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Original Article

Effects of KTJ740, a Novel Antithrombotic Agent, on Platelet-Derived Growth Factor-Induced Rat Aortic Smooth Muscle Cell Proliferation and Cell Cycle Progression

Kim, Tack-Joong PhD*,1; Jeon, Jinseon MS*,1; Jin, Yong-Ri PhD; Son, Dong-Ju PhD*; Yoo, Hwan-Soo PhD*; Hong, Jin-Tae PhD*; Ryu, Chung-Kyu PhD; Shin, Hwa-Sup PhD§; Lee, Kwang-Ho PhD§; Yun, Yeo-Pyo PhD*

Author Information
Journal of Cardiovascular Pharmacology: May 2007 - Volume 49 - Issue 5 - p 280-286
doi: 10.1097/FJC.0b013e3180399448
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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.

Chemical structure of KTJ740.

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).

DNA Laddering

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).

Western Blotting

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.

Statistical Analysis

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.

Inhibitory effect of KTJ740 on the PDGF-BB-stimulated proliferation of RASMCs. A, Cell counting. PDGF-BB-stimulated RASMCs were pretreated with the indicated concentrations of KTJ740 for 24 hours. RASMCs were counted by hemocytometry. B, DNA synthesis. PDGF-BB (50 ng/mL)-stimulated RASMCs were treated with or without the indicated concentrations of KTJ740 for 24 hours. [3H]-thymidine (2 μCi/mL) was added to medium and RASMCs were incubated for 4 hours. Radioactivity was determined using a liquid scintillation counter. Results represent 6 different experiments. Data are expressed as means ± SDs. *P < 0.05, **P < 0.01 vs PDGF-BB-stimulated RASMCs. C, Apoptosis or necrosis. Cells were precultured in serum-free medium in the presence or absence of KTJ740 (5 μM) for 24 hours and then stimulated with 50 ng/mL PDGF-BB for 24 hours. Apoptotic (Annexin V+/PI−) or necrotic cells (Annexin V+/PI+) were identified by means of Annexin V binding and PI dye detected by flow cytometry. D, DNA fragmentation. DNA fragmentation was analyzed by electrophoresis on 1.8% agarose gel as described in Materials and Methods. Lane M, 100-bp DNA marker; Lane 1, DMSO; Lane 2, KTJ740 (5 μM); Lane 3, PDGF-BB + DMSO; Lane 4, PDGF-BB + KTJ740 (5 μM).
Cell Viability in KTJ740-Treated RASMCs

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).

Inhibitory effect of KTJ740 on the cell cycle progression in the PDGF-BB-stimulated RASMCs. Serum-starved RASMCs were treated with or without 5 μM KTJ740 for 24 hours. RASMCs were then stimulated with 50 ng/mL PDGF-BB for 24 hours, harvested, washed with 1 × PBS, fixed with ice-cold ethanol, and digested with RNase; then cellular DNA was stained with propidium iodide. Flow cytometric analysis of DNA contents was then performed. Results shown are representative of 3 different experiments. *P < 0.05 vs PDGF-BB-stimulated RASMCs.

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 ERK1/2, Akt, and PLCγ1 activations in the PDGF-BB-stimulated RASMCs. A-C, Serum-starved RASMCs were treated with or without the indicated concentrations of KTJ740 for 24 hours and then stimulated with 50 ng/mL PDGF-BB for 5 minutes (A, ERK1/2 activation; C, PLCγ1 activation) or 30 minutes (B, Akt activation). RASMC lysates and immunoblotting were performed as described in Materials and Methods. Results shown are representative of 3 different experiments. The phospho-ERK1/2, phospho-Akt, or phospho-PLCγ1 was normalized by the total ERK1/2, Akt, or PLCγ1 values, respectively. *P < 0.05, **P < 0.01 vs PDGF-BB-stimulated RASMCs.

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).

Effect of KTJ740 on PDGF-Rβ activation in the PDGF-BB-stimulated RASMCs. Serum-starved RASMCs were treated with or without the indicated concentrations of KTJ740 for 24 hours. RASMCs were then stimulated with 50 ng/mL PDGF-BB for 1 minute. RASMC lysates were prepared and immunoblotting was performed as described in Materials and Methods. The results shown are representative of 3 different experiments. The phospho-PDGF-Rβ was normalized by the total PDGF-Rβ values, respectively. *P < 0.05, **P < 0.01 vs PDGF-BB-stimulated RASMCs.


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).


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      KTJ740; rat aortic smooth muscle cell; platelet-derived growth factor; vascular disorder

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