Electric stimulation in physical therapy has been used for many years in repairing non-healing soft and hard tissue. DC mode ES can redistribute cell membrane receptors such as integrin via electrophoresis and electroosmosis mechanism[16] even in the 3-D environment [17]. The EF-induced integrin
redistribution serves as the site for formation of a focal adhesion by recruiting other molecules such as actin-binding proteins and signaling molecules[19] and influences the cell migration directionality.
Based on these proposed mechanisms, we first hypothesize that different
waveform (direct current, diadynamic current, faradic current, sinusoidal current, and high voltage pulsed galvanic stimulation) caused different integrin distribution and induced different fibroblast cell behavior (cell migration, orientation). Therefore, we investigated the effects of physical therapy electrical stimulation on ligament cell migration and morphology. Most of the waveforms we tested resulted in enhanced fibroblast migration speed, while their effects on cell migration directionality were noticeably different. As we have previously shown, bovine ACL fibroblast and chondrocytes both exhibit different EF threshold response in motility and
directionality[4, 5]. Furthermore, ACL fibroblast exhibit frequency dependent
migration behaviors [4]. Using different waveform and field strengths, we discovered a decoupling of cell shape and directionality, which may suggest disparate mechanisms in the two responses [36]. Faradic ad diadynamic stimulation promoted the most significant response in both speed and directionality. Faradic stimulation has been shown to promote collagen organization while diadynamic stimulation promotes skin wound healing [7, 12]. Interestingly, while HVPGS has been demonstrated to increase proliferation, collagen synthesis and skin wound healing, no significant effect of increased cell migration is shown here [37].
In terms of cell migration behavior under different field strengths DC EF, interestingly, no directionality was found with the constant 0.19 V EF group even though it has similarly enhanced migration speed with the 30 V group. In here, we hypothesize disparate mechanism control migrate speed and directionality. Zhao et al.
[38] demonstrated using pharmacological inhibitors to find that directionality and migration rate are regulated by separable pathways. To determine the effects of the electric waveforms on cell migration behavior and the major factors control migration mechanism, we investigated EF-induced integrin redistribution.
When analyzing the asymetric index, we found out the asymmetric value between groups are related to EF-induced directionality. We further look into the
cause and effect between integrin redistribution and EF-induced directionality to clarify the signaling mechanism.
We investigated the time dependent redistribution effectiveness firstly.
Asymmetric distribution of integrin receptors labeled with specific antibodies was evident as early as 5 min after EF onset. Integrin redistribution occurs within minutes, which is the early and proximal event like EF-induced cell directionality. Furthermore, we decreased cell membrane fluidity by cholesterol hemisuccinate to reduce
EF-induced integrin redistribution. Membrane lipid fluidity depends mainly on the cholesterol content as well as on the degree of saturation of phospholipid acyl chains and phospholipid polar head group composition [39, 40] . Therefore we modify the physical state of membrane lipid matrix (i.e., the membrane lipid fluidity or microviscosity) by alteration of the cholesterol content. The fluorescence recovery after photobleaching (FRAP) experiment shows ChH-treated cell membrane diffusion coefficient is lower than the normal cell membrane. Moreover the ChH-treated
membrane eliminated polarization of integrin in the EF, and decreased EF-induced directionality, but did not affect the migration speed. Since the ChH treatment is the non-specific way to reduce the integrin redistribution, we further blocked integrin function specifically by anti-integrin antibody. The Ab-treated cell also decreased EF-induced directionality but did not affect speed; its effect on migration behavior is
same as ChH-treated cell. When using confocal microscopy to measure EF-induced integrin distribution, we found that integrin also redistributed toward cathode. Even though applied EF polarized the antibody-bound integrin, the functional blocking treatment abolished the directional response. Taken together, our results indicate that activation and polarization of α2β1 integrin mediates EF-induced directionality.
EF not only directs cathodal redistribution of integrin but up-regulates the integrin receptor [30]. Numerous steps have been taken toward understanding the Rho GTPases during cell polarization and directional migration, and Rho GTPases have emerged as key regulators in cell migration directionality [43].This introduces EF activates one of the major signaling molecules, which is downstream factor of mediating migration from the integrin asymmetrically.
