DC Electric Fields Induce Distinct Preangiogenic Responses
in Microvascular and Macrovascular Cells
Huai Bai, Colin D. McCaig, John V. Forrester, Min Zhao
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Objective—Electrical stimulation induces significant angiogenesis in vivo. We have shown recently that electrical
stimulation induces directional migration, reorientation, and elongation of macrovascular endothelial cells. Because
angiogenesis occurs mainly in the microvasculature, we have extended this observation to include human microvascular
endothelial cells (HMEC-1s) and compared the responses with that of vascular fibroblasts and smooth muscle cells and
human umbilical vein endothelial cells.
Methods and Results—Four types of vascular cells were cultured in electric fields (EFs). Dynamic cell behaviors were
recorded and analyzed with an image analyzer. EFs of 150 to 400 mV/mm induced directed migration, reorientation,
and elongation of all the vascular cells. HMEC-1s showed the greatest directional migration (migration rate of 11 ␮m/h
and directedness of 0.35 at 200 mV/mm). Most intriguingly, HMEC-1s migrated toward the cathode, whereas the other
cell types migrated toward the anode.
Conclusions—Endothelial cells derived from angiogenic microvascular as opposed to nonangiogenic macrovascular
tissues were more responsive to electrical stimulation. This intriguing directional selectivity indicates that a DC
electrical signal as a directional cue may be able to play a role in the spatial organization of vascular structure.
(Arterioscler Thromb Vasc Biol. 2004;24:1234-1239.)
Key Words: vascular cells 䡲 electrical stimulation 䡲 angiogenesis 䡲 heterogeneity 䡲 cell migration
䡲 alignment 䡲 orientation
M
pig skin arises as soon as the wound occurs, and this persists
until re-epithelialization is complete.19 –21 This laterally oriented EF is attributable to the immediate flow of current
driven by the transepithelial potential difference, which is
sustained distant from the wound but has been short circuited
at the wound site. At a corneal wound, these endogenous EFs
guide epithelial cell migration and direct nerve sprouting
toward the wound edge.22–25 A small EF also affects cell
proliferation rates and the orientation of the axis of cell
division.25,26 Endogenous EFs also exist in and around the
vasculature. For example, ␨-potentials arise from the flow of
blood in large blood vessels and are 100 to 400 mV at the
blood endothelial cell interface.27 Injured and ischemic tissues also are polarized electrically, and this can produce a DC
EF of ⬇5.8 mV/mm across an 8-mm zone at the boundary
with undamaged tissue.11
New blood vessel formation is based on the capacity of
human microvascular endothelial cells (HMEC-1s) to migrate, proliferate, elongate, and organize in 3 dimensions into
tubules. Certain growth factors (eg, basic fibroblast growth
factor, VEGF) can stimulate endothelial cell migration and
proliferation and therefore promote angiogenesis. Small applied EFs may upregulate some growth factor receptors and
odulation of new blood vessel formation, either to
increase the blood supply to ischemic tissue or to
inhibit blood supply to undesired neoplasm such as cancer,
offers great hope for treatment of a vast spectrum of diseases.1 Electrical stimulation has emerged recently as a novel
approach to induce angiogenesis in vivo, and this is mediated
by enhanced local expression of vascular endothelial growth
factor (VEGF) by muscle cells.2– 4 More recently, we and
others have shown that electrical stimulation also has significant direct effects on endothelial cells to induce reorientation
of the long axis of the cell, directional cell migration, and to
stimulate cell elongation.5– 8 This may be of physiological
significance because endogenous electric fields (EFs) have
been found to be associated with circulation, tissue damage,
and abnormal cell proliferation.9 –14
Endogenous EFs are widespread, have been measured
directly in animals and in humans, and may be important for
development and wound healing.15–17 For example, a steady
DC EF of 450 to 1600 mV/mm has been measured across the
wall of the amphibian neural tube during early neuronal
development,16 and disrupting this perturbs neural development.18 At surface wounds, a steady DC EF of at least 40
mV/mm in bovine cornea and 100 to 200 mV/mm in guinea
Received March 17, 2004; revision accepted April 1, 2004.
From the Departments of Biomedical Sciences (H.B., C.D.M., M.Z.) and Ophthalmology (J.V.F.), Institute of Medical Sciences, University of
Aberdeen, Aberdeen AB25 2ZD, Scotland, UK.
Correspondence to Dr. M Zhao, Department of Biomedical Sciences, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD,
Scotland, UK. E-mail [email protected]
© 2004 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
1234
DOI: 10.1161/01.ATV.0000131265.76828.8a
Bai et al
Responses of Vascular Cells to Electric Fields
1235
passage 10 for HUVECs. Fibroblasts were used for experiments at
passages 4 through 10.
