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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 towards the cathode. The progenitor cells also align and elongate in an EF. Inhibition of vascular endothelial growth factor (VEGF) receptor signalling 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 signalling in vitro. Applied EFs may be used to control behaviours of EPCs in tissue engineering, in homing of EPCs to wounds and to an injury site in the vasculature.
Directional migration of endothelial progenitor cells (EPCs) is critical in vasculogenesis, angiogenesis, and vasculature repair. In embryos, EPCs migrate, differentiate, and coalesce into clusters of hemangioblasts to form new blood vessels (vasculogenesis). In adults, EPCs may move to sites of injury or a site of neovascularization and then differentiate into mature endothelial cells, thus contributing to re-endothelialization and neo-vascularization (angiogenesis) [1–4]. Injury to vascular endothelium leads to the loss of the anti-thrombotic properties of the vessel wall, which could result in thrombosis, intimal hyperplasia and stenosis [1,5]. Small damages may be repaired by migration and proliferation of surrounding mature endothelial cells. Larger damages require migration and differentiation of EPCs that are marked in general by CD133+/CD34+/ vascular endothelial growth factor receptor 2+ (VEGFR-2)/VE-cadherin− [2,6–10].
Directional migration of EPCs is meditated by varied chemokines, extracellular matrix, growth factors and membrane receptors [7,10]. Vascular endothelial growth factor (VEGF) is the most important growth factor in angiogenesis, controlling and increasing blood vessel formation [6,11].
Endogenous electric fields (EFs) occur naturally at wounds and around vasculature, which may be an important signal in guiding cell migration [12–15]. Applied EFs induced important pre-angiogenic responses in mature endothelial cells in culture. Human umbilical vein endothelial cells, bovine aortic endothelia cells, human dermal microvascular endothelial cells respond to applied EFs by directional migration, elongation and alignment [16–18].
Importantly, electrical stimulation has been demonstrated as an effective approach to induce angiogenesis in ischemia and wound healing [18–24]. Electrical stimulation significantly enhances angiogenesis in ischemic and non-ischemic rat limbs, possibly mediated by VEGF and hepatocyte growth factor (HGF) expressed in muscle cells [20,22,24,25]. Small EFs directly stimulate VEGF production by endothelial cells and direct reorientation, elongation and migration of endothelial cells in culture, through phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB or Akt), Rho-ROCK signalling pathways [17,18].
It has not been reported whether behaviours of EPCs can be regulated by applied EFs. To be able to manipulate cellular behaviours of EPCs, and to direct EPCs to sites where angiogenesis is required, may have significant clinical implication in tissue engineering, and in controlling angiogenesis and many angiogenesis-related diseases. Directed migration of endothelial cells in an EF appears to be cell-type and origin specific. The endothelial cells from human microvasculature (HMEC-1 cells) or cultured bovine aortic migrated toward the cathode, whilst the endothelial cells derived from human umbilical vein migrated toward the anode [16,17]. It is therefore necessary to determine the responses of EPCs. We report here that DC EFs are a very powerful signal to direct migration, orientation, and elongation of three different types of EPCs. The EF-guided cell migration appears to be mediated by VEGF receptors.
We used the MFLM-4, AEL-deltaR1 and AEL-deltaR1/Runx1cells, because they are relatively well studied murine endothelial progenitor cells. Using those cells offer a basis to do further in vitro and in vivo genetic studies with mouse models. Progenitor cell marker CD133, and endothelial cell markers VEGFR-2 and von Willebrand Factor (vWF) were used to confirm the endothelial progenitor cell nature. These combined proteins are the markers used to identify EPCs. We confirmed that the three cell lines (MFLM-4, AEL-deltaR1 and AEL-deltaR1/Runx1) are all positive with specific stem cell marker-CD133 and endothelial cell markers-vWF and VEGFR-2 (Fig. 1).
MFLM-4 cells migrated towards the cathode in EFs of 150–400 mV/mm (Fig. 2; Supplementary Video 1). Significant directional migration occurred at a field strength of 150 mV/mm with migration directedness of 0.24 ± 0.07 (n = 101; P < 0.01 compared with that of no EF control: −0.01 ± 0.06, n = 141, Fig. 2E). When the EF polarity was reversed, cells rapidly changed direction to move towards the new cathode (Fig. 2C, D). This reversal of the migration direction can be observed ~15 minutes after reversing the polarity of the applied EF. The cell directedness was voltage-dependent (P < 0.05; Fig. 2E). Cell migration speed along the X axis (Dx/T) significantly increased when exposed to EFs of 150–400 mV/mm. Straight-line migration speed (Td/T) also significantly increased in EFs of 200–400 mV/mm (P < 0.05 compared with no EF control; Fig. 3A).