Our current data show that this occurs in parallel with a cathodal redistribution of RhoA. We report on the involvement of α2β1 integrin mediated RhoA polarization in EF-induced migration. α2β1 integrin specifically binds to the main ligament matrix component, type I collagen, and α2 integrin has been shown to modulate ligament fibroblast migration. By functionally blocking α2β1 integrin with its antibody, we inhibited directionality without changing motility. This response coincides with the lack of RhoA polarization. Inhibition of RhoA also abolishes EF-induced
directionality.
The mechanism underlying the key role between RhoA and integrin
redistribution is not clearly understood. One likely possibility role on redistributing and activating RhoA is PKA. RhoA is a PKA substrate [41] and PKA activity at the leading edge exhibited a close temporal and spatial correlation with the formation of
activation of RhoA and cell protrusions [42]. We report on the inhibition of PKA activity by inhibitor also diminished EF-induced directionality, but EF-induced RhoA polarization still existed. Even though applied EF polarized the RhoA, the PKA inhibitor treatment abolished the directional response.
In summary, we have shown that EF stimulates the formation of adhesion molecules redistribution during cell migration, and cell migration directionality is dependent on integrin and RhoA polarization. Moreover RhoA redistribution is dependent on integrin-mediated signals. These findings have contributed to the understanding of some of the modes of action of EF in cell migration directionality.
Finally, our studies additionally identified waveform-induced disparate migration speed and directionality, and signaling molecules polarization under EF that will be mediators for EF-induced directionality. Results from this study may benefit our understanding the electro-therapy treatment on cell behavior and provide further treatment options.
Figure 1 The composition of a galvanotaxis chamber. (a) Cells are put on the center of glass slide and then fit into the chamber. The transparent chamber permits direct observation for cell behaviors with a microscope. (b) The chamber is connected to saline reservoirs with 2% agarose salt bridge. We establish stable electric field across the chamber with external power supply. (Adaped from Chao PH, Roy R, Mauck RL, Liu W, Valhmu WB, & Hung CT (2000) J Biomech Eng 122, 261-267.)
Figure 2 Constant direct current (DC) EF was applied at either 30 V or 0.19 V across the chamber using a Keithley SourceMeter. Other waveforms that were applied using a custom stimulator (Dynaprog, MingQuo, Taiwan) as illustrated in Figure 2.
All waveforms and the constant 0.19 V group except for HVPGS were controlled to have the same amount of total current flow (1.9×10-5As). Faradic current (Far) represented a graduation series of triangular waves peaking at 15 V at 50 Hz.
High-voltage pulsed galvanic stimulation (HVPGS) consists of monophasic,
twin-spike pulses that have a fixed pulse duration of 100 μs and max intensity at 100 V with a frequency of 2.5 Hz. Sinusoidal waves (sin) have a peak intensity of 1.2 V at 50 Hz and the diadynamic waves (diadyn) are rectified monophasicc sinusoidal waves
Speed
Figure 3A Under electrical stimulation, ACL fibroblasts exhibited significantly different responses in migration. All groups except for the HVPGS group exhibited enhanced migration speed. When compared with the groups subjected to constant DC EFs, all the other groups were slower except for the diadynamic group (* means p<0.05 vs 0V control, § means p<0.05 vs. 30 V group, n=53-99 cells).
0 2 4 6
0V 30V 0.19V Far HVPGS sin diadyn
Speed ( μ m/hr)
* *
* * *
§ §
§
Directional velocity
Figure 3B In terms of directional velocity, all groups except for the HVPGS group exhibited enhanced migration directional velocity. When compared with the groups subjected to constant 30V DC EFs, all the other groups were slower (*
means p<0.05 vs 0V control, § means p<0.05 vs. 30 V group, n=53-99 cells).
Interestingly, no directionality was found with the constant 0.19 V EF group even though it has similarly enhanced migration speed with the 30 V group.
-6 -4 -2 0
Di rect iona l V el oc ity (μ m/hr )
*
§
§
* §
* §
* §
0V 30V 0.19V Far HVPGS sin diadyn
Figure 4 Morphological examination revealed differential cell shape responses.