EF Stimulation
The cell culture, experimental set-up, and field exposure were similar
to those reported previously.33
Cell Behavior Quantification
Serial pictures were taken immediately before EF application and
then hourly for up to 5 hours to quantify migration, or at 4, 8, and 24
hours to quantify orientation and elongation as described in detail
previously.7,33 Individual frames were recorded and analyzed using
an image analyzer (Q500MC; Leica). All single visually viable cells
were analyzed except those that merged into cell sheets or made
contact with a reference scratch mark during EF exposure.
Directional Migration
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Figure 1. Applied EFs directed different types of vascular cells
to migrate in different directions. HMEC-1s migrated cathodally
(a); however, BPAF cells (b), MASMCs (c), and HUVECs (d)
moved in the opposite direction, anodally (200 mV/mm). Lamellipodia extended preferentially in the direction in which cells
moved. Cells are identified by numbering. Bottom panels show
the outlines of the cells at the beginning and end of each experiment, with migration direction indicated by the arrows.
Bar⫽50 ␮m.
Mean migration rate and directedness were quantified during a
5-hour period.33,34 The migration rate was defined as D/t, where D is
the distance of a straight line connecting starting and end position of
a cell over this period of time, and t is the duration of time. The angle
(␪) that each cell moved with respect to the imposed EF vector was
measured. The cosine of this angle (defined as directedness) is 1 for
cells moving directly toward the cathode, 0 for cells moving
perpendicular to the EF vector, and ⫺1 for cells moving directly
toward the anode. Averaging the cosines (⌺cos␪i/n, where ␪i is the
angle between the field vector and the direction of movement for an
individual cell measured in the group of cells, and n is the total
number of cells) yields average directedness of cell movement.
Perpendicular Orientation
increase growth factor release.
These changes are important for migration of many cell types, including human
umbilical vein endothelial cells (HUVECs) and bovine aortic
endothelial cells.5,7,8 Because angiogenesis occurs mainly in
the microvasculature usually at post-capillary venules and not
in large blood vessels,30 we studied the effects of a DC EF on
an HMEC-1 line.31 In addition, because the vascular wall
contains different endothelial and other cell types that are
likely to be exposed to the same EF in vivo or when EFs are
applied exogenously, we compared the responses of
HMEC-1s with those of vascular fibroblasts, vascular smooth
muscle cells (SMCs), and the previously studied HUVECs.
We show that endothelial cells derived from angiogenic
microvasculature as opposed to macrovascular tissues moved
fastest and in the opposite direction in a small DC EF. Thus,
different cell types from a common tissue source responded
differently to an applied DC EF. This intriguing directional
selectivity indicates that a DC electric signal as a directional
cue may be able to play a role in the spatial organization of
vascular structure.
2,28,29
Methods
Reagents and Cell Culture
DMEM, FBS, and other cell culture reagents were from GIBCO/
BRL. HMEC-1s (from Dr. F.J. Candal, Centers for Disease Control,
Bethesda, Md) were cultured as described previously in growth
factor complete endothelial basic medium (EBM; Clonetics) supplemented with FBS (10%).31 Murine aorta SMC (MASMC) line (from
Dr. Nixon, University of Aberdeen, UK), HUVEC (from American
Type Culture Collection), and primary culture of bovine pulmonary
artery fibroblasts (BPAFs)32 were cultured in complete DMEM
(DMEM with 10% FBS, 100 U/mL penicillin, and 100 ␮g/mL
streptomycin) at 37°C in CO2 incubator. All experiments were done
within passage 20 for HMEC-1s, passage 12 for MASMCs, and
Cell orientation was quantified as an orientation index (Oi).7,33 Oi of
a cell with respect to the EF was defined as a function of cos(2␣),
where ␣ is the angle formed by the long axis of each cell with a line
drawn perpendicular to the field vector, which was measured using
the image analyzer. This Oi varies from ⫺1 to 1. A cell lying parallel
to the EF vector has an Oi of ⫺1, and a cell perpendicular to the EF
vector has an Oi of 1 (Figure 4i). Average Oi for a cell population [⌺
cos (2␣i) /n] was calculated, where ␣i is the angle formed by the long
axis of a cell with a line drawn perpendicular to the field vector in the
group of cells, and n is the total number of cells. A population of
cells with each cell oriented in random direction would give an Oi
value of 0, whereas with more and more cells aligned more and more
perpendicular to EF vector, the Oi value would increase and
approach 1. The significance of this orientation was calculated using
Rayleigh distribution.35 The probability that the population is ran2
–4
domly oriented is given by P⫽e-(L n)(10 ), where L⫽{[⌺
2
2 1/2
sin(2␣i)] ⫹[⌺ cos(2␣i)] } /n (0.01), and n is the total number of
cells. A probability level of 0.001 was used as the limit for
significant perpendicular orientation.