MFLM-4 cells cultured without an EF had flat spindle-shaped morphology, with the long axis of the cell body oriented randomly (e.g. 0h in Fig. 2A; Fig. 3B). In contrast, cells cultured in DC EFs were re-orientated with their long axes aligning perpendicular to the vector of the applied EF (e.g. 4h in Fig. 2A; Fig. 3B). The orientation index increased gradually when the strength of the applied EFs increased from 150 to 400 mV/mm (P < 0.05 compared with no EF control; Fig. 3B). EF had no effect on MFLM-4 cell shape, as assessed by long/short axis ratio (Fig. 3C).
Next, AEL-deltaR1 and AEL-deltaR1/Runx1cells were tested. In the absence of an applied EF, AEL-deltaR1 cells migrated randomly, with an average net directedness of 0.04 ± 0.07 and displacement speed along the X axis of 0.23 ± 0.75 µm/hour. At an EF of 300 mV/mm, cells had clear response toward the cathode, with an average net directedness of 0.66 ± 0.05 and displacement speed along the X axis of 7.35 ± 0.72 µm/hour (P < 0.001 compared with no EF control; Fig. 4A–C; Fig. 5A; Supplementary Video 2). Cells extended cathode-directed lamellipodia and began directed migration towards the cathode within 5 minutes of switching the EF on (Fig. 4A). The cells reoriented to align perpendicular to the EF vector (Fig. 5B). Migrating cells extended membrane protrusions preferentially toward the cathode, either from the leading edge, or at both ends of the long axis (Fig. 4A; Supplementary Video 2). EF exposure significantly induced cell elongation (P < 0.001 compared with no EF control; 3 h in Fig. 4A; Fig. 5C; Supplementary and Video 2).
AEL-deltaR1/Runx1 cells also migrated toward the cathode at an EF of 300 mV/mm, with an average net directedness of 0.47 ± 0.05 and displacement speed along the X axis of 10.60 ± 1.20 µm/hour (P < 0.001 compared with no EF control directedness of 0.01 ± 0.06 and displacement speed along the X axis of 0.13 ± 1.49 µm/hour; Fig. 4D–F; Fig. 5D; Supplementary Video 3). Cells reoriented to align perpendicular to the EF vector like AEL-deltaR1 (Fig. 4D; Fig. 5E; Supplementary Video 3) but EF exposure did not induce AEL-deltaR1/Runx1 cell elongation (P > 0.05 compared with no EF control; Fig. 4D; Fig. 5F; Supplementary Video 3).
VEGF receptor signalling is critical in the control of many endothelial cell behaviours and angiogenesis. Our previous work has shown that electric field-directed human umbilical vein endothelial cell migration is mediated through VEGFR signalling . In the present study, the activity of VEGFR-2 was investigated. Addition of VEGF (100 ng/ml) significantly enhanced directional MFLM-4 cell migration towards the cathode. Both the directedness (113%; P < 0.05) and displacement speed along the X axis (124%; P < 0.05) were significantly increased in culture medium containing VEGF (100 ng/ml) (Fig. 6; Supplementary Video 4). Cell orientation and elongation were not influenced by VEGF (Fig. 7A, B; Supplementary Video 4).
Inhibition of VEGFR-2 with a specific VEGFR-2 Kinase Inhibitor II (VEGFR-2I) at 50 µM completely abolished the directness (4%; P < 0.001) and decreased migration speed (Tt/T 53%; P < 0.001; Td/T 41%; P < 0.001; Dx/T 1%; P < 0.001) of MFLM-4 cells in an EF (Fig. 6). The inhibition of the directness and migration speed was dose-dependent (Fig. 6; Supplementary Video 5 and Video 6).
The VEGFR-2 kinase inhibitor (50 µM) completely abolished the MFLM-4 cell orientation response (Fig. 7A; Supplementary Video 6). The Oi (orientation index) values of the cells treated with VEGFR-2 inhibitor were 0.12 ± 0.07 and –0.08 ± 0.07 in EF for 1 µM and 50 µM, respectively. The latter is significantly different from the non-inhibitor-treated values of 0.18 ± 0.07 (P < 0.01; Fig. 7A).