In the constant 30 V group, ACL fibroblasts exhibited significant elongation and aligned perpendicular to the constant applied EF groups. All other groups did not exhibit any significant elongation or orientation (*p<0.05 vs. the 0V control, §p<0.05 vs. the 30 V group, n= 41-156 cells)
0V 30V 0.19V Far HVPGS sindiadyn
A spect Ratio
26
+
-0.788 (a)
(b) 0.7 1.45
(C)
Figure 5 Under electrical field, most of waveforms redistributed integrin toward cathode except for HVPGS, 0.19V DC EF and sine wave. Sine wave induce integrin move toward anode. (a) immunostaining for integrin α2β1 (b) normalized integrin distribution (c) asymmetric Index (AI: I cathode- I anode). *p<0.05 vs. the 0V control,
§p<0.05 vs. the 30 V group, n= 8-30 cells
-1.5
-1 -0.5 0 0.5 1
0V 30V 0.19V Far HVPGS sin diadyn
*
* *
* §
§
§
§
Figure 6 effect of cholesterol treatment on FRAP in membrane. Typical FRAP curves (a) of DiI labeled membrane, with (red) or without cholesterol hemisuccinate (blue). Average T 1/2 values are shown (n= 15–20). Mean ± s.e.m. (n= 15–20) of the diffusion coefficient (D= A/ 4T1/2) are shown on the lower-left panels (*P< 0.05).
Cholesterol induced a decrease in the diffusion rates and fluorescence recovery intensity, accompanied by lower diffusion coefficient (b) and relative intensity (c).
0
Diffusion coefficient Relative intensity
Normalized fluorescence intensity
Figure 7 In 30V and sine group, both control and integrin antibody treatment can induce integrin redistribution, but cholesterol treatment cause integrin distribute randomly (a) (★p<0.05 vs. 0V, ※p<0.05 vs. the 30V control, §p<0.05 vs. the sine control group, n= 8-30 cells). Both ChH and antibody treatment, the migration speed is significant faster on the 30V DC EF and sine wave group (b), but they slower EF-induced directionality (c). (★p<0.05 vs. 0V, ※p<0.05 vs. the 30V control,
§p<0.05 vs. the sine control group, n= 71-194 cells) -1.5
control ChH Antibody
★
★
Directional Velocity (μm/hr)
※
Figure 8 In short-term EF expose, both 5 min and 10 min period affect integrin redistribute toward cathode (* p< 0.05 to 0V control, § p< 0.05 to 10 min period 30V DC EF, n= 15-30 cells).
0V 30V (5min) 30V (10min) 30V (1hr)
0.803
Figure 9 For 1hr DC EF, the RhoA redistributed toward cathode. Other than EF- induced RhoA cathodal distribution, sine wave induce RhoA move toward anode (*means p<0.05 vs. 0V, n= 10-20 cells). According to AI of RhoA and integrin distribution, the EF-induced RhoA redistribution coincides with integrin
-4
AI of RhoA distribution
sine 0V 30V
Figure 10 For 1hr exposure to 30V DC EF, the RhoA redistributed toward cathode, furthermore, inhibition of RhoA activation had a similar effect on RhoA redistribution in applied EF, but anti-integrin antibody treatment caused RhoA distribute randomly. (*means p<0.01 vs. all other groups, § means p<0.01 vs. DC group, n=10-20 cells)
-0.5 0 0.5 1 1.5 2 2.5 3
no EF DC DC+AB DC+KT5720
*
*§
AI
Figure 11 Inhibition of RhoA and PKA activation by C3 transferase and KT 5720 had similar effect on cell migration. The migration speed is significant faster on the 30V DC EF (a) but they slower EF-induced directionality (b). (*p<0.05 vs. 0V,
§p<0.05 vs. other treatment groups, n= 35-118 cells) 0
control C3 transferase KT5720
Directional Velocity (μm/hr)
* *
*§
(a)
(b)
*§
Reference
1. Kloth, L.C., Electrical stimulation for wound healing: a review of evidence
from in vitro studies, animal experiments, and clinical trials. Int J Low Extrem
Wounds, 2005. 4(1): p. 23-44.