Cell Elongation: Long:Short Axis Ratio
The distance between the 2 points on the edge that are farthest apart
and the greatest width measured perpendicular to that line are
defined as the long and short axis of a cell. Long and short axes were
traced manually with the interactive software of the image analyzer
for those cells with distinct long and short axes (Figure 4). A
long:short axis ratio was calculated and gives an objective assessment of elongation of the cells tested.
Statistical analysis was made using unpaired, 2-tailed Student t
test or Welch unpaired t test when SDs were significantly different
from each other. Data are expressed as mean⫾SEM.
Results
Microvascular and Macrovascular Cells Migrate
in Different Direction in EFs
When cultured without EFs, all 4 types of vascular cells
migrated in random directions, although MASMCs moved
very little. When cultured in a physiological EF, the 4 types
1236
Arterioscler Thromb Vasc Biol.
July 2004
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of fibronectin or collagen and optimized medium for each
type of cells increased the migration speed significantly, the
difference in migration direction remained. In EBM (the
original medium used for HMEC-1s), HMEC-1s migrated
toward the cathode as in DMEM, the standard medium we
use for EF stimulation for all types of cells. However, the
migration speed was increased significantly to ⬇18 ␮m/h.
BPAF cells in Eagle’s minimum essential medium with 15%
FBS migrated toward the anode. Fibronectin and collagen
coating significantly increased the migration rate of HUVECs
and BPAF cells, respectively (⬇14 ␮m/h). This confirmed
the directional difference in microvascular and macrovascular
cells.
To exclude possible chemotaxis effects in the chamber
toward an electrically induced molecular gradient, we did
control experiments with fluid flow perpendicular to the EF
lines. A cross-flow of medium (0.6 mL/min) perpendicular to
the EF through the chamber did not have significant effects
on migration directedness of HMEC-1s. Cells still migrated
toward the cathode in DC EF at 300 mV/mm (directedness
0.35⫾0.07; n⫽58). Thus, the applied EFs rather than any
secondarily induced chemical gradient directed cell
migration.
Figure 2. Scatter plots show cumulated cell migration of vascular cells in applied EFs. Each point represents a single cell,
located initially at the center of the circular graph (0 hour) and
plotted using their final location after 5 hours. The radius of
each circle is 150 ␮m, and the directedness is indicated.
HMEC-1s migrated toward the cathode (left), whereas vascular
fibroblasts, SMCs, and HUVECs moved anodally (to the right).
of cells showed evident directional migration. Strikingly,
HMEC-1s migrated toward the cathode, whereas BPAFs,
MASMCs, and HUVECs migrated anodally (Figure 1a
through 1d). HMEC-1s and BPAF cells were the most
actively migrating cells in our experimental conditions, with
directional migration obvious within 3 hours (Figure 1a and
1b). Cells extended lamellipodia in the direction of migration
within 1 hour after the onset of the EF. The size and shape of
HMEC-1s changed more frequently than BPAF cells in EFs.
MASMCs and HUVECs migrated more slowly, although as
shown in Figure 1c, MASMCs did migrate toward the anode
very slowly (3.2 ␮m/h).
The difference in the migration direction in EFs among the
4 types of vascular cells was confirmed and quantified with
detailed analysis of the time lapse images at different EF
strengths (Figures 2, 3a, and 3c). As described in Methods,
cells moving toward the cathode (left) have directedness
values approaching 1, whereas cells moving toward the anode
have a directedness approaching ⫺1. In an EF of 150
mV/mm, HMEC-1s showed significant cathodal directedness, whereas BPAF cells, MASMCs, and HUVECs migrated
anodally (Figures 2, 3a, and 3c). The difference in polarity of
directed movement was striking (Figure 3a and 3c).
The vascular cells in our culture conditions migrate slowly
compared with another report.36 To confirm the directional
difference in migratory responses of different vascular cells
to DC EFs, several different culture media and substratum
coating conditions were tested. Although substratum coating
Directional Migration of Vascular Cells Cultured
in EFs Was Voltage Dependent
We quantified the directional migration of the 4 types of
vascular cells at different field strengths. The directedness of
HMEC-1s migrating to the cathode and of BPAF cells,
MASMCs, and HUVECs migrating to the anode all peaked at
150 to 200 mV/mm (Figure 3a). Additional increases of EF
strength ⬎200 mV/mm did not increase directedness, but
instead directed migration of cells decreased (Figure 3a).
Figure 3a indicates that vascular cells respond to EFs with a
threshold ⱕ150 mV/mm.
Small EFs Stimulated Migration of Vascular Cells
Exposure to EFs significantly increased the translocation
speed of cells; HMEC-1s increased the most (Figure 3b). At
200 mV/mm, HMEC-1s migrated fastest at ⬇11 ␮m/h. This
was ⬇2⫻ the rate of HUVECs (5.6 ␮m/h), 3⫻ that of
MASMCs (3.2 ␮m/h), and 20% higher than the rate of
fibroblasts (9.2 ␮m/h; Figure 3b, 3d, and 3e). The increase in
mean translocation rates for HMEC-1s and HUVEC showed
a clear voltage dependency (Figure 3b).
