Inhibition of angiogenesis mediated by extremely low-frequency magnetic fields (ELF-MFs).

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.

ELF-MF transiently increases skeletal myoblast migration: possible role of calpain system.

PURPOSE: Cell migration is crucial for myogenesis since it is required for the alignment and fusion of myoblast. Ca(2+) signals are involved in regulating myoblast migration and an extremely low frequency (ELF) magnetic field (MF) increases intracellular calcium levels in C2C12 myoblast. This study was aimed at investigating whether ELF-MF could affect myoblast migration. As calpains contribute to the regulation of myoblast motility, the effect of ELF-MF on µ- and m-calpain was also investigated. MATERIALS AND METHODS: The effect of ELF-MF (1 mT; 50 Hz) on C2C12 cell motility was observed by wound-healing assay. Protein expression of calpains, calpastatin, myristoylated alanine-rich C-kinase substrate (MARCKS) and vinculin were examined by Western blot analysis. Casein zymography and immunofluorescence analysis were carried out to evaluate, respectively, activity levels of calpains and intracellular distribution of calpains, calpastatin and actin. RESULTS: Exposure to ELF-MF resulted in a transient but significant increase of myoblast migration. This stimulatory effect was associated with a marked increase of µ- and m-calpain activity followed by the concomitant variation in their subcellular localization. No significant changes in intracellular distribution and protein levels of calpastatin were detected. Finally, a significant decrease of MARCKS expression and modifications of actin dynamics were reported. CONCLUSIONS: This study clearly outlines an involvement of calpains in ELF-MF-mediated myoblast migration.

Involvement of mitochondrial activity in mediating ELF-EMF stimulatory effect on human sperm motility.

It has recently been reported that the exposure of human spermatozoa to an extremely low frequency (ELF) electromagnetic field (EMF) with a square waveform of 5 mT amplitude and frequency of 50 Hz improves sperm motility. The functional relationship between the energy metabolism and the enhancement of human sperm motility induced by ELF-EMF was investigated. Sperm exposure to ELF-EMF resulted in a progressive and significant increase of mitochondrial membrane potential and levels of ATP, ADP and NAD(+) that was associated with a progressive and significant increase in the sperm kinematic parameters. No significant effects were detected on other parameters such as ATP/ADP ratio and energy charge. When carbamoyl cyanide m-chlorophenylhydrazone (CICCP) was applied to inhibit the oxidative phosphorylation in the mitochondria, the values of energy parameters and motility in the sperm incubated in the presence of glucose and exposed to ELF-EMF did not change, thus indicating that the glycolysis was not involved in mediating ELF-EMF stimulatory effect on motility. By contrast, when pyruvate and lactate were provided instead of glucose, the energy status and motility increased significantly in ELF-EMF-treated sperm. Under these culture conditions, the inhibition of glycolitic metabolism by 2-deoxy-D-glucose (DOG) again resulted in increased values of energy and kinematic parameters, indicating that gluconeogenesis was not involved in producing glucose for use in glycolysis. We concluded that the key role in mediating the stimulatory effects exerted by ELF-EMF on human sperm motility is played by mitochondrial oxidative phosphorylation rather than glycolysis.

Extremely low frequency electromagnetic fields (ELF-EMFs) induce in vitro angiogenesis process in human endothelial cells.

Effects of extremely low frequency (ELF) electromagnetic fields (EMFs) on activation of angiogenesis were analysed using cultured umbilical human vein endothelial cells (HUVECs). The cultures were exposed to a sinusoidal EMF to intensity of 1 mT, 50 Hz for up to 12 h. EMFs increased the degree of endothelial cell proliferation and tubule formation, coupled by an acceleration in the process of wound healing. Since this process is physiologically accompanied by a large modification in the structural organization of actin and focal adhesions, we analyzed the rearrangement of some cytoskeleton elements demonstrating a major reorganization of the fibres and of the focal adhesion complexes after EMF exposure. Finally, Western blot analysis revealed a significant increase in phosphorylation as well as the overall expression of VEGF receptor 2 (KDR/Flk-1) suggesting that EMFs may modulate in vitro some endothelial functions correlated to angiogenesis through signal transduction pathways dependent on VEGF.

