Directing migration of endothelial progenitor cells with applied DC electric fields

Abstract

Naturally-occurring, endogenous electric fields (EFs) have been detected at skin wounds, damaged tissue sites and vasculature. Applied EFs guide migration of many types of cells, including endothelial cells to migrate directionally. Homing of endothelial progenitor cells (EPCs) to an injury site is important for repair of vasculature and also for angiogenesis. However, it has not been reported whether EPCs respond to applied EFs. Aiming to explore the possibility to use electric stimulation to regulate the progenitor cells and angiogenesis, we tested the effects of direct-current (DC) EFs on EPCs. We first used immunofluorescence to confirm the expression of endothelial progenitor markers in three lines of EPCs. We then cultured the progenitor cells in EFs. Using time-lapse video microscopy, we demonstrated that an applied DC EF directs migration of the EPCs toward the cathode. The progenitor cells also align and elongate in an EF. Inhibition of vascular endothelial growth factor (VEGF) receptor signaling completely abolished the EF-induced directional migration of the progenitor cells. We conclude that EFs are an effective signal that guides EPC migration through VEGF receptor signaling in vitro. Applied EFs may be used to control behaviors of EPCs in tissue engineering, in homing of EPCs to wounds and to an injury site in the vasculature.

Figure 1. Expression of progenitor markers

The three cell lines MFLM-4 (A), AEL-deltaR1/Runx1 (B), and AEL-deltaR1 (C) express markers for endothelial progenitor cells: CD133 (green), VEGFR2 (red), and von Willebrand Factor (green). Blue is DAPI staining for nucleus. Scale bars = 10 µm.

Figure 2. Electric field-directed migration of MFLM-4 cells

A DC EF directs EPCs (MFLM-4) to migrate to the cathode. Reversing the polarity of the EF reversed the cell migration direction. (A) Time lapse photographs of cell migration in an EF for 6 hours. Arrows show direction of the cells migrated. Outlines (A’) of the labelled cells from 3–6 hr highlight cell migration. Cells are traced starting from 3 hour for clarity. (B) Composite graphs of cell trajectories without an EF. The trajectories of the cells are arranged with the starting position of each cell placed at the origin. Cells migrated without preferred direction. (C) Application of an EF induced directional cell migration to the cathode on the right. (D) Reversing the EF reversed the directional cell migration to the new cathode on the left. Values of mean directedness ± SEM are shown. (E) Voltage dependence of the directional cell migration. All data were calculated from three independent experiments. **: P < 0.01 compared with no EF. Scale bars = 50 µm.

Figure 3. MFLM-4 cell response in small physiological EFs

(A) Trajectory speed (Tt/T) and voltage dependence of displacement speed (Td/T) and displacement speed along the X axis (Dx/T). (B) Voltage dependence of orientation index. (C) Long/short axis ratio in different EFs. All data were calculated from three independent experiments. *: P < 0.05; **: P < 0.01 compared with no EF.

Figure 4. An applied EF directs migration of two other EPC cell lines

(A–C) AEL-deltaR1 cell response in small physiological EFs (300 mV/mm). (D–F) AEL-deltaR1/Runx1 cell response in small physiological EFs (300 mV/mm). (A, D) Time lapse photos of cell migration in an EF. (B, E) Composite graphs of migration trajectories of the two progenitor cells. Cells migrate to the cathode at the right in an EF. (C, F) Directedness of cell migration. All data were calculated from three independent experiments. **: P < 0.01 compare with no EF. Scale bar = 50 µm.

Figure 5. AEL-deltaR1 and AEL-deltaR1/Runx1 cell response in an EF (300 mV/mm)

(A–C) AEL-deltaR1 cells. (D–F) AEL-deltaR1/Runx1 cells. (A, D) Trajectory speed (Tt/T), displacement speed (Td/T), and displacement speed along the X axis (Dx/T). (B, E) Orientation index. (C, F) Long/short axis ratio. All data were calculated from three independent experiments. **: P < 0.01 compared with no EF.

Figure 6. Activation of VEGFR-2 is required for electrotactic response of EPCs

(A) Directedness of cell migration was abolished when VEGFR-2 was inhibited with VEGFR2I. (B) Trajectory speed, displacement speed, and displacement speed along the X axis of cell migration were also affected with VEGFR2I. VEGFR2I = VEGF receptor 2 inhibitor. All data were calculated from three independent experiments. *: P < 0.05; **: P < 0.01 compared with control (no EF).

Figure 7. Activation of VEGFR-2 is required for response of EPCs to an applied EF

(A) Orientation index. (B) Long/short axis ratio. VEGFR2I = VEGF receptor 2 inhibitor. All data were calculated from three independent experiments. *: P < 0.05: **: P < 0.01 compared with control (no EF).