2. Aaron, R.K., D.M. Ciombor, and B.J. Simon, Treatment of nonunions with
electric and electromagnetic fields. Clin Orthop Relat Res, 2004(419): p. 21-9.
3. Akai, M., et al., Electrical stimulation of ligament healing. An experimental
study of the patellar ligament of rabbits. Clin Orthop Relat Res, 1988(235): p.
296-301.
4. Chao, P.H., et al., Effects of applied DC electric field on ligament fibroblast
migration and wound healing. Connect Tissue Res, 2007. 48(4): p. 188-97.
5. Carley, P.J. and S.F. Wainapel, Electrotherapy for acceleration of wound
healing: low intensity direct current. Arch Phys Med Rehabil, 1985. 66(7): p.
443-6.
6. Campbell, C.E., D.V. Higginbotham, and T.J. Baranowski, Jr., A constant
cathodic potential device for faradic stimulation of osteogenesis. Med Eng
Phys, 1995. 17(5): p. 337-46.
7. Ciullo, J.V. and B. Zarins, Biomechanics of the musculotendinous unit:
relation to athletic performance and injury. Clin Sports Med, 1983. 2(1): p.
71-86.
8. Philipson, T., et al., [The effect of diadynamic current on chronic soft-tissue
pain in the neck and shoulder girdle]. Ugeskr Laeger, 1983. 145(7): p. 479-81.
9. Lisinski, P., W. Zapalski, and W. Stryla, [Physical agents for pain
management in patients with gonarthrosis]. Ortop Traumatol Rehabil, 2005.
7(3): p. 317-21.
10. Romanenko, S.G., O.P. Tokarev, and S. Vasilenko Iu, [Electrostimulation of
laryngeal muscles with fluctuating currents in the treatment of patients with
unilateral laryngeal paralysis]. Vestn Otorinolaringol, 2001(3): p. 52-4.
11. Rusiaev, V.F., Z.N. Salienko, and V.F. Pavlenko, [Effect of a diadynamic
current on the development of thromboembolism]. Patol Fiziol Eksp Ter,
1983(1): p. 10-4.
12. Dobrova, A.M., et al., [Use of diadynamic currents to treat suppurating
wounds]. Sov Med, 1979(9): p. 55-8.
13. Brown, M., et al., High-voltage galvanic stimulation on wound healing in
guinea pigs: longer-term effects. Arch Phys Med Rehabil, 1995. 76(12): p.
1134-7.
14. Gogia, P.P., R.R. Marquez, and G.M. Minerbo, Effects of high voltage
galvanic stimulation on wound healing. Ostomy Wound Manage, 1992. 38(1):
p. 29-35.
15. Hynes, R.O., Integrins: versatility, modulation, and signaling in cell adhesion.
Cell, 1992. 69(1): p. 11-25.
16. Cho, M.R., et al., Integrin-dependent human macrophage migration induced
by oscillatory electrical stimulation. Ann Biomed Eng, 2000. 28(3): p. 234-43.
17. Sun, S. and M. Cho, Human fibroblast migration in three-dimensional
collagen gel in response to noninvasive electrical stimulus. II. Identification of
electrocoupling molecular mechanisms. Tissue Eng, 2004. 10(9-10): p.
1558-65.
18. Cho, M.R., et al., Induced redistribution of cell surface receptors by
alternating current electric fields. FASEB J, 1994. 8(10): p. 771-6.
19. Giancotti, F.G. and E. Ruoslahti, Integrin signaling. Science, 1999. 285(5430):
p. 1028-32.
20. Calderwood, D.A., S.J. Shattil, and M.H. Ginsberg, Integrins and actin
filaments: reciprocal regulation of cell adhesion and signaling. J Biol Chem,
2000. 275(30): p. 22607-10.
21. Carman, C.V. and T.A. Springer, Integrin avidity regulation: are changes in
affinity and conformation underemphasized? Curr Opin Cell Biol, 2003. 15(5):
p. 547-56.
22. Yamada, K.M. and S. Miyamoto, Integrin transmembrane signaling and
cytoskeletal control. Curr Opin Cell Biol, 1995. 7(5): p. 681-9.
