Nutr Res Pract.  2014 Oct;8(5):521-525. 10.4162/nrp.2014.8.5.521.

Artemisinin attenuates platelet-derived growth factor BB-induced migration of vascular smooth muscle cells

• Lee KP
• Park ES
• Kim DE
• Park IS
• Kim JT
• Hong H

• KMID: 2313773
• DOI: http://doi.org/10.4162/nrp.2014.8.5.521

Abstract

BACKGROUND/OBJECTIVES
Artemisinin (AT), an active compound in Arternisia annua, is well known as an anti-malaria drug. It is also known to have several effects including anti-oxidant, anti-inflammation, and anti-cancer activities. To date, the effect of AT on vascular disorders has not been studied. In this study, we investigated the effects of AT on the migration and proliferation of vascular smooth muscle cells (VSMC) stimulated by platelet-derived growth factor BB (PDGF-BB).
MATERIALS/METHODS
Aortic smooth muscle cells were isolated from Sprague-Dawley rats. PDGF-BB stimulated VSMC migration was measured by the scratch wound healing assay and the Boyden chamber assay. Cell viability was determined by using an EZ-Cytox Cell Viability Assay Kit. The production of reactive oxygen species (ROS) in PDGF-BB stimulated VSMC was measured through H2DCF-DA staining. We also determined the expression levels of signal proteins relevant to ROS, including measures of extracellular signal-regulated kinase (ERK) 1/2 measured by western blot analysis and matrix metalloproteinase (MMP) 9 measured by reverse transcription-polymerase chain reaction (RT-PCR).
RESULTS
AT (10 microM and 30 microM) significantly reduced the proliferation and migration of PDGF-BB stimulated VSMC in a dose-dependent manner. The production of ROS, normally induced by PDGF-BB, is reduced by treatment with AT at both concentrations. PDGF-BB stimulated VSMC treated with AT (10 microM and 30 microM) have reduced phosphorylation of ERK1/2 and inhibited MMP9 expression compared to untreated PDGF-BB stimulated VSMC.
CONCLUSIONS
We suggest, based on these results, that AT may exert an anti-atherosclerotic effect on PDGF-BB stimulated VSMCs by inhibiting their proliferation and migration through down-regulation of ERK1/2 and MMP9 phosphorylation.

Keyword

Artemisinin; vascular smooth muscle cells; platelet-derived growth factor-BB; ERK1/2; MMP9

MeSH Terms

Blotting, Western
Cell Survival
Down-Regulation
Muscle, Smooth, Vascular*
Myocytes, Smooth Muscle
Phosphorylation
Phosphotransferases
Platelet-Derived Growth Factor
Rats, Sprague-Dawley
Reactive Oxygen Species
Wound Healing
Phosphotransferases
Platelet-Derived Growth Factor
Reactive Oxygen Species

Figure

• 

  Fig. 1 The effect of artemisinin on proliferation in PDGF-BB-stimulated VSMC. (A) The chemical structure of artemisinin. (B) VSMC were seeded (2 × 103 cells/well) and incubated for 12 h. VSMC were incubated in serum-free media for 24 h and then cells were treated with the indicated concentrations of AT (10 µM and 30 µM) in the presence or absence of PDGF-BB for 24 h. The graph indicated the representative cell viability from three independent experiments. Data are mean ± standard error. Values with the same superscript letter are not significantly different by Duncan's multiple range test (P < 0.05).

• 

  Fig. 2 Effects of AT on scratch wound healing in PDGF-BB-stimulated VSMC. VSMC were treated with AT (10 µM and 30 µM) and PDGF-BB (10 ng/mL) for 24 h. (A) Dotted white lines indicate the wounded area from the initial scratches. Magnification, × 200. Bar = 500 µm. The number in the top right of each image is concentration of PDGF-BB/concentration of AT. (B) Amount of migration relative to the untreated control (%). Results are presented as mean ± standard error. Values with the same superscript letter are not significantly different by Duncan's multiple range test (P < 0.05).

• 

  Fig. 3 Effects of AT on migration in PDGF-BB-stimulated VSMC. VSMC were treated with AT (10 µM and 30 µM) and PDGF-BB (10 ng/mL) for 90 min. (A) Migrating VSMC on membranes, the spots are Diff quick-stained cells. The number at the top right corner of each image is the concentration of PDGF-BB/concentration of AT. Magnification, × 200. Bar = 500 µm. (B) Migrated cell counts indicated relative to the untreated control (%). Results are presented as mean ± standard error. Values with the same superscript letter are not significantly different by Duncan's multiple range test (P < 0.05).

• 

  Fig. 4 Effects of AT on migration in H2O2-stimulated VSMC. (A) ROS production was assessed using 1 µg/mL DAPI (upper panel) or H2DCFDA (lower panel) in PDGF-BB-induced proliferating VSMC, Magnification, × 400. Bar = 50 µm. (B) Migrating VSMC on membranes, the spots are Hoescht stained cells. VSMC were treated with AT (10 µM and 30 µM) and H2O2 (100 µM) for 90 min. The number at the top right corner of each image is the concentration of H2O2/concentration of AT. Magnification, 200 ×. Bar = 500 µm. (C) Migrating cell number relative to the untreated control (%). Results are presented as mean ± standard error. Values with the same superscript letters are not significantly different by Duncan's multiple range test (P < 0.05).

• 

  Fig. 5 Effect of AT on PDGF-BB-induced phosphorylation of ERK1/2 and MMP9 transcription in VSMC. (A) VSMC were pretreated with or without AT (10 µM and 30 µM) for 1 h and then stimulated with 10 ng/mL of PDGF-BB for 15 min. For statistical analysis, densitometry of the band representing the phosphorylated form of ERK1/2 normalized to the total expression of ERK is considered 100%. (B) The levels of MMP9 mRNA were determined by RT-PCR. VSMC were pretreated with or without AT (10 µM and 30 µM) for 1 h and then stimulated with 10 ng/mL of PDGF-BB for 6 h. cDNA was synthesized using 1 µg of total RNA each treatment group. After PCR and electrophoresis on a 1% agarose gel, the bands were measured using densitometry of the ethidium bromide stained DNA under UV light. The background intensity is considered 100%, and the expression of MMP9 is defined relative to the GAPDH control. Results are presented as the mean ± standard error of three independent experiments. Values with the same superscript letters are not significantly different by Duncan's multiple range test (P < 0.05).

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