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Physiological electric fields control the G1/S phase cell cycle checkpoint to inhibit endothelial cell proliferation¹

Department of Biomedical Sciences, Institute of Medical Sciences, and
^* Department of Ophthalmology, Medical School, University of Aberdeen, Aberdeen AB25 2ZD, UK

²Correspondence: Department of Biomedical Sciences, Institute of Medical Sciences, Aberdeen AB25 2ZD, Scotland, UK. E-mail: c.mccaig{at}abdn.ac.uk

SPECIFIC AIMS

Endogenous electrical fields (EFs) are common in areas where angiogenesis occurs during development, wound healing, and tumor growth and have profound influences on cell migration and the axis of cell division. We aimed to determine whether a physiological EF regulates proliferation of vascular endothelial cells (VECs).

PRINCIPAL FINDINGS

1. A physiological EF inhibited proliferation of cultured VECs
We applied a physiological EF in culture and determined its effect on proliferation. Cell number and cell density (cell number/cm²) of cultures were assessed and mean cell density was calculated for specific time points. Cell growth rates were calculated at 24, 48, and 72 h as [(Ni - N0)/N0] ·100%, where N0 was the initial cell number before EF exposure (0 h time point) and Ni was the cell number at a specific time after EF exposure. Cells also were stained with an anti--tubulin antibody to label mitotic spindles and the numbers of cells in mitosis was counted. A mitosis index (proportion of mitotic cells per 1000 cells) was obtained. In cultures with no EF, cell density increased with time. In an EF of 200 mV/mm, VEC density increased only marginally even over 72 h (Fig. 1 A). An EF of 200 mV/mm also reduced the mean cell growth rate (Fig. 1B, C ) and decreased the mitotic index of VECs. Mitotic indices of cells at 200 mV/mm were 1.86% (12 h) and 1.78% (24 h), significantly lower than the mitotic indices of control cultures, which were 3.2% (12 h) and 3.6% (24 h; P<0.01). By contrast, VECs exposed to a lower EF (50 or 100 mV/mm) proliferated at normal rates and showed the same levels of increasing growth rate as control cultures (Fig. 1C ).

View larger version (21K): [in this window] [in a new window]   Figure 1. An applied EF reduced cell density and cell growth rate. Cell density (A) and growth rate (B, C) were reduced after EF exposure of 200 mV/mm, but not at 50 or 100 mV/mm. The apoptosis inhibitor Z-VAD-FMK did not prevent the reduction of cell proliferation induced by EF exposure of 200 mV/mm (B). EF exposure of 50 mV/mm or 100 mV/mm did not significantly influence cell growth ability (C). *P < 0.01 compared with control without EF exposure. Data are expressed as mean ± SE. Numbers of fields of view assessed from a minimum of 2 cultures were 34 (control), 44 (EF exposure of 50 mV/mm), 44 (EF exposure of 100 mV/mm), 29 (EF exposure of 200 mV/mm), and 34 (EF exposure of 200 mV/mm with Z-VAD-FMK), respectively.

2. A physiological EF did not induce apoptosis
The lack of significant increase in cell numbers in VEC cultures at 200 mV/mm could have been due to apoptosis offsetting proliferation rather than an inhibition of proliferation. To test this, cultures were exposed to 200 mV/mm in the presence of the caspase apoptosis inhibitor Z-VAD-FMK (20 µM). These cultures still failed to increase cell numbers and were indistinguishable quantitatively from those at 200 mV/mm with no drug (Fig. 1B ). To quantify any apoptosis, we stained for caspase-3. VECs exposed to an EF of 200 mV/mm for 4, 8, 12, or 24 h did not show caspase-3 staining, but caspase-3 staining was prominent in VEC cultures treated with staurosporine as a positive control.

Flow cytometry also was used to determine whether apoptosis had occurred. The sub-G1 peak, indicative of apoptosis, was not found on the DNA content histogram in EF-exposed VECs. Apoptosis cannot account for the EF-induced inhibition of VEC proliferation.

3. A physiological EF induced cell cycle arrest
Flow cytometry showed that an EF of 200 mV/mm inhibited cell cycle progression through the G1/S transition. Compared with control cells, the percentage of the cell population in the S and G2/M phases in EF-exposed cells decreased significantly whereas the percent of the cell population in the G₁ phase increased significantly. Thus, EF-induced inhibition of VEC proliferation resulted from cell cycle arrest or G1 block.

4. A physiological EF controlled expression of cell cycle regulators
Western blot analysis of VECs showed that EF exposure 1) induced a significant decrease in the expression of cyclin E, 2) did not influence expression of Cdk2, and 3) increased significantly the expression of p27^kip1, an inhibitor of the cyclin E/Cdk2 complex. The level of p27^kip1 increased significantly after 6 h EF exposure and maintained a high level for up to 24 h compared with control (Fig. 2 ).

