Abstract: In this study, we studied electromagnetic field effect on angiogenesis. Ross fertilized eggs were used that divided into 3 random groups 42 consisted of control, sham-exposed and a test group which treated by 0.04 T electromagnetic field. In day 10 members of test group was occurred in 0.04 T electromagnetic fields for 4 h. Members of sham-exposed group was occurred in the same electromagnetic field but in turn off condition. In day 12 chorioalantoic membranes were examined and photographed in all cases. The numbers and lengths of vessels in four random same areas were measured and compared with each other by t-test (p<0.05). Comparison between average number and length of vessels in controls and sham-exposed didn't show any significant differences. In test group we saw a significant decrease in average number (26.69±7.88) and length (44.41±9.88 cm) of vessels in comparison with controls. As a result 0.04 T electromagnetic field has an inhibitory effect on angiogenesis in chick chorioalantoic membrane.
INTRODUCTION
Angiogenesis, the sprouting of capillaries from pre-existing venules, orchestrated by a plenitude of different molecules and pathways, including Vasculo-Endothelial Growth Factor (VEGF) (Breier, 2006), Stem Cell Factor (SCF), Epithelial Growth Factor (EGF), Transforming Growth Factor β (TGFβ) family (Lioyd et al., 2003), FGF, extracellular matrix (ECM) (Li et al., 2003), angiopoietins and other cytokines (Hiromatsu and Toda, 2003). In the adult organism, new blood vessel formation is tightly controlled and occurs only under certain physiological and pathological conditions, such as pregnancy, wound healing (Li et al., 2003) diabetic retinopathy, or solid tumor growth (Srivastava et al., 2002; Breier, 2006). Balasubramanian and Reed (2003) showed that following embryonic and postnatal development, blood vessel endothelial cells proliferate and may remain quiescent for several years, in physiological conditions, a fine balance of pro- and anti-angiogenic factors is maintained as part of normal homeostatic mechanisms.
Electromagnetic fields, constant and alternating, are a static element of the environment. They originate from both natural and man-made sources. Depending on the type of the field, its intensity and time of activity, they exert different effects on the natural world (plants and animals). Some animals utilize magnetic field of the earth for their own purposes (Rochalska, 2007). Several reports have shown that weak, Extremely-Low-Frequency (ELF), Pulsed Magnetic Fields (PMFs) can adversely affect the early embryonic development of the chick, PMFs can induce irreversible developmental alterations and the pulse waveform can be a determinant factor in the embryonic response to ELF magnetic fields (Ubeda et al., 1994). In vitro or in vivo studies in nonhuman species can be used to study mechanisms and the effects that have been suggested by human investigations. The studies dealing with mutagenesis, cell death and cell proliferation using in vitro systems do not indicate that EMFs have the potential for deleteriously affecting proliferating and differentiating embryonic cells at the exposures to which populations are usually exposed. Of course, there is no environmental agent that has no effect, deleterious or not, at very high exposures. The animal and in vitro studies dealing with the reproductive effects of EMF exposure are extensive (Brent, 1999).
Several mechanisms, both thermal and nonthermal, are were established by which electromagnetic fields can interact with biological systems. Thermal mechanisms are related to heating of tissue and generate when electrical energy change to heat. Nonthermal mechanisms directly depend on electromagnetic field itself and low frequency electric fields interfere to cell membrane stimulation and decrease it (Litvak et al., 2002). A report was published in 2004 that referred to effect of 0.2 T electromagnetic field on angiogenesis in chick chorioallantoic membrane (Ruggerio et al., 2004). In this study we analyze the effects of 0.04 T electromagnetic field on angiogenesis with this hypothesis that if 0.04 T electromagnetic field has inhibitory effect on angiogenesis.
MATERIALS AND METHODS
This research was executed in Research Laboratory in Biology Department of Mashhad Islamic Azad University (Mashhad, Iran, 2008-2009). We used 42 fertilized Ross eggs that hold in an incubator with 38°C temperature and 65% moisture.