Intrafollicular expression of matrix metalloproteinases and their inhibitors in normally ovulating women compared with patients undergoing in vitro fertilization treatment.

OBJECTIVE: To assess possible differences in the activity of matrix metalloproteinases (MMPs), MMP-2 and MMP-9, and their inhibitors, the tissue inhibitors of MMPs, TIMP-1 and TIMP-2, in follicular fluid (FF) of women undergoing in vitro fertilization (IVF) treatment and of normally ovulating women. DESIGN: Prospective study. METHODS: MMP-2 and MMP-9 activity was analyzed by gelatin zymography and MMP-2, MMP-9, TIMP-2, TIMP-1 and 17beta-estradiol levels were measured in FF by ELISA. RESULTS: We found significantly reduced MMP levels in FF of women undergoing IVF treatment when compared with those of normally ovulating women. In contrast, the TIMP-1 levels were found significantly increased in FF from IVF patients vs normally ovulating women. No significant differences were found for TIMP-2 between the two groups. CONCLUSIONS: These findings underline a marked difference in MMPs and their inhibitors in the IVF women and the control group. Therefore we assume MMPs depend on hormonal steroidogenesis modulation induced by the gonadotropin protocol for IVF treatment.

Ca2+-independent protein kinase C signalling in mouse eggs during the early phases of fertilization.

Protein kinase C (PKC), an enzyme playing a central role in signal transduction pathways, is activated in fertilized mouse eggs downstream of the fertilization Ca2+ signal, to regulate different aspects of egg activation. Given the presence of Ca2+-independent PKC isoforms within the egg, we investigated whether fertilization triggers PKC stimulation in mouse eggs by activating Ca2+-independent signalling pathways. An increase in PKC activity was detected as early as 10 min after the beginning of insemination, when about 90% of eggs had fused with sperm and the first Ca2+ rise was evident in most of the eggs. A similar level of activity was found 20 min later, when about 60% of eggs had resumed meiosis. When the Ca2+ increase was buffered by an intracellular Ca2+ chelating agent, PKC stimulation was not blocked but only slightly reduced. Confocal microscopy analysis revealed that the increase in PKC activity at fertilization coincided with the translocation of PKCdelta, a Ca2+-independent and diacylglycerol-dependent PKC isoform, to the meiotic spindle. When, in the absence of the Ca2+ signal, metaphase-anaphase transition was inhibited, PKCdelta moved to the meiotic spindle but still maintained a sustained cytoplasmic distribution. In summary, our results indicate that: 1) PKC activation is an early event of egg activation; 2) both Ca2+-dependent and Ca2+-independent pathways contribute to increased PKC activity at fertilization; 3) PKCdelta is one of the isoforms participating in this signalling process.

Spectrin and Ankyrin-like Proteins in the Egg of Discoglossus pictus (Anura): Their Identification and Localization in the Site of Sperm Entrance versus the Rest of the Egg: (spectrin/ankyrin/anuran eggs/fertilization).

The Discoglossus pictus egg has a specific site of sperm-egg interaction, the dimple, which has a well-defined cytoskeleton. We studied whether there are cytoskeletal and cytoskeleton-related proteins typically involved in the polarization of plasma membrane proteins. The identity and the localization of the molecules cross-reacting with antispectrin, antifodrin and antiankyrin antiobodies were investigated by immunofluoresecence and immunoblotting of the proteins of the dimple (D) and of the rest of the egg (dimple-less-egg; DLE). Two polypeptides of about 254-and 246-kD were detected in the D and DLE, and localized in the egg cortex. A third molecule, weakly cross-reacting with antispectrin and antifodrin, was found in the subcortical cytoplasm. The 246-kD polypeptide was labile in samples prepared for SDS-PAGE; a mild prefixation of eggs prevented its dispersion. Mild fixation was also needed to retain antispectrin reactivity in cryostat sections of the DLE cortex, while this is not necessary in D. A molecule of about 204-kD, cross-reacting with antiankyrin, was detected in the cortex of the whole egg. These data and the finding that the concentrations of both the 254-kD polypeptide and ankyrin are about 12-fold higher in D than in the DLE, suggest that, in D, spectrin has a specific organization.