Low-amplitude electric field (EF) is usually an important component of wound-healing response and can promote vascular tissue repair; however, the mechanisms of action on endothelium remain ambiguous. MAPK/JNK and MAPK/p38 pathways activation was observed. The endothelial response to EF did not require VEGF binding to VEGFR2 receptor. EF-induced MEK phosphorylation was reversed in the presence of MEK and Ca2+ inhibitors, reduced by endothelial nitric oxide synthase inhibition, and did not depend on PI3K pathway activation. The results provide evidence for a novel intracellular mechanism for EF rules of endothelial angiogenic response via frequency-sensitive MAPK/ERK pathway activation, with important ramifications for EF-based therapies for vascular tissue regeneration. angiogenesis in both ischaemic and non-ischaemic rat limbs [5,12,13] and in mouse wound healing [14]. Migration, tubular formation, proliferation and vascular endothelial growth factor (VEGF) manifestation in human umbilical cord endothelial cells (HUVECs) were stimulated by direct current (DC) as well as pulsed electromagnetic fields [10,15C17]. Importantly, the majority of previous studies have used in-plane DC field configuration, where exposure to the DC EF resulted in dramatic cell reorientation and directional migration (electrotaxis) [10], as well as an altered pattern of integrin receptor clustering and the associated actin reorganization in endothelial cells and fibroblasts [2,18,19]. However, there is usually significant variability in EF-induced cell migration, not 23076-35-9 manufacture only between cells of different types [2], but also between endothelial cells of different source. Thus, bovine aortic endothelial cells migrate towards cathode [18], while HUVECs migrate towards anode [10]. Overall, experimental evidence suggests that the mechanisms responsible for EF-mediated angiogenic endothelial cell activation may be different from those that 23076-35-9 manufacture govern electrotaxis. Therefore, activation of electrotaxis alone may not necessarily result in an overall enhanced angiogenic response and improved wound healing. This is usually consistent with the results of the clinical studies that suggest that a pulsed (not DC) EF may be the most efficient modality in the treatment of chronic wounds [7,8,20] and in alleviating the symptoms of multiple sclerosis [9,21,22]. Importantly, mechanistic understanding of EF effects on endothelial cells is usually essential for the informed choice of the field parameters for wound-healing therapies. Among the intracellular responses that may be mediated by EF, mitogen-activated protein kinase (MAPK) signalling cascade family [23] is usually the main candidate. Of this family, extracellular signal-regulated kinase (ERK), c-Jun NH2-airport terminal kinase (JNK) and stress-activated protein kinase-2 (p38) pathways are known to be involved in angiogenic as well as stress-activated signalling in the absence of EF [24C30]. There is usually also evidence that these pathways can be activated in response to EF. It has been reported that 900 MHz mobile phone radiation activated the warmth shock protein 27 (Hsp27)/p38MAPK stress response pathway in human endothelial cells [31], while a 50 Hz sinusoidal magnetic field affected the cellular distribution of Hsp27 and increased DGKD Hsp70, but not Hsp27 mRNA in aortic endothelial cells [32]. Also, DC EF activated ERK, JNK and p38 in embryonic stem cells and induced endothelial differentiation [33]. Different types of electromagnetic fields have been shown to impact the activation of ERK, JNK and p38 in several non-endothelial cell types [34C36]. However, the role of different EF modalities on MAPK activation in endothelial cells is usually not comprehended. Previous studies have shown that EF-induced intracellular responses in non-endothelial cells may depend on the field frequency [2,37]; however, the possible role of this parameter in angiogenic responses of endothelial cells to EF is usually not known. It has been suggested [38] that at frequencies below 100 MHz, the cell (including cytoplasm and nucleus) can be considered as a conductive media surrounded by high capacitance membrane, which results in excluding the field from the cell cytoplasm. In contrast, at higher frequencies (gigahertz range), the low membrane impedance allows the current to circulation through intracellular space (dielectric behaviour), which results in the field penetration across the membrane. The experimental evidence in this area remains 23076-35-9 manufacture limited. The objective of this study was to elucidate the possible intracellular mechanisms for EF-mediated angiogenic responses in endothelial cells in a controlled establishing in the absence of electrotaxis, to allow direct mechanistic meaning of the data. We tested the hypothesis that EF with amplitudes in the physiological range regulates endothelial angiogenic response via activation of MAPK/ERK pathway. Experiments were conducted by using a custom-engineered multi-component system for microvascular endothelial cell exposure to EF with spatially controlled field distribution, combined with cell culture, microscopy and molecular.