Regulation of endothelial MAPK/ERK signalling and capillary morphogenesis by low-amplitude electric field

Abstract

Low-amplitude electric field (EF) is an important component of woundhealing response and can promote vascular tissue repair; however, the mechanisms of action on endothelium remain unclear. We hypothesized that physiological amplitude EF regulates angiogenic response of microvascular endothelial cells via activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway. A custom set-up allowed non-thermal application of EF of high (7.5 GHz) and low (60 Hz) frequency. Cell responses following up to 24 h of EF exposure, including proliferation and apoptosis, capillary morphogenesis, vascular endothelial growth factor (VEGF) expression and MAPK pathways activation were quantified. A db/db mouse model of diabetic wound healing was used for in vivo validation. High-frequency EF enhanced capillary morphogenesis, VEGF release, MEK-cRaf complex formation, MEK and ERK phosphorylation, whereas no 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 Ca 2þ 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 regulation of endothelial angiogenic response via frequency-sensitive MAPK/ERK pathway activation, with important implications for EF-based therapies for vascular tissue regeneration.

Modulation of cell function by electric field: a high-resolution analysis

“…Our numerical model demonstrates that by non-contact application of an EF, we can induce a sufficient EF in the cell membrane and cytoplasm to manipulate protein expression and activate intracellular pathways as supported by our experimental results [3]. Such numerical modelling of EF -cell interactions is an essential and necessary part of the clinical translation process; it helps to establish therapeutic concepts and to advance approaches toward the development of non-pharmacological EF therapies [106][107][108], which can be applied locally and without direct contact with tissue [101,105,[109][110][111][112].…”

“…The model follows the design of our recent experimental in vitro studies [3]. The electrodes used to generate the EF are isolated from the medium by dielectric spacers (figure 1a) and modelled as infinitely conducting planes.…”

“…Electric fields (EFs) can regulate a variety of cell functions, including growth, adhesion, reorganization of cytoskeleton, contractility, differentiation, proliferation, activation of intracellular pathways, secretion of proteins and gene expression [1][2][3][4][5][6][7]. The regulatory effect of EFs has been demonstrated on different cell types such as neurons, osteoblasts, myoblasts, fibroblasts, endothelial cells, muscle cell and epithelial cells [8][9][10][11][12][13][14].…”

“…The cell is modelled as a semi-spherical nonconducting shell separating two conducting regions, the culture medium and the cytoplasm, in direct contact with a flat dielectric substrate. To recapitulate our experimental configuration [3], the electrodes supplying the EF are isolated from the medium. The EF is therefore coupled to the cell and its environment capacitively, which eliminates electrochemical processes in the medium and reduces the electric current and associated ionic flow effects on the cell membrane.…”

Angiogenic microenvironment augments impaired endothelial responses under diabetic conditions

“…Diabetes-induced cardiac remodeling is associated with in vivo microvascular pathologies such as reduced expression of angiogenic growth factors and decreased endothelial cell infiltration and neovascularization (16,47). In vitro cell responses such as cytokine expression, cell proliferation, and migration, as well as Ca 2ϩ regulation are also altered under hyperglycemic conditions (8,13,16,33,53,60,65). However, there is a paucity of information regarding the differential effects of short-term hyperglycemic spikes vs. long-term exposure on vascular endothelial cells (14), and more studies are needed regarding the comprehensive effect of diabetic conditions on the angiogenic responses of cardiac endothelial cells at molecular and cellular levels.…”