Cardiovascular diseases, which are a leading cause of mortality and morbidity in the Western world, are associated with a multitude of pathophysiologic conditions, such as inflammation, pulmonary hypertension, and restenosis after balloon angioplasty.1 Vascular smooth muscle cell (VSMC) proliferation is induced in response to vessel injury, and platelet-derived growth factor (PDGF) is also known to be a prerequisite for the intimal thickening that is observed after angioplasty.2,3 Much evidence indicates that the expression of PDGF is increased in atherosclerotic lesions1,25 and that PDGF is a principal regulator of mitogenesis in VSMCs.1,33 Therefore, it is an important pharmacologic approach for inhibition of PDGF-induced VSMC proliferation during lesion development.3
The signaling pathway involved in PDGF-β receptor (PDGF-Rβ)-induced mitogenesis has been relatively well characterized. The binding of PDGF-BB to the PDGF-Rβ leads to its phosphorylation at multiple tyrosine residues. This activated PDGF-Rβ is associated with a number of SH2 domain-containing proteins, including the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K) and phospholipase C (PLC)γ1.4 Several molecules have been implicated in mitogen-activated protein kinase (MAPK) signaling pathways, such as extracellular regulated kinases 1 and 2 (ERK1/2) by triggering activation of Raf-1. Moreover, ERK1/2 mediate several apparently conflicting cellular responses, such as proliferation, apoptosis, growth arrest, and differentiation, in a cell-type-dependent manner.5,6 The PI3K-Akt pathway is another important pathway triggered by PDGF-BB. PI3K is activated by the binding of its p85 regulatory subunit to tyrosine-phosphorylated c-Met, and Akt kinase, a downstream target of PI3K, is recruited to the plasma membrane by direct binding to PI3K-produced phospholipids.7 The PI3K-Akt pathway was recently shown to be involved in the antiapoptotic activity of PDGF-BB in VSMCs.8 Moreover, approaches using PDGF-Rβ antagonism or protein kinase inhibitor reduced VSMC proliferation in vitro and prevented cardiovascular problems in several animal experiments.9-11
Compounds with the naphthoquinone backbone are known to have pronounced biological effects. In particular, they have been credited with antitumor, antiviral, antifungal, antimycobacterial, and antiplatelet activities.12-16 Additionally, some naphthoquinone analogues, 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone, were reported to inhibit tumor cell growth and to arrest cell cycle progression at G1 and G2/M, potently.34 In our previous study, we reported that KTJ740 is a newly synthesized naphthoquinone derivative, displaying a potent antithrombotic effect in mice in vivo and antiplatelet activity in vitro and ex vivo.17 However, the effect and mechanism regulating proliferation of VSMC by this compound remain unclear. Therefore, we investigated the inhibitory effect and mechanism of KTJ740, a synthetic naphthoquinone analogue, on rat aortic smooth muscle cells (RASMCs) proliferation.
In the present study, we provide evidence that KTJ740 potently inhibits RASMC proliferation and that it inhibits the ERK1/2, Akt, and PLCγ1 signaling pathways through PDGF-Rβ suppression in RASMCs. Our results reveal that KTJ740 may be a potential agent for the prevention and treatment of vascular disorders like atherosclerosis and restenosis.
KTJ740 [2-chloro-3-(4-(ethylcarboxy)-phenyl)-amino-1,4-naphthoquinone] was synthesized and characterized as previously described (Fig. 1).35 It was dissolved in dimethylsulfoxide (DMSO) and added to Dulbecco's modified Eagle's medium (DMEM) with a maximum final DMSO concentration of 0.05%. Cell culture materials were purchased from Gibco-BRL (Maryland, USA). Antiphospho-ERK1/2, antiphospho-Akt, antiphospho-PLCγ1, anti-ERK1/2, anti-Akt, and anti-PLCγ1 antibodies were from New England Biolabs (Massachusetts, USA). PDGF-BB, antiphospho-PDGF-Rβ, and anti-PDGF-Rβ polyclonal antibody were obtained from Upstate Biotechnology (New York, USA). Other chemicals were of analytical grade.
Isolation and Culture of Rat Aortic Smooth Muscle Cells
RASMCs were isolated by enzymatic dispersion as previously described.18 RASMCs were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin, 8 mM HEPES, and 2 mM L-glutamine at 37°C in a humidified 95% air and 5% CO2 incubator. The purity of RASMCs in culture was confirmed by the Western blotting of α-smooth-muscle actin. RASMCs were used at passages 4-8.
