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 wound-healing 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(2+) 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.

Figure 1.

Experimental set-up for microvascular endothelial cells exposure to EF. (a) High-frequency (7.5 GHz) EF set-up: an insert with endothelial cells was placed in the small plastic dish inside the cavity resonator to which EF was delivered from a vector network analyser (VNA) via a coaxial line. Cell culture medium flowed continuously between the cell insert and reservoir outside the resonator. (b) Low-frequency (60 Hz) EF set-up: plastic dish with the cell insert was placed between the plates of a parallel-plate capacitor. Red arrows in (a) and yellow arrows in (b) indicate direction of the electric field component, which was perpendicular to the cell surface in both configurations. Both set-ups operated inside a cell culture incubator (37°C, 5% CO₂). (c,d) Numerical calculation of the EF distribution using a three-dimensional model of the apparatus and ANSYS HFSS software demonstrated that while the EF distributions outside the insert were different between two configurations, EF distributions at the location of the cells were similar (c) and uniform in the central part of the insert (d). (e) Temperature of the sample medium remained constant at 37°C during EF exposure.

Figure 2.

Electric field enhances angiogenic response by microvascular endothelial cells. (a) Cell exposure to high-frequency EF significantly enhanced capillary morphogenesis, with clear multi-cellular tubule formation (20× magnified image, green: endothelial cells, blue: cell nuclei), resulting in larger characteristic network size (b) when compared with low-frequency and no-EF groups (n = 10, p < 0.001). Scale bar, 100 µm. (c) High-frequency EF stimulation resulted in significantly higher levels of VEGF released into the culture medium, when compared with the no-EF group (n = 3, p < 0.01).

Figure 3.

Angiogenic effects of EF do not require VEGF binding to VEGFR2 receptor. The effects of EF on capillary morphogenesis and VEGF release into the medium were retained in the presence of VEGFR2 blocking antibody (n = 4, p < 0.05), suggesting that EF-mediated stimulation of angiogenesis does not require VEGF ligand–receptor binding (a). Treatment of endothelial cells with a potent VEGFR2 inhibitor SU5416 effectively abolished capillary morphogenesis and VEGF release (b), but not high-frequency EF-induced MEK phosphorylation, which was significantly higher in the high-frequency group even in the presence of SU5416, when compared with low-frequency and no-EF controls (c). Interestingly, the relative magnitude of high-frequency EF-induced MEK phosphorylation normalized to no-EF (0.41 ± 0.09), no-EF + SU5416 (0.25 ± 0.04), no-EF + SU5426 + VEGF (0.44 ± 0.30), respectively, did not depend on the presence of SU5416 or exogenous VEGF and remained 1.5- to twofold higher than no-EF levels (p < 0.05, n = 3). This effect was not present in the low-frequency group.

Figure 4.

MAPK/ERK pathway is involved in EF-mediated angiogenic response. (a) Treatment with MEK inhibitor U0126 resulted in decreased characteristic network size in all groups (n = 5, p < 0.05), and effectively abolished the increase in capillary morphogenesis in the high-frequency EF group, when compared with low-frequency EF and no-EF groups (n = 5, p < 0.001), suggesting that angiogenic effects of EF involve the MEK/ERK pathway. (b) Similarly, the VEGF release by endothelial cells in the presence of U0126 was significantly reduced in all groups, with VEGF levels significantly smaller in high-frequency EF than no-EF group (n = 4, p < 0.001).

Figure 5.

High-frequency EF increases the phosphorylation of ERK, but not JNK or p38 MAP kinase in microvascular endothelial cells. (a) Exposure to EF did not alter the total protein levels of ERK, JNK or p38. (b) High-frequency EF significantly increased phosphorylation of ERK, when compared with low-frequency or no-EF groups (n = 6, p < 0.001). In contrast, no effect of high-frequency EF on JNK or p38 phosphorylation relative to no-EF controls was observed. In the low-frequency EF group, phosphorylated levels of ERK and p38 were significantly decreased when compared with the no-EF group (n = 6, p < 0.001).

Figure 6.

High-frequency EF enhances MEK phosphorylation and MEK–cRaf complex formation in microvascular endothelial cells. (a) Cell exposure to high-frequency EF resulted in significantly higher protein levels of phosphorylated MEK, while the total levels of MEK remained unchanged with EF exposure both in the absence or presence of MEK inhibitor U0126. (b) Cell exposure to high-frequency EF significantly increased protein levels and phosphorylation of MEK-cRaf complex, when compared with low-frequency or no-EF groups (n = 7, p < 0.001). This was consistent with low levels of free (unbound) MEK in the high-frequency EF group, when compared with those in low-frequency and no-EF groups (n = 7, p < 0.05). Free MEK levels in the low-frequency group were significantly larger than the values in the high-frequency and no-EF groups (n = 7, p < 0.05). (c) In contrast to the EF effects observed in the absence of U0126 in (b), cell exposure to high-frequency EF in the presence of MEK inhibitor resulted in significantly reduced protein levels and phosphorylation of MEK-cRaf complex, as well as increased free MEK levels (n = 7, p < 0.05), when compared with low-frequency or no-EF groups (n = 7, p < 0.01).

Figure 7.

Effects of PI3K and eNOS inhibition and Ca²⁺ chelation on EF-mediated MEK phosphorylation: (a) addition of PI3K inhibitor LY294002 did not abolish EF-induced increase in pMEK levels, when compared with no-EF controls (n = 4, p < 0.05), suggesting that this pathway may not be critical for EF-mediated angiogenic cell responses. (b) Interestingly, addition of Ca²⁺ chelator BAPTA resulted in significantly reduced pMEK levels, when compared with no-EF controls, which was similar to the trends in cell responses observed in the presence of MEK inhibitor (figure 4). (c) eNOS inhibition using l-NAME did not affect pMEK levels in low-frequency and no-EF groups, and effectively abolished high-frequency EF-induced increase in MEK phosphorylation (n = 4, p < 0.05). These results indicate the involvement of Ca²⁺ and eNOS pathways in EF-mediated MEK pathway activation.

Figure 8.

In vivo exposure to high-frequency EF enhances VEGF expression in diabetic wounds: (a) In vivo EF exposure set-up consisted of two antennae, which were connected to the EF source through a flexible co-axial cable. The antennae were approximately 5 mm away from the wounds created on the back of the diabetic mice. EF stimulation was applied for 1 h per day for 7 days. (b) Wound treatment with high-frequency 7.5 GHz EF significantly increased VEGF protein levels in the wound tissue when compared with no-EF treated control wounds (n = 5, p < 0.05).

Figure 9.

(a) Angiogenic signalling pathways and (b) proposed mechanism for high-frequency EF regulation of angiogenic response. Our results demonstrate that high-frequency EF enhances capillary morphogenesis and VEGF release via the MEK–cRaf step in the ERK pathway, with a possible involvement of Ca²⁺—and, to a lesser extent, eNOS-mediated MEK activation. The observed pro-angiogenic effects of EF are frequency-sensitive and are independent of VEGF binding to VEGFR2 receptor and PI3K signalling. A potential mechanism for these effects is that high-frequency EF may directly regulate the interaction of MEK protein with its binding partner cRaf (b), consistent with the possibility of gigahertz EF penetrating the cell membrane and affecting intracellular signalling. Thus, 7.5 GHz EF may enhance interaction between MEK and its binding partner (c-Raf or U0126), resulting in an increased complex formation, increased phosphorylation of MEK protein and activation of downstream MAPK/ERK pathway, ultimately leading to stimulation of angiogenic responses in the absence of U0126, or inhibition of angiogenic responses when U0126 is present.