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First published online 16 December 2003
doi: 10.1242/jcs.00868

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Electrical stimulation directly induces pre-angiogenic responses in vascular endothelial cells by signaling through VEGF receptors

Min Zhao^*,, Huai Bai^*, Entong Wang, John V. Forrester and Colin D. McCaig

Departments of Biomedical Sciences and Ophthalmology, Institute of Medical Sciences, University of Aberdeen, Aberdeen, AB25 2ZD, UK

View larger version (108K): [in a new window]   Fig. 1. Perpendicular orientation and elongation of endothelial cells in a small physiological EF. (A) Control HUVEC cells cultured in the same chamber without EFs showed a typical cobblestone morphology and random orientation. (B) Cells exposed to small applied EFs showed dramatic elongation and perpendicular orientation in the EF. (C) Cells treated with a VEGFR inhibitor that completely abolished perpendicular orientation and significantly inhibited elongation in an applied EF (72 hours, 100 mV mm–1). (G-I) Most actin filaments (red) and microtubules (green) became aligned along the long axis of the cells (12 hours at 150 mV mm–1). (D-F) No-field controls showed no obvious alignment and cell elongation. (A,B) Images taken with Hoffman modulation optics. (C) Image taken with phase-contrast optics. (F,I) Merged images.

View larger version (78K): [in a new window]   Fig. 2. Endothelial cells reoriented, elongated and migrated directionally in a small physiological EF. Obvious elongation and orientation of cells can be seen after 8 hours in an EF. Directional lamellipodial extension and cell migration were evident at 12 hours and 24 hours after EF exposure. Scratches made on the culture dish as static reference points can be seen to the left and top of each frame.

View larger version (26K): [in a new window]   Fig. 3. Time and voltage dependency of orientation of endothelial cells in a small physiological EF. (A) Orientation index as a function of time and voltage (n=65-598 from at least two independent experiments). (B) Effects of various drugs on EF-induced perpendicular orientation. Inhibition of PI3K (LY), Akt (Akt-i), Rho (Y27632) and actin polymerization (Latrunc) significantly decreased orientation responses, whereas inhibition of VEGFR (VEGFR-i), or combined inhibition of both Akt and Rho (Akt-i + Y) completely abolished the orientation response. VEGFR-i, VEGFR inhibitor (50 µM); LY, PI3K inhibitor LY294002 (50 µM); Akt-i, Akt inhibitor (50 µM); Y27632, Rho inhibitor (50 µM); Akt-i + Y27632, Akt and Rho inhibitors (10 µM each); Latrunc, Latrunculin (50 µM). Endothelial cells were subjected to EFs of 200 mV mm–1 for 24 hours. n=47-343 from at least two independent experiments. **, P<0.0001 compared with cells exposed to 200 mV mm–1 without drug treatment.

View larger version (20K): [in a new window]   Fig. 4. Exposure to a physiological EF increased VEGF release from endothelial cells. HUVEC cells were cultured in serum-free DMEM and exposed to an EF of 200 mV mm–1. VEGF in the medium was quantified by ELISA. Values are means±s.e.m.

View larger version (28K): [in a new window]   Fig. 5. Time and voltage dependency of elongation of endothelial cells in a small physiological EF. (A) Cell elongation as a function of time and voltage (n=65-598 from at least two independent experiments). (B) Effects of various drugs on EF-induced cell elongation. Inhibition of PI3K (LY), Akt (Akt-i), Rho (Y27632) and actin polymerization (Latrunc) significantly decreased the elongation response, as did inhibition of VEGR (VEGFR-i), and the combined inhibition of both Akt and Rho (Akt-i + Y). VEGFR-i, VEGFR inhibitor (50 µM); LY, PI3K inhibitor LY294002 (50 µM); Akt-i, Akt inhibitor (50 µM); Y27632, Rho inhibitor (50 µM); Akt-i + Y27632, Akt and Rho inhibitors (10 µM each); Latrunc, Latrunculin (50 µM). Cells were subjected to EFs of 200 mV mm–1 for 24 hours. n=47-343 from more than two independent experiments. *, P<0.002 compared with cells exposed to 200 mV mm–1 without drug treatment; **, P<0.0001 compared with cells exposed to 200 mV mm–1 without drug treatment; #, P<0.001 compared with same drug treatment but not exposed to EFs; ##, P<0.0001 compared with same drug treatment but not exposed to EFs.

View larger version (104K): [in a new window]   Fig. 6. Directional endothelial cell migration in a small physiological EF. (A) Endothelial cells in culture exposed to an EF of 100 mV mm–1 migrated directionally towards the anode. Cells migrated slowly but steadily toward the anode over 24 hours. Movement is evident using the static scratch on the culture dish as a reference (right margin). Notice that lamellipodia extended preferentially toward the anode. (B) Scatter plot showing biased migration of endothelial cells in an EF. Cells started at the origin and each dot represents the position of each cell 4 hours later. The distribution of the cells shifted towards the anode. Radius=7 µm.

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