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, 8 (11), e79309
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Inhibition of Angiogenesis Mediated by Extremely Low-Frequency Magnetic Fields (ELF-MFs)

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Inhibition of Angiogenesis Mediated by Extremely Low-Frequency Magnetic Fields (ELF-MFs)

Simona Delle Monache et al. PLoS One.

Abstract

The formation of new blood vessels is an essential therapeutic target in many diseases such as cancer, ischemic diseases, and chronic inflammation. In this regard, extremely low-frequency (ELF) electromagnetic fields (EMFs) seem able to inhibit vessel growth when used in a specific window of amplitude. To investigate the mechanism of anti-angiogenic action of ELF-EMFs we tested the effect of a sinusoidal magnetic field (MF) of 2 mT intensity and frequency of 50 Hz on endothelial cell models HUVEC and MS-1 measuring cell status and proliferation, motility and tubule formation ability. MS-1 cells when injected in mice determined a rapid tumor-like growth that was significantly reduced in mice inoculated with MF-exposed cells. In particular, histological analysis of tumors derived from mice inoculated with MF-exposed MS-1 cells indicated a reduction of hemangioma size, of blood-filled spaces, and in hemorrhage. In parallel, in vitro proliferation of MS-1 treated with MF was significantly inhibited. We also found that the MF-exposure down-regulated the process of proliferation, migration and formation of tubule-like structures in HUVECs. Using western blotting and immunofluorescence analysis, we collected data about the possible influence of MF on the signalling pathway activated by the vascular endothelial growth factor (VEGF). In particular, MF exposure significantly reduced the expression and activation levels of VEGFR2, suggesting a direct or indirect influence of MF on VEGF receptors placed on cellular membrane. In conclusion MF reduced, in vitro and in vivo, the ability of endothelial cells to form new vessels, most probably affecting VEGF signal transduction pathway that was less responsive to activation. These findings could not only explain the mechanism of anti-angiogenic action exerted by MFs, but also promote the possible development of new therapeutic applications for treatment of those diseases where excessive angiogenesis is involved.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Exposure system.
A) Experimental apparatus employed for oscillating magnetic field generation at the centre of the solenoid. B) B versus I(eff) at the centre of the solenoid as experimentally determined. The best fitting straight line of the measured values was obtained using the method of the least squares.
Figure 2
Figure 2. Effect of MF on the proliferation of HUVECs.
A) Histograms show the proliferative rate of HUVECs exposed to MFs. Each rate value was calculated respect to CTR at 1 h. The left histogram shows effects of a MF of 1 mT intensity from1 h to 24 h of treatment. The right histogram shows the effects of a MF of 2 mT intensity from 1 h to 24 h of treatment. B) Histogram shows the growth rate of MS-1 cells exposed or not to MF (2 mT, 50 Hz) for 72 h. Statistical differences were indicated for P<0.05.
Figure 3
Figure 3. Effect of MF on tubule formation of HUVEC cells.
A) Effect of MF (2 mT, 50 Hz) on angiogenesis in vitro. HUVECs were plated on Matrigel-coated culture plates, exposed for 1 h, 6 h and 12 h to MF (D, E, F) and photographed 16 h after plating. Unexposed control cells (CTR) were represented by HUVECs incubated in the absence of any stimulation for 1 h, 6 h and 12 h (A, B, C). B) Quantitative analysis for the tube length n = 5/treatment; C) Quantitative analysis of images for the branch point. n = 5/treatment CTR = unexposed control cells; MF = MF exposed cells. Data were expressed as a percentage of MF respect to unexposed control cells. Statistical differences were indicated for P<0.05.
Figure 4
Figure 4. Inhibition of tumour size in mice inoculated with MS-1.
A) MF (2 mT, 50 Hz) exposed cells (right) in comparison with unexposed cells (left). B) The size of the tumour was reduced in mice inoculated with MF treated MS-1 cells compared to control mice (inoculated with control cells). N = 5 mice per group, * P<0.05. C) Histology of tissue samples from a mouse inoculated with control cells showed a vascular tumour with large blood-filled lumens (a). The density and size of vessels is reduced in samples from mouse bearing MF-treated cells (b). White arrows indicate some blood filled structures. White asterisks indicate a blood filled diameter. Bar = 0.1 mm.VEGFR2 immunofluorescence micrographs of tissue sections from mice inoculated with control (c) and MF treated cells (d) Bar = 0.2 mm.
Figure 5
Figure 5. Effect of MF (2 MT, 50 Hz) on VEGFR expression and activation.
Protein of equal quantity was separated by 8% SDS-page and electroblotted on to a PVDF membrane. Representative blots show effects exerted by MF (2 mT, 50 Hz) on VEGFR2 total expression and phosphorylation after 1 h, 6 h and 12 h of exposure A). Histograms show values obtained by means ± SD of relative protein bands calculated in 3 experiments after incubation with anti-VEGFR2 antibody B) and with anti-pVEGFR2 antibody C). All protein bands were verified using an anti-tubulin antibody. Asterisks indicate statistical differences * P<0.05 D) Immunofluorescence of HUVECs exposed (bottom panels) or not (upper panels to MF for 1 h, 6 h and 12 h. Phosphor-VEGFR2 receptors were stained with a secondary antibody FITC-conjugated. Images shows that after MF exposure, the density of the phosphorylated receptors resulted decreased at each time point investigated.
Figure 6
Figure 6. Effect of MF (2 mT, 50 Hz) on ERK activation and phosphorylation levels of HUVECs.
A) Representative blots of ERK expression and phosphorylation after 1 h, 6 h and 12 h of exposure to MF of HUVECs. Histograms show values obtained by means ± SD of relative protein bands calculated in at least 3 experiments after incubation with anti-pERK antibody, anti-ERK1,2 antibody. MF = MF exposed cells; CTR = control unexposed cells. Analysis of protein expression levels of MS-1 cells (B) Representative blots of VEGFR2 phosphorylation and expression showed a marked decrease of VEGFR2 active form combined with a more slight reduction of VEGFR2 total form. HSP70 and HSP90 expression after 72 h of exposure to MF in MS-1 cells seemed markedly reduced. Histograms show values obtained by means ± SD of relative protein bands calculated in 3 experiments after incubation with anti-VEGFR2 and anti-pVEGFR2 antibodies and HSP70 and HSP90 antibodies. All protein bands were verified using an anti-tubulin antibody.

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Grant support

This work was supported by MURST ex-60% Grant from “Ministero dell’Universita’e della Ricerca Scientifica e Tecnologica”. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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