DC EFs are present naturally at wounds and are a powerful cue that directs polarization and migration of epithelial cells, fibroblast cells, and vascular endothelial cells [12,17,18,26]. Electrical stimulation induces pre-angiogenic responses in endothelial cells and therefore may contribute to the regulation of angiogenesis [19–24]. EPCs are a key player in vascular repair and angiogenesis [2,6–10,27]. We report the novel effects of applied EFs on three independent endothelial progenitor cell lines. We confirmed the progenitor nature of the cells. All three EPCs respond to an applied EF by directional migration towards the cathode. Such responses depended on VEGF receptor signalling.
EPCs circulate in the blood stream and contribute to re-endothelialization of injured vessels as well as neovascularization of ischemic lesions. A decrease in the number of EPCs is an independent predictor of morbidity and mortality of cardiovascular diseases. It is therefore suggested that EPCs play a major role in the pathogenesis of atherosclerosis and cardiovascular diseases [1,27]. EPCs may come from the stem cell pool in the circulating blood and migrate or be recruited to the wound site [4,7,10,27,28].
Many factors may control homing of EPCs to damaged or angiogenesis sites. The specific mechanisms are not clear. Chemokines and cytokines, such as VEGF and many others, affect the migration and population of EPCs [7,10,28–31]. Understanding the controlling mechanisms of EPC homing to damaged or angiogenesis sites and developing effective approaches to facilitate EPC migration will be of great clinical significance.
Vasculature is associated with many electric activities. In many cases, angiogenesis occurs in the presence of endogenous EFs, which are generated by active ion transport across polarized epithelia and endothelia [12,13,15,32–34]. There are several types of electric potential differences around the endothelium of blood vessels that might be involved in regulating the development of thromboses . The ζ (streaming) potentials, for example, are created at the endothelial cell wall by blood flow in both the aorta and vena cava, and they range from 100 to 400 mV . Injured and ischemic tissue also becomes electrically polarized relative to surrounding normal tissue because cells become depolarized and extracellular K+ ions build up in the damaged areas. In the heart, this leads to a flow of injury current intracellularly through junctionally-coupled cells, with an extracellular return loop, and is thought to be involved in arrhythmogenesis . Extracellular injury currents, which create directly measured DC EFs of 58 mV/cm, extend over about 8 mm at the boundary between ischemic and normal tissue . Electrical stimulation significantly enhances angiogenesis in ischemic and non-ischemic rat limbs [20,22,24,25].
Directed migration of cells in an EF appears to be cell-type specific, since some cells types migrate cathodally and others anodally [12,13]. Mature vascular endothelial cells have been reported to respond differently. The endothelial cells from human microvasculature (HMEC-1 cells) or cultured bovine aortic migrated toward the cathode, whilst the endothelial cells derived from human umbilical vein migrated toward the anode [16,17]. Mouse adipose-derived stromal cells (mASC) migrated toward the cathode in response to EFs . However, human induced pluripotent stem cells (hiPS) show anode directed migration . It is therefore necessary to determine the responses of EPCs. We demonstrate here that three independent lines of EPCs (MFLM-4, AEL-deltaR1 and AEL-deltaR1/Runx1) respond to applied EFs by directional cell migration towards the cathode (Fig. 2–5). Our data therefore confirm the DC EF as a novel, extracellular, perhaps physiological cue for directing the migration of EPCs. These results suggest that application of an EF may be a good candidate to locally modulate recruitment of EPCs.
VEGFR-2 mediates the major actions of VEGF [6,11]. A selective and potent VEGFR-2 inhibitor for receptor-2 tyrosine kinase (50% inhibitory concentrations of 70 nM for VEGFR-2) abolished the EF-directed migration of EPCs . Inhibition of VEGFR-2 abolished the directional migration of progenitor cell MFLM-4 (Fig. 7). PI3Kγ mediates the chemokine-induced migration of human EPCs. PI3Kγ is involved in the in vivo homing of progenitor cells to ischemic sites in the mouse model of hindlimb ischemia. PI3Kγ is also essential for the in vivo neovascularization-promoting capacity of progenitor cells in ischemic sites . Kawasaki and colleagues found that Ras signalling downstream of VEGFR-2 is involved in specifying endothelial differentiation of VEGFR-2 positive vascular progenitor cells . Electrotaxis of endothelial progenitor cells is likely to share the same signalling pathway of chemotaxis.