| Fig. 1: | The electromagnetic system with incubator. A: Electrical coiling, B: Incubator, C: Resistant, D: Condenser and E: Amper meter |
| Fig. 2: | Chorioalantoic membrane in experimental groups. (a) Control case and (b) test case (treated by 0.04T electromagnetic field). Black quadrate show one of the measured area |
In day 2 of incubation, windows were opened on eggs under sterile condition (Ruggerio et al., 2004; Laminair flow, Teslar AV-100, Spain) and eggs were considered in 3 random groups include of, 1) control, that hold in normal condition; 2) sham-exposed, that were placed in electromagnetic field, but in turn of position and 3) test, treated by 0.04 electromagnetic field. In day 10 members of group 2 were occurred in 0.04 T electromagnetic field (made in biology research lab of Islamic Azad university of Mashhad, Iran) for 4 h, but in turn off position (Fig. 1) and members of group 3 were occurred in this electromagnetic field in turn on position (0.04 T for 4 h). Chorioalantoic membrane were examined daily and photographed (by photo-stereomicroscope, Zeiss, Germany) at day 12 in 0.65x10x4 magnification (Fig. 2a, b). Four equal random area (quadrate shape) were selected and data were analyzed for the number and length of angiogenic blood vessels by software programs and t-test.
RESULTS AND DISCUSSION
As shown in Table 1, there was not any differences between the average number (42.00±7.26) and length (57.25±5.05 cm) of blood vessels in control and average number (42.93±6.73) and length (58.22±6.40 cm) of blood vessels in sham-exposed, so electromagnetic field in turn off position has not any effects on blood vessel formation. In test group which treated with 0.04 T electromagnetic field (MF) there was a significant decrease in average number (26.69±7.88) and length (44.41±9.88 cm) of vessels in comparison with control.
In the extremely low frequency (ELF, <300 Hz) part of the electromagnetic spectrum, experimental therapies have been emerging for a variety of medical conditions, such as non-union bone fractures, skin ulcers, migraines and degenerative nerves (Shupak, 2003). Williams and his co-workers reported reduction of angiogenesis in breast adenocarcinoma cells treated by 10, 15 and 20 μT electromagnetic fields, Okano et al. (2007) showed that 10 and 15 mT static electromagnetic fields reduce angiogenesis in rats with experimental hypertension (McKay et al., 2007). Okano et al. (2007) demonstrated that 120 mT Static Magnetic Field (SMF) significantly reversed the inhibitory effects of TGF-β1 arteriogenesis in vitro, suggesting that SMF could have potential to modify tubular formation, depending on the origin of the cells and the experimental conditions, including angiogenesis inhibitors or stimulators in the medium used for incubation, field intensity, localization of exposure, exposure duration and heterogeneous or homogeneous magnetic fields (Okano et al. (2007)).
| Table 1: | Average number and length of vessels in experimental groups (±SD), control, sham-exposed and test (treated with 0.04 T electromagnetic field) |
Del Monache et al. (2008) showed that some important functions of human microvascular endothelial cells (in vitro), including proliferation, migration and tubule formation increased under the influence of a sinusoidal electromagnetic field (1 mT, 50 Hz) and the organization of the actin and local adhesion inside the cell, the state of activation and the distribution of VEGF receptors were also affected. It was reported that angiogenesis increased in rat subcutaneous tissue when exposed to 0.1 mT for 30 min twice daily for 8 or 12 weeks (Weber et al., 2004). Tepper et al. (2004) showed that 1.2 mT pulsed electromagnetic fields (PEMF) increased angiogenesis in transgenic mice (8 h day-1 whole body exposure for 3, 10 or 14 days) (Tepper et al., 2004). There are several factors that make different effects of electromagnetic fields on the basis of intensity and exposure time of electromagnetic field and the hereditary characteristics of the treated cases too (Lahijani and Sajadi, 2004). In this study we found when eggs were exposed to 0.04 T electromagnetic field, angiogenesis was inhibited and the number of blood vessels were decreased. Bare (2004) suggested that by creating a synergism of biochemical, electrochemical and electronic principles, the practitioner should be able to achieve a superior treatment outcome; reporting that present chemotherapy regimens can cause permanent damage to various vital organs including the heart, lung and kidneys; even without such permanent damage, short term toxicity manifested as repression of the hematopoietic system and other type of physical unpleasantness.
CONCLUSION
The 0.04 T sinusoidal electromagnetic field can inhibit angiogenesis in chick chorioalantoic membrane which appear as a significant decrease in numbers and lengths of blood vessels. Based on different results in different studies, we purpose that electromagnetic fields with different densities and shape of waves can use for treated some pathological conditions that involve with angiogenesis as solid tumor and metastasis (inhibitory effect) or wound healings (stimulatory effect).