Cell Count, Viability, and [3H]-Thymidine Incorporation Assay
RASMCs were then seeded into 12-well plates and cultured in DMEM containing 10% FBS at 37°C for 24 hours. Under these conditions, a cell confluence of ∼70% was reached. Twelve-well plates were precultured in serum-free media with or without KTJ740. RASMCs were stimulated with PDGF-BB (50 ng/mL) for 24 hours, trypsinized, and counted using a hemocytometer. Cell viability was measured by trypan blue exclusion at 24 and 48 hours after addition of KTJ740 (1-5 μM). For [3H]-thymidine incorporation experiments, RASMCs were plated on 24-well culture plates at 5,000 cells/well and then allowed to grow for 3-4 days in DMEM. They were then placed in serum-free media with or without KTJ740 (1-5 μM) for 24 hours and exposed to 50 ng/mL PDGF-BB media. After 20 hours, RASMCs were pulsed with 2 μCi/well [3H]-thymidine (Amersham Pharmacia Biotech, USA) for 4 hours. The labeling reaction was terminated by aspirating the medium and subjecting cultures to sequential washes on ice with 1 × phosphate-buffered saline (PBS) containing 10% trichloroacetic acid and ethanol/ether (1:1 v/v). Acid-insoluble [3H]-thymidine was extracted into 250 μL of 0.5 M NaOH per well, and 100 μL of the extract obtained was mixed with a liquid cocktail and counted in a scintillation counter (model LS3801, Beckman).
Cell Cycle and Annexin V Assay by Flow Cytometry
RASMCs in 60 mm2 dishes were incubated in DMEM without serum (Gibco-BRL) with or without 5 μM KTJ740 for 24 hours. The RASMCs were then treated with or without 50 ng/mL PDGF-BB for 24 hours and then were trypsinized and centrifuged at 1,500 × g for 7 minutes. Centrifuged pellets were suspended in 1 mL 1 × PBS, washed twice, and recentrifuged. Pellets were suspended in 70% ethanol and fixed overnight at 4°C. The fixed RASMCs were briefly vortexed and centrifuged at 15,000 × g for 5 minutes. Ethanol was discarded and pellets were stained with 0.4 mL propidium iodide (PI) solution (50 μg/mL PI in buffer containing 100 μg/mL of RNase A). Before analysis by flow cytometry, samples were incubated for 1 hour at room temperature. The complex of PI-DNA in each cell nucleus was measured using a FACSCalibur (Becton & Dickinson Co., USA). The rate of G0/G1, S, and G2/M phases were determined using a computer program ModFitLT (Verity House Software, Topsham, Maine, USA). Apoptosis was determined by staining with annexin V (Annexin-V-FLUOS Staining Kit, Roche, USA). After the incubations, floating and adherent cells that were later trypsinized were pooled and centrifuged for 5 minutes at 1000 × g. Pelleted cells were washed in cold PBS. Thereafter, cells were centrifuged again for 5 minutes at 1000 × g and resuspended in 100 μL annexin-binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4) yielding a cell density of 1 × 106 cells/mL. Five microliters of annexin V conjugate and 1 μL of 100 μg/mL PI working reagent were added to each 100 μL of cell suspension. The cells were incubated at room temperature for 15 minutes. After the incubation period, 400 μL of annexin-binding buffer was added and mixed gently, and the samples were kept on ice. The stained cells were analyzed by flow cytometry, where the fluorescence emission was measured at 530 nm (Alexa Fluor 488). The rate of cells was determined using a computer program ModFitLT (Verity House Software, Topsham, Maine, USA).
Genomic DNA was extracted using G-DEX Genomic DNA extraction kits according to the manufacturer's instructions (Intron Biotech, Seoul, Korea). In brief, cells were trypsinized, collected, rinsed twice with cold PBS, and lysed at 65°C for 10 minutes using lysis buffer. The obtained DNA was then incubated with RNase A (100 μg/mL) at 37°C for 1 hour, treated with 100% isopropanol, and precipitated by centrifugation at 12,000 × g for 30 minutes at 4°C. DNA pellets were then washed with 70% ethanol and dissolved in DNA hydration buffer containing TE buffer. DNA concentrations were determined by spectrophotometry at 260 nm. The 20 μg of DNA was electrophoresed for 3 hours at 30 V in a 1.8% agarose gel containing ethidium bromide. DNA fragmentation bands were photographed under ultraviolet light (Hoffer, San Francisco, California, USA) and images were captured on Polaroid film (Fuji Film, Japan).
SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) was performed on RASMCs lysates using 10% acrylamide gels, as described by Laemmli.19 Proteins were transferred to PVDF membranes (Millipore Corp., USA) and membranes were blocked overnight at 4°C in Tris-buffered saline containing 0.1% Tween 20 (TBS/T) and 5% skim milk powder and then incubated with 1:2000 dilution of antiphospho-ERK1/2, antiphospho-Akt, antiphospho-PLCγ1, anti-ERK1/2, anti-Akt, anti-PLCγ1, antiphospho-PDGF-Rβ, and anti-PDGF-Rβ antibodies. Blots were then washed with TBS/T and incubated with a 1:5000 dilution of horseradish peroxidase-conjugated antimouse immunoglobulin G (IgG) or antirabbit IgG antibodies (New England Biolabs, Massachusetts, USA). The proteins were detected using an enhanced chemiluminescence (ECL) Western blotting detect reagent (Amersham Biosciences, USA). The phospho-ERK1/2, phospho-Akt, phospho-PLCγ1 or phospho-PDGF-Rβ was normalized by the total ERK1/2, Akt, PLCγ1, or PDGF-Rβ values, respectively. The intensities of the bands were quantified using a Scion-Image for the Windows program.
Experimental results are expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) was used for multiple comparisons, and this was followed by Dunnett's test. A P value less than 0.05 was regarded as significant.
Effect of KTJ740 on PDGF-BB-Stimulated RASMC Proliferation
To examine the inhibitory effect of KTJ740 on PDGF-BB-stimulated RASMC proliferation, we performed direct cell countings and [3H]-thymidine incorporation assays. Figure 2A shows the inhibitory effect of KTJ740 on PDGF-BB-stimulated cell numbers. Compared with control cells, stimulation of PDGF-BB significantly increased cell number, and this increase was inhibited by KTJ740 in a concentration-dependent manner (inhibition percentages were 30.79%, 73.01%, and 96.82% at concentrations of 1, 3, and 5 μM, respectively). The incorporation of [3H]-thymidine into DNA was also measured as an index of cell growth. The incorporation of [3H]-thymidine into DNA was found to be significantly increased by adding PDGF-BB to RASMCs, and KTJ740 significantly inhibited [3H]-thymidine incorporation into DNA in a concentration-dependent manner (inhibition percentages were 14.67%, 43.08%, and 92.32% at concentrations of 1, 3, and 5 μM, respectively) (Fig. 2B). To evaluate whether the reduction in the cell proliferation assay was caused by induction of cell death, the cytotoxicity of KTJ740 (1-5 μM)-treated RASMCs after 24 or 48 hours was assessed by trypan blue exclusion method. The percentages of cell viability were not altered significantly by these treatments (Table 1). Because antiproliferation is often accompanied by apoptosis, we also measured the apoptotic effects of KTJ740 on RASMCs using annexin V binding and DNA laddering assay, which are major indexes of apoptosis. RASMCs were pretreated with KTJ740 (5 μM) in serum-free medium for 24 hours and then were stimulated with PDGF-BB (50 ng/mL) for 24 hours. KTJ740 at 5 μM did not induce annexin V binding (Fig. 2C) and DNA laddering (Fig. 2D) in RASMCs. These results indicate that KTJ740 blocks RASMC proliferation without inducing apoptotic or necrotic cell death.
Effect of KTJ740 on PDGF-BB-Stimulated RASMC Cycle Progression
Cell cycle analysis was measured by flow cytometry for effect of KTJ740 on PDGF-BB-induced RASMCs. When RASMCs were grown in serum-starved DMEM for 24 hours with or without KTJ740 (5 μM), the proportions of cells in the G0/G1, S and G2/M phases were 89%, 2%, and 9% in the absence of KTJ740, but 91%, 1%, and 8% in the presence of KTJ740. However, when RASMCs were stimulated for 24 hours with PDGF-BB (50 ng/mL), S and G2/M phase proportions were significantly increased to 8.3% and 12.5%, whereas the proportions of S and G2/M phases were significantly inhibited in the presence of KTJ740 to 4.6% and 6.7%, respectively (Fig. 3).