DC EFs exist in vivo and have profound influences on cell migration, orientation, and proliferation [12–14]. Disrupting the EFs impairs development and wound healing [12,33]. Angiogenesis plays a major role in development, wound healing and tumor growth, and EPCs are expected to experience endogenous EFs. These include: ζ (streaming) potentials created by blood flow, extracellular injury currents produced at the boundary between ischemic and normal tissue, and altered surface charges that are a hallmark of the rapid, accelerated proliferation of tumor cells. The close association of angiogenesis with EFs in vivo and the effect of EFs on pre-angiogenic responses of EPCs through one of the most important VEGFR-2 signalling pathways suggest that EFs might play an important role in angiogenesis in vivo. The threshold of applied DC EFs to induce these cellular behaviours in MFLM-4 is 150 mV/mm. The experimentally measured EFs near the cuts are 40 mV/mm and 100–200 mV/mm in bovine cornea and guinea-pig skin, respectively [15,43]. The field strength decreases exponentially within the extracellular space away from the wound. Further studies of proliferation and differentiation with EPCs will determine the EF threshold needed to induce the responses of the EPCs that are mainly involved in angiogenesis and provide further evaluation of possible roles physiological EFs might have in vivo.
In conclusion, small DC EFs induce angiogenic responses in EPCs, causing significant directional migration and orientation. This is a primary response of the EPCs and does not require any other cell type. These responses are mediated by VEGFR-2 activation.
Dulbecco’s modified eagle’s medium (DMEM) and CO2 independent cell culture medium were obtained from Invitrogen. VEGF, fetal bovine serum (FBS), poly-l-lysine and gelatin were purchased from Sigma-Aldrich. Dishes coated with collagen I and fibronectin were from BD BioCoat. VEGF receptor 2 inhibitor was from Calbiochem.
MFLM-4 cells (kindly provided by Dr Ann L. Akeson, The Children's Hospital Medical Center, Ohio, USA), were maintained in DMEM (supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin B and 1% L-glutamine) at 37°C in 5% CO2. This cell line was isolated and cloned from murine E14.5 lung mesenchyme as described [2,8]. AEL-deltaR1 and AEL-deltaR1/Runx1 cells (kindly supplied by Dr Takahiko Hara, The Tokyo Metropolitan Institute of Medical Science, Japan) were maintained in DMEM (with the same supplemented chemicals as for MFLM-4, except without amphotericin B) at 32.5°C in 5% CO2. AEL-deltaR1 cell line was established from the aorta-gonad-mesonephros region of Runx1-null mouse embryos . AEL-deltaR1 cell line transfected with Runx1 cDNA were named AEL-deltaR1/Runx1; Runx1 is associated with endothelial cell differentiation . In our pre-experimental observations, these three progenitor cell lines migrated faster on collagen I than on tissue culture plastic dishes or dishes coated with fibronectin, poly-l-lysine, or gelatin (data not shown). All the results presented are from cells cultured on collagen I.
For immunofluorescence staining, cells were fixed in 4% paraformaldehyde (15 minutes), permeabilized (5 minutes in 0.2% Triton X-100), and blocked for 30 minutes with blocking solution (10% goat serum, 1% BSA and 0.02% NaN3 in PBS). A polyclonal antibody against CD133 (1:200, Abcam) was used to label CD133 (stem cell marker) for 1 hour at room temperature. Antibodies against von Willebrand Factor (vWF, or Factor VIII-related antigen) (1:200, Sigma) and monoclonal antibodies against VEGF receptor-2 (KDR) (1:500, Sigma) were used to confirm the cell endothelial nature. Cells were incubated for 1 hour in solution with the primary antibodies at room temperature. After washing, the cells were incubated with phalloidin-FITC (1:100 Sigma) and Texas Red-conjugated secondary antibodies (1:200 Jackson Immuno Research Laboratories) for 1 hour at room temperature. Nuclei were stained with DAPI. Images were obtained with a Zeiss inverted fluorescence microscope (Axiovert 100) controlled with MetaMorph software. A 40x Fluor oil-immersion lens was used.