23. Ridley, A.J. and A. Hall, The small GTP-binding protein rho regulates the
assembly of focal adhesions and actin stress fibers in response to growth
factors. Cell, 1992. 70(3): p. 389-99.
24. Hotchin, N.A. and A. Hall, The assembly of integrin adhesion complexes
requires both extracellular matrix and intracellular rho/rac GTPases. J Cell
Biol, 1995. 131(6 Pt 2): p. 1857-65.
25. Yang, B., et al., p190 RhoGTPase-activating protein links the beta1
integrin/caveolin-1 mechanosignaling complex to RhoA and actin remodeling.
Arterioscler Thromb Vasc Biol. 31(2): p. 376-83.
26. Whitehead, I.P., et al., Dbl family proteins. Biochim Biophys Acta, 1997.
1332(1): p. F1-23.
27. Ren, X.D., W.B. Kiosses, and M.A. Schwartz, Regulation of the small
GTP-binding protein Rho by cell adhesion and the cytoskeleton. EMBO J,
1999. 18(3): p. 578-85.
28. Barry, S.T., et al., Requirement for Rho in integrin signalling. Cell Adhes Commun, 1997. 4(6): p. 387-98.
29. Chong, L.D., et al., The small GTP-binding protein Rho regulates a
phosphatidylinositol 4-phosphate 5-kinase in mammalian cells. Cell, 1994.
79(3): p. 507-13.
30. Clark, E.A., et al., Integrin-mediated signals regulated by members of the rho
family of GTPases. J Cell Biol, 1998. 142(2): p. 573-86.
31. Pozo, D., et al., Identification of G-protein coupled receptor subunits in
normal human dental pulp. J Endod, 2000. 26(1): p. 16-9.
32. Kumanogoh, H., et al., Biochemical and morphological analysis on the
localization of Rac1 in neurons. Neurosci Res, 2001. 39(2): p. 189-96.
33. Carmena, M.J., et al., Cholesterol modulation of membrane fluidity and VIP
receptor/effector system in rat prostatic epithelial cells. Regul Pept, 1991.
33(3): p. 287-97.
34. Axelrod, D., et al., Mobility measurement by analysis of fluorescence
photobleaching recovery kinetics. Biophys J, 1976. 16(9): p. 1055-69.
35. Sprague, B.L., et al., Analysis of binding reactions by fluorescence recovery
after photobleaching. Biophys J, 2004. 86(6): p. 3473-95.
36. McLaughlin, S. and M.M. Poo, The role of electro-osmosis in the
electric-field-induced movement of charged macromolecules on the surfaces of
cells. Biophys J, 1981. 34(1): p. 85-93.
37. Bourguignon, G.J. and L.Y. Bourguignon, Electric stimulation of protein and
DNA synthesis in human fibroblasts. FASEB J, 1987. 1(5): p. 398-402.
38. Zhao, M., et al., Membrane lipids, EGF receptors, and intracellular signals
colocalize and are polarized in epithelial cells moving directionally in a
physiological electric field. FASEB J, 2002. 16(8): p. 857-9.
39. van Ginkel, G., H. van Langen, and Y.K. Levine, The membrane fluidity
concept revisited by polarized fluorescence spectroscopy on different model
membranes containing unsaturated lipids and sterols. Biochimie, 1989. 71(1):
p. 23-32.
40. Yeagle, P.L., Lipid regulation of cell membrane structure and function.
FASEB J, 1989. 3(7): p. 1833-42.
41. Lang, P., et al., Protein kinase A phosphorylation of RhoA mediates the
morphological and functional effects of cyclic AMP in cytotoxic lymphocytes.
EMBO J, 1996. 15(3): p. 510-9.
42. Machacek, M., et al., Coordination of Rho GTPase activities during cell
protrusion. Nature, 2009. 461(7260): p. 99-103.
43. Fukata, M., M. Nakagawa, and K. Kaibuchi, Roles of Rho-family GTPases in
cell polarisation and directional migration. Curr Opin Cell Biol, 2003. 15(5):
p. 590-7.