Effects of KTJ740 on PDGF-BB-Stimulated ERK1/2, Akt, and PLCγ1 Activation in RASMCs
The MAPK (ERK1/2), Akt, and PLCγ1 pathways play the central roles in PDGF-BB-regulated RASMCs growth.5,6,8 To investigate the inhibitory mechanism of KTJ740 on RASMC proliferation, we examined whether KTJ740 affects the activation of ERK1/2, Akt, and PLCγ1 signaling molecules, which is known to regulate VSMC proliferation. In our previous studies, we determined the maximum phosphorylation in 5 minutes for ERK1/2 and PLCγ1 and 30 minutes for Akt by PDGF-BB (50 ng/mL) stimulation of RASMCs.36 Stimulation with PDGF-BB for 5 minutes significantly induced ERK1/2 phosphorylation, whereas pretreatment with KTJ740 significantly inhibited this induced phosphorylation in a concentration-dependent manner (inhibition percentages were 28%, 71%, and 92% at concentrations of 1, 3, and 5 μM, respectively) (Fig. 4A). To further investigate whether the Akt pathway is involved in the RASMC growth inhibition by KTJ740, we tested the effect of KTJ740 on the activation of Akt. In the presence of various concentrations of KTJ740, activated Akt was significantly inhibited by KTJ740 in a concentration-dependent manner (inhibition percentages were 14%, 43%, and 87% at concentrations of 1, 3, and 5 μM, respectively) (Fig. 4B). We next examined the effect of KTJ740 on the activation of PLCγ1. The PDGF-BB-stimulated PLCγ1 phosphorylation was found to be decreased by KTJ740 in a concentration-dependent manner. The inhibition percentages were 43%, 72%, and 89% at concentrations of 1, 3, and 5 μM, respectively (Fig. 4C).
Effect of KTJ740 on PDGF-Rβ Tyrosine Kinase Activation in PDGF-BB-Stimulated RASMCs
KTJ740 was found to inhibit the downstream components of PDGF-BB, such as ERK1/2, Akt, and PLCγ1 phosphorylation, in a similar manner, indicating that PDGF-Rβ, an upstream component of MAPK in the PDGF-BB signaling pathway, may be a direct target of KTJ740. Our results showed that PDGF-Rβ tyrosine kinase phosphorylation was inhibited by KTJ740 in a concentration-dependent manner. The inhibition percentages were 15%, 27%, and 65% at concentrations of 1, 3, and 5 μM, respectively (Fig. 5).
During atherosclerotic lesion progression, VSMC proliferation is of particular pathophysiologic importance.20 In atherosclerotic lesions, VSMCs are exposed to mitogenic substances, such as PDGF and endothelin.19,21 Moreover, the association between PDGF and VSMCs proliferation has been demonstrated in animal experiments, in which increases and augmentations of PDGF-BB after arterial injury were found to be correlated with neointimal cellular proliferation.22 In the present study, we investigated the antiproliferative effect of KTJ740 on PDGF-BB-induced RASMCs and a possible signaling pathway related to this antiproliferative effect. KTJ740 was found to inhibit RASMC proliferation and DNA synthesis induced by PDGF-BB in a concentration-dependent manner (Fig. 2), and these results agreed with those of other studies, which showed that carvedilol, a well-known therapeutic agent for cardiovascular disease, inhibited RASMC proliferation.23,24 Moreover, because antiproliferative effects are often accompanied by apoptosis, we also measured the apoptotic effects of KTJ740 on RASMCs under the same conditions. However, KTJ740 at the concentrations (1-5 μM) used in the present study did not reduce RASMC viability or increase apoptosis significantly, as assessed by trypan blue exclusion (Table 1) and DNA fragmentation (Fig. 2D), indicating that KTJ740 blocks RASMC proliferation without inducing apoptosis or necrosis. We also elucidated the mechanism underlying the antiproliferative effect of KTJ740. The activations of ERK1/2, Akt, and PLCγ1 are initiated by ligand-binding to the cell surface receptor, activation of the receptor, and binding of adapter molecules to phosphotyrosine residues in the activated receptor. This is followed by the activation of the small GTP-binding protein Ras by a guanine nucleotide exchange factor.25,26 To investigate the signaling pathway involved in KTJ740 inhibition, we examined the activations of ERK1/2, Akt, and PLCγ1. These proteins were found to be rapidly activated (5 minutes for ERK1/2 and PLCγ1, 30 minutes for Akt) after the PDGF-BB stimulation of RASMCs. Moreover, ERK1/2, Akt, and PLCγ1 activations were significantly inhibited by KTJ740 in a concentration-dependent manner (Fig. 4). These findings suggest that the antiproliferative effect of KTJ740 on RASMCs is related to the inhibition of the activation of MAPK family member, the Akt and PLCγ1 protein. PDGF-Rβ activates multiple signaling pathways that lead to cellular transformation. Therefore, we hypothesized that PDGF-Rβ is a direct target for KTJ740 and that this leads to the inhibition of RASMC proliferation. PDGF-BB is a potent RASMC mitogen, and the phosphorylation of PDGF-Rβ occurs early in the mitogenic signaling cascade.25,26 PDGF-BB is postulated to act through the autophosphorylation of PDGF-Rβ, which leads to the activation of tyrosine kinase, the phosphorylation of intracellular proteins, and DNA synthesis.27 Our results show that KTJ740 acts via suppressing PDGF-BB-stimulated PDGF-Rβ tyrosine kinase activation (Fig. 5).