Cell motility was assayed using an electrotaxis apparatus as previously detailed . Briefly, a DC EF was applied through agar-salt bridges connecting silver/silver chloride electrodes in beakers of Steinberg's solution to pools of culture medium on either side of the chamber. A roof of No. 1 coverglass was applied and sealed with silicone grease (Corning DC4). The final dimensions of the chamber, through which current was passed, were 40 mm × 10 mm × 0.2 mm. Immediately before EF application, CO2-independent medium (including 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin) was transferred into the culture chambers. Cells were exposed to an EF for 2–6 hours at 37°C in a temperature-controlled chamber on an inverted microscope stage. Serial time-lapse images were recorded using MetaMorph.
We analyzed and quantified the directional cell migration (directedness) and migration rates by tracing the positions of cell nuclei at a frame interval of 5 minutes using MetaMorph software . The directedness of migration was defined as cosine θ, where θ is the angle between the EF vector and a straight line connecting the start and end position of a cell. A cell moving directly along the field lines toward the cathode would have a directedness of 1; a cell moving directly toward the anode would have a directedness of −1. The cosine θ will provide a number between −1 and +1 and the average of all of the separate cell events yields an average directedness index. The average directedness of a population of cells gives an objective quantification of how directional cells have moved in relation to the EF vector. A mean value close to 0 for a polulation of cells represents random cell movement.
The trajectory speed (Tt/T) is the total length of the trajectory (Tt) a cell migrated divided by the time (T). The displacement speed (Td/T) is the straight-line distance between the start and end positions of a cell (Td) divided by time (T). Displacement along the X axis (Dx/T) is the projection of the cell trajectory on the X axis (Dx) divided by the time (T), which represents the ability of cells to migrate along the EF vector.
Cell orientation was quantified as an orientation index (Oi=cos2(α)) [13,44], where α is the angle formed by the long axis of a cell with a line drawn perpendicular to the field lines. A cell with its long axis parallel to the vector of the EF will have an Oi of −1, and a cell with its long axis exactly perpendicular to the EF vector will have an Oi of +1. A randomly oriented population of cells will have an average Oi (defined as [Σncos2(α)] ÷ n) of 0.
Results are presented as mean ± standard error of the mean (SEM). Differences between mean values were compared using a two-sample Student’s t test, performed with equal or unequal variance according to an f test. In graphs, asterisks indicate significant difference (P value in figure legend); absence of an asterisk indicates no significant difference.
>We tested electrotaxis of endothelial progenitor cells (EPCs). > EPCs migrated towards the cathode in electric fields (EFs). > We conclude that EFs are an effective cue that guides EPC migration.
A DC EF directs migration of MFLM-4 cells. Time-lapse video corresponding to Fig. 2A shows that MFLM-4 cells migrate directionally towards the cathode to the right. The recording time is 6 hours with a frame interval of 10 minutes. EF = 300 mV/mm.
Directional migration of AEL-deltaR1 cells in a DC EF of 300 mV/mm. Time-lapse video corresponding to Fig. 4A shows that AEL-deltaR1 cells migrate directionally towards the cathode to the right. The recording time is 3 hours with a frame interval of 5 minutes.
Directional migration of AEL-deltaR1/Runx1 cells in a DC EF of 300 mV/mm. Time-lapse video corresponding to Fig. 4D shows that AEL-deltaR1/Runx1 cells migrate directionally towards the cathode to the right. The recording time is 3 hours with a frame interval of 5 minutes.
Addition of VEGF (100 ng/ml) increases the directional migration of MFLM-4 cells in a DC EF of 300 mV/mm. The recording time is 2 hours with a frame interval of 5 minutes.
Effect of VEGFR2I (1 µM) on migration of MFLM-4 cells in a DC EF of 300 mV/mm. The recording time is 2 hours with a frame interval of 5 minutes.
VEGFR2I (50 µM) inhibits migration of MFLM-4 cells in a DC EF of 300 mV/mm. The recording time is 2 hours with a frame interval of 5 minutes.
This work is supported by a grant from the California Institute of Regenerative Medicine RB1-01417 (to MZ). MZ is also supported by NIH 1R01EY019101, NSF MCB-0951199, and the UC Davis Dermatology Developmental Fund. Dr Ann L. Akeson kindly provided the endothelial progenitor cell line MFLM-4, and Dr Takahiko Hara kindly supplied the AEL-deltaR1 and AEL-deltaR1/Runx1 cells. We are grateful to Erica V. Whitney (School of Medicine, University of California, Davis) for editorial assistance.
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