View larger version (62K): [in this window] [in a new window]   Figure 2. Western blot analyses for expression of cyclin E, Cdk2, and p27kip1. Human VECs were exposed to a direct current EF of 200 mV/mm. At 6, 12, and 24 h after EF exposure, respectively, the cells were lysed. Cells without EF exposure were controls. Identical amounts of protein lysates were resolved by SDS-PAGE, followed by electroblotting onto nitrocellulose membranes. The membranes were probed with anti-cyclin E, anti-Cdk2, and anti-p27kip1 antibodies, respectively. After incubation with antibodies conjugated with HRP, immunoblots were detected by the enhanced chemiluminescence (ECL) detection system. EF exposure down-regulated the expression of cyclin E and up-regulated the expression of cyclin E/Cdk inhibitor p27kip1, but did not influence the expression of Cdk 2.

CONCLUSIONS AND SIGNIFICANCE

EFs exist in vivo and have a profound influence on cell migration and cell division. Applied EFs have effects both in vivo and in vitro. For example, weak electrical stimulation induced an increase in capillary density in the ischemic limb of rat by enhancing cell migration and cell proliferation. Others have shown that EF exposure induced a strength-dependent reduction in the growth rate of plant root cells and mammalian cells. The effects of an applied EF on cell proliferation therefore depend on the precise stimulation parameters and on cell type. Here, an EF of 50 or 100 mV/mm had no effect on proliferation of VECs, but at 200 mV/mm proliferation was inhibited. The threshold for reducing proliferation therefore is 100-200 mV/mm, or 2.5-5 mV extracellularly, across a cell 25 µm in diameter.

Many antiproliferate factors trigger apoptosis. Caspases are mediators of the execution phase of apoptosis, and caspase-3 is a major player. Z-VAD-FMK is a cell-permeable inhibitor of caspase-3 and inhibits the induction of apoptosis. We found, however, that Z-VAD-FMK (20 µM) did not prevent the lack of VEC proliferation induced at 200 mV/mm. This indicates that the EF-induced prevention of cell proliferation was not due to enhanced apoptosis.

Many of the signals controlling proliferation act during G1 phase. A physiological EF inhibited VEC proliferation by inhibiting cell cycle progression at the G1/S transition. Cell proliferation is controlled by various cyclin/Cdk complexes. The Cdk2-cyclin E complex is a pivotal controller of G1/S transition. Cyclin E, a G1-specific cyclin, is necessary and rate-limiting for the passage of mammalian cells through G₁ phase. In EF-exposed cells, cyclin E decreased markedly but the expression level of Cdk2 did not change. Without their cyclin partners, the Cdks are inactive; therefore, reduced expression of cyclin E will inactivate the Cdk2-cyclin E complexes and prevent passage through G1.

Cdk inhibitors prevent cell proliferation by negatively regulating cyclin-Cdk complexes. Many extracellular stimuli exert checkpoint control by inducing members of the Cip/Kip families of cell cycle kinase inhibitors, such as p27^kip1. p27^kip1 activity increases in response to growth inhibitory signals and decreases in response to positively acting growth factors. In quiescent cells, the level of p27^kip1 is relatively high. p27^kip1 protein associates with G1-specific cyclin-Cdk complexes, inhibits the activity of cyclin E/Cdk2, and induces G1 arrest. Here, inhibition of VEC proliferation by a physiological EF was accompanied by a significant increase in the expression of p27^kip1 within 6 h, which was sustained through 24 h. A physiological EF therefore arrested the cell cycle of VECs at G1 by selectively up-regulating p27^kip1 expression and down-regulating cyclin E expression.

This work has both physiological and pathological significance. The existence of electrical potential differences around blood vessel endothelium and of zeta potentials created at the blood-endothelial membrane interface by the flow of blood indicates that endogenous EFs from various sources and with varying temporal patterns could exert control over VEC proliferation. This could occur during normal cell turnover and in angiogenesis. For example, the cell cycle of VECs is much shorter at bifurcations, where flow is more turbulent, than in long uninterrupted vessels, where laminar shear stress is greater. Cell cycle control in these situations depends on cell shape; shear-stressed/elongated cells turn over more slowly. However, the slow proliferation of endothelial cells in a high flow situation is also correlated with the local presence of elevated electrical signals. Areas of high shear stress, where endothelial cells become elongated, are areas where high streaming potentials and high zeta potentials exist and angiogenesis is low. The opposite is true in the postcapillary venous system, where lower shear stress, lower electrical signals, and fewer elongated cells result in greater cell proliferation and more angiogenesis. Intriguingly, an EF also induces an elongated shape in VECs, and so the separate contribution of mechanical and electrical influences in controlling the cell cycle is unclear.

Spatial variations in extracellular potentials exist between damaged and healthy tissue, and these give rise to steady dc EFs. These extracellular EFs have been used diagnostically to locate tumors. Since an EF induces slower proliferation and directed cell migration, there may be a direct link between oriented angiogenesis toward tumors and wounded regions. Finally, these data raise the possibility of devising in vivo strategies in which EFs are applied to promote or inhibit directed angiogenesis into specific regions.

View larger version (40K): [in this window] [in a new window]   Figure 3. Schematic diagram illustrating the effects of physiological electric fields on the cell cycle control of endothelial cells.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0510fje; to cite this article, use FASEB J. (January 22, 2003) 10.1096/fj.02-0510fje

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