Recently, we reported that luteolin and kaempferol, 2 flavonoid compounds, prevent the PDGF-BB-stimulated proliferation of RASMCs by inhibiting PDGF-Rβ phosphorylation.30,31 These results agree with the findings of the present study. When RASMCs are stimulated by mitogens, the entry into the S and G2/M phases are major points of cell proliferation in mammalian cells.2,32 To investigate the effect of KTJ740 on the cell cycle, flow cytometry was performed. Pretreatment of RASMCs with vehicle for 24 hours showed 85%, 1%, and 9% cells in the G0/G1, S, and G2/M phases, respectively. Stimulation of PDGF-BB resulted in a significant increase of RASMCs in the S and G2/M phase, whereas pretreatment with KTJ740 for 24 hours before stimulation by PDGF-BB almost totally prevented S-phase entry and effectively arrested RASMC cycle progression, indicating that the inhibitory effect of KTJ740 on RASMC cycle progression may be mediated by blocking of the PDGF-Rβ (Fig. 3).
Taken together, these results provide evidence that KTJ740 inhibits PDGF-BB-stimulated RASMC proliferation and cell cycle progression, which is mediated by inhibiting PDGF-Rβ autophosphorylation.
This work was supported by the program of the Research Center for Bioresource and Health of the Korean Ministry of Science & Technology and the Korea Science & Engineering Foundation and by a Korean Research Foundation Grant funded by the Korean Government (MOEHRD, Basic Research Promotion Fund, KRF-2005-005-J15002). TJ Kim is the recipient of a postdoctoral grant from the Brain Korea 21 project (Frontier Pharmaceutical Technology for Biotopia).
1. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature
2. Braun-Dullaeus RC, Mann MJ, Dzau VJ. Cell cycle progression: new therapeutic target for vascular proliferative disease. Circulation
3. King SB 3rd, Williams DO, Chougule P, et al. Endovascular beta-radiation to reduce restenosis after coronary balloon angioplasty; results of the Beta Energy Restenosis Trial (BERT). Circulation
4. Muller DW. The role of proto-oncogenes in coronary restenosis. Prog Cardiovasc Dis
5. Hommes DW, Peppelenbosch MP, van Deventer SJ. Mitogen activated protein (MAP) kinase signal transduction pathways and novel anti-inflammatory targets. Gut
6. Robinson MJ, Cobb MH. Mitogen-activated protein kinase pathways. Curr Opin Cell Biol
7. Downward J. Signal transduction. New exchange, new target. Nature
8. Higaki M, Shimokado K. Phosphatidylinositol 3-kinase is required for growth factor-induced amino acid uptake by vascular smooth muscle cells. Arterioscler Thromb Vasc Biol
9. Waltenberger J. Modulation of growth factor action. Implications for the treatment of cardiovascular diseases. Circulation
10. Myllarniemi M, Frosen J, Calderon Ramirez LG, et al. Selective tyrosine kinase inhibitor for the platelet-derived growth factor
receptor in vitro inhibits smooth muscle cell proliferation after reinjury of arterial intima in vivo. Cardiovasc Drugs Ther
11. Lipson KE, Pang L, Huber LJ, et al. Inhibition of platelet-derived growth factor
and epidermal growth factor receptor signaling events after treatment of cells with specific synthetic inhibitors of tyrosine kinase phosphorylation. J Pharmacol Exp Ther
12. Chlebowski RT, Dietrich M, Akman S, et al. Vitamin K3 inhibition of malignant murine cell growth and human tumor colony formation. Cancer Treat Rep
13. Kar S, Carr BI. Growth inhibition and protein tyrosine phosphorylation in MCF7 breast cancer cells by a novel K vitamin. J Cell Physiol
14. Rodriguez S, Wolfender JL, Hakizamungu E, et al. An antifungal naphthoquinone, xanthones and secoiridoids from Swertia calycina. Planta Med
