydrolysis and therefore bioavailability. If this is true, dehydrozingerone would provide an attractive alternative to curcumin in the treatment of the aforementioned conditions. However, a major shortcoming of this paper is that the data presented here are from in vitro studies only. It is important that these in vitro data be substantiated with in vivo studies before such claims can be made.
In summary, this is the first report to show that dehydrozingerone, half-analog of curcumin, is a potent inhibitor of the PDGF signal transduction a stimulated by PDGF and H2O2. Aortic SMC migration, proliferation, and collagen synthesis, shown here to be inhibited by dehydrozingerone, are crucial events underlying the complications of vascular injury. On the basis of these data, it is tempting to speculate that dehydrozingerone may be of therapeutic value in preventing or treating of vascular diseases.
1. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature
2. Schwartz RS, Holmes DR Jr, Topol EJ. The restenosis paradigm revisited: an alternative proposal for cellular mechanisms. J Am Coll Cardiol
3. Strauss BH, Chisholm RJ, Keeley FW, et al. Extracellular matrix remodeling after balloon angioplasty injury in a rabbit model of restenosis. Circ Res
4. Heldin CH, Westermark B. Mechanism of action and in vivo role of platelet-derived growth factor. Physiol Rev
5. Raines EW. PDGF and cardiovascular disease. Cytokine Growth Factor Rev
6. Rhee SG. Cell signaling. H2
, a necessary evil for cell signaling. Science
7. Yang X, Thomas DP, Zhang X, et al. Curcumin inhibits platelet-derived growth factor-stimulated vascular smooth muscle cell function and injury-induced neointima formation. Arterioscler Thromb Vasc Biol
8. Kuo PC, Damu AG, Cherng CY, et al. Isolation of a natural antioxidant, dehydrozingerone from Zingiber officinale and synthesis of its analogues for recognition of effective antioxidant and antityrosinase agents. Arch Pharm Res
9. Subramanian M, Sreejayan N, Rao MN, et al. Diminution of singlet oxygen-induced Dna damage by curcumin and related antioxidants. Mutat Res
10. Sreejayan N, Rao MN. Curcuminoids as potent inhibitors of lipid peroxidation. J Pharm Pharmacol
11. Sreejayan N, Lin Y, Hassid A. No attenuates insulin signaling and motility in aortic smooth muscle cells via protein tyrosine phosphatase 1B-mediated mechanism. Arterioscler Thromb Vasc Biol
12. Yigzaw Y, Poppleton HM, Sreejayan N, et al. Protein-tyrosine phosphatase-1B (Ptp1b) mediates the anti-migratory actions of Sprouty. J Biol Chem
13. Sarkar R, Meinberg EG, Stanley JC, et al. Nitric oxide reversibly inhibits the migration of cultured vascular smooth muscle cells. Circ Res
14. Sundaresan M, Yu ZX, Ferrans VJ, et al. Requirement for generation of H2O2 for platelet-derived growth factor signal transduction. Science
15. Heffetz D, Bushkin I, Dror R, Zick Y. The insulinomimetic agents H2O2 and vanadate stimulate protein tyrosine phosphorylation in intact cells. J Biol Chem
16. Kappert K, Sparwel J, Sandin A, et al. Antioxidants relieve phosphatase inhibition and reduce PDGF signaling in cultured VSMCs and in restenosis. Arterioscler Thromb Vasc Biol
17. Meng TC, Fukada T, Tonks NK. Reversible oxidation and inactivation of protein tyrosine phosphatases in vivo. Mol Cell
18. Majesky MW, Reidy MA, Bowen-Pope DF, et al. PDGF ligand and receptor gene expression during repair of arterial injury. J Cell Biol
19. Ferns GA, Raines EW, Sprugel KH, et al. Inhibition of neointimal smooth muscle accumulation after angioplasty by an antibody to PDGF. Science
20. Sirois MG, Simons M, Edelman ER. Antisense oligonucleotide inhibition of Pdgfr-beta receptor subunit expression directs suppression of intimal thickening. Circulation
21. Park SD, Jung JH, Lee HW, et al. Zedoariae rhizoma and curcumin inhibits platelet-derived growth factor-induced proliferation of human hepatic myofibroblasts. Int Immunopharmacol
22. Cai H. Nad(P)H oxidase-dependent self-propagation of hydrogen peroxide and vascular disease
. Circ Res
23. Nedeljkovic ZS, Gokce N, Loscalzo J. Mechanisms of oxidative stress and vascular dysfunction. Postgrad Med J
. 2003;79:195-199; quiz, 198-200.
24. Tonks NK. Redox redux: revisiting PTPS and the control of cell signaling. Cell
25. Persson C, Savenhed C, Bourdeau A, et al. Site-selective regulation of platelet-derived growth factor beta receptor tyrosine phosphorylation by T-cell protein tyrosine phosphatase. Mol Cell Biol
26. Buchdunger E, O'Reilly T, Wood J. Pharmacology of imatinib (Sti571). Eur J Cancer
. 2002;38(Suppl 5):S28-S36.
27. Maresta A, Balducelli M, Latini R, et al. Starc II, a multicenter randomized placebo-controlled double-blind clinical trial of trapidil for 1-year clinical events and angiographic restenosis reduction after coronary angioplasty and stenting. Catheter Cardiovasc Interv
28. Maheshwari RK, Singh AK, Gaddipati J, Srimal RC. Multiple biological activities of curcumin: a short review. Life Sci
29. Aggarwal BB, Kumar A, Bharti AC. Anticancer potential of curcumin: preclinical and clinical studies. Anticancer Res
30. Lim GP, Chu T, Yang F, et al. The curry spice curcumin reduces oxidative damage and amyloid pathology in an Alzheimer transgenic mouse. J Neurosci
31. Egan ME, Pearson M, Weiner SA, et al. Curcumin, a major constituent of turmeric, corrects cystic fibrosis defects. Science
32. Tamvakopoulos C, Dimas K, Sofianos ZD, et al. Metabolism and anticancer activity of the curcumin analogue, dimethoxycurcumin. Clin Cancer Res