15. Shen AY, Huang MH, Teng CM, et al. Inhibition of 2-p-mercaptophenyl-1,4-naphthoquinone on human platelet function. Life Sci
16. Ko FN, Sheu SJ, Liu YM, et al. Inhibition of rabbit platelet aggregation by 1,4-naphthoquinones. Thromb Res
17. Yuk DY, Ryu CK, Hong JT, et al. Antithrombotic and antiplatelet activities of 2-chloro-3-[4-(ethylcarboxy)-phenyl]-amino-1,4-naphthoquinone, a newly synthesized 1,4-naphthoquinone derivative. Biochem Pharmacol
18. Kim TJ, Zhang YH, Kim YS, et al. Effect of apigenin on the serum- and platelet derived growth factor-BB-induced proliferation of rat aortic VSMCs. Planta Med
19. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature
20. Gordon D, Reidy MA, Benditt EP, et al. Cell proliferation in human coronary arteries. Proc Natl Acad Sci USA
21. Gutstein WH, Teresi JA, Wu JM, et al. Increased serum mitogenic activity for arterial smooth muscle cells associated with relaxation and low educational level in human subjects with high but not low hostility traits: implications for atherogenesis. J Psychocom Res
22. Uchida K, Sasahara M, Morigami N, et al. Expression of platelet derived growth factor beta-chain in neointimal smooth muscle cells of balloon injured rabbit femoral arteries. Atherosclerosis
23. Ohlstein EH, Douglas SA, Sung CP, et al. Carvedilol, a cardiovascular drug, prevents vascular smooth muscle cell proliferation, migration, and neointimal formation following vascular injury. Proc Natl Acad Sci USA
24. Sung CP, Arleth AJ, Ohlstein EH. Carvedilol inhibits vascular smooth muscle cell proliferation. J Cardiovasc Pharm
25. Majesky MW, Reidy MA, Bowen-Pope DF, et al. PDGF ligand and receptor gene expression during repair of arterial injury. J Cell Biol
26. Casscells W. Smooth muscle cell growth factors. Prog Growth Factor Res
27. Thyberg J, Hedin U, Sjolund M, et al. Regulation of differentiated properties and proliferation of arterial smooth muscle cells. Arteriosclerosis
28. Wilden PA, Agazie YM, Kaufman R, et al. ATP-stimulated smooth muscle cell proliferation requires independent ERK and PI3K signaling pathways. Am J Physiol
29. Bacqueville D, Casagrande F, Perret B, et al. Phosphatidylinositol 3-kinase inhibitors block aortic smooth muscle cell proliferation in midlate G1 phase: Effect on cyclin-dependent kinase 2 and the inhibitory protein p27kip1. Biochem Bioph Res Comm
30. Kim SY, Jin YR, Lim Y, et al. Inhibition of PDGF beta-receptor tyrosine phosphorylation and its downstream intracellular signal transduction in rat aortic vascular smooth muscle cells by kaempferol. Planta Med
31. Kim JH, Jin YR, Park BS, et al. Luteolin prevents PDGF-BB-induced proliferation of vascular smooth muscle cells by inhibition of PDGF beta-receptor phosphorylation. Biochem Pharmacol
32. Fang F, Newport JW. Evidence that the G1-S and G2-M transitions are controlled by different cdc2 proteins in higher eukaryotes. Cell
33. Sachinidis A, Locher R, Vetter W, et al. Different effects of platelet-derived growth factor
isoforms on rat vascular smooth muscle cells. J Biol Chem
34. Tamura K, Southwick EC, Kerns J, et al. Cdc25 inhibition and cell cycle arrest by a synthetic thioalkyl vitamin K analogue. Cancer Res
35. Ryu CK. Synthesis of anticoagulant 2-chloro-3(N-arylamino)-1,4-naphthoquinones. Yakhak Hoeji
36. Fang LH, Zhang YH, Ma JJ, et al. Inhibitory effects of tetrandrine on the serum- and platelet-derived growth factor
-BB-induced proliferation of rat aortic smooth muscle cells through inhibition of cell cycle progression, DNA synthesis, ERK1/2 activation and c-fos expression. Atherosclerosis