Small EFs Induced Alignment of Vascular Cells
When cultured in EFs, HMEC-1s, BPAF cells, MASMCs,
and HUVECs aligned with their long axis perpendicular to
the EF vector (Figure 4b, 4d, 4f, and 4h). In control
cultures (no EF), the long axis of cells aligned randomly
(Figure 4a, 4c, 4e, and 4g). We quantified cell alignment
using the Oi: Oi⫽cos 2␣ (see Methods and Figure 4i). The
orientation response of all 4 types of vascular cells was
time dependent in EFs, with a linear increase of Oi in EFs
of 200 mV/mm (Figure Ia, available online at http://
atvb.ahajournals.org). Orientation of each cell type was
evident after 4 to 5 hours in the EF. BPAF cells, MASMCs,
Bai et al
Responses of Vascular Cells to Electric Fields
1237
Figure 3. Voltage dependence of EF-directed
migration (a) and enhanced migration rate of
vascular cells (b). Directedness and rates of cell
migration of HMEC-1s, HUVECs, MASMCs, and
BPAFs cultured in the same medium (see Methods) were calculated during a 5-hour period.
Cathodal migration of HMEC-1s and anodal
migration of BPAFs, MASMCs, and HUVECs
were voltage dependent (a). Irrespective of
migration direction, the increase in migration rate
also was voltage dependent (b). Data are
expressed as mean⫾SEM. Cell numbers
included in the analysis at individual field
strengths of 0 to 400 mV/mm are 51 to 59 for
HMEC-1s, 57 to 222 for HUVECs, 56 to 70 for
MASMCs, and 51 to 68 for BPAFs. Different
vascular cells migrated in different directions (c)
and responded with different enhancement in
migration (d) and percentage change in migration rate (e) at 200 mV/mm. *P⬍0.05; **P⬍0.01;
***P⬍0.001, when compared with that in corresponding control with no EF (0 mV). Values are
means⫾SEM.
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and HUVECs showed a more robust response at 8 hours
and 24 hours than HMEC-1s (Figure Ia).
The orientation response of the vascular cells also showed
voltage dependence. BPAF, MASMC, and HUVEC cells had
similar robust voltage dependency, whereas HMEC-1s
showed a less dramatic increase in Oi, with time in an EF of
200 mV/mm (Figure Ib). However, for all the 4 types of
vascular cells, the orientation response had a threshold
between 50 and 150 mV/mm.
Small EFs Induced Elongation of Vascular Cells
Whereas the 4 types of vascular cells migrated and aligned in
an EF, they also elongated in response to the EF (Figure 4b,
4d, 4f, and 4h). In contrast, cells cultured without applied EFs
retained a typical nonpolarized morphology (Figure 4a, 4c,
4e, and 4g). We quantified the elongation of cells using the
long:short axis ratio (see Methods). A perfectly round cell has
a long:short axis ratio of 1. As cells elongate, the ratio
increases. The elongation response to the EF happened much
later than the directional migration and orientation of the long
axis of the cell (Figure I). Cultured in an EF of 200 mV/mm,
cells did not show significant elongation after 5 hours;
however, 24 hours after onset of continuous EF, HMEC-1s,
BPAF cells, MASMCs, and HUVECs showed a significant
elongation response when compared with control cells (0 mV
for 24 hours; P⬍0.01 to 0.0001; Figure II, available online at
http://atvb.ahajournals.org).
Discussion
DC EFs induce directional migration and a significant increase in cell migration rates of HMEC-1s and HUVECs, and
of fibroblasts from bovine aorta and MASMCs. Intriguingly,
HMEC-1s migrated in the opposite direction from endothelial
cells, fibroblasts, and SMCs derived from large vessels. Each
of the vascular cells tested showed EF-stimulated perpendicular orientation and cell elongation. Migration, orientation,
and elongation of vascular cells are important cellular behaviors underlying angiogenesis and vascular remodeling. Our
observation that applied EFs induced distinct heterogeneous
responses in different vascular cells may be important in
understanding the potential electrical control of angiogenesis
by endogenous or exogenously applied electrical stimulation.
Blood flow to ischemic tissues can be re-established by 2
main mechanisms: angiogenesis and collateral circulation.
Angiogenesis involves mainly endothelial sprouting from
capillaries and venules. There is morphological and functional heterogeneity between endothelial cells from different
parts of the body, from different sizes of vessels, in different
organs, and in different regions of the same organ.37 One
example is the different responsiveness of microvascular
versus macrovascular endothelial cells to interleukin 8 (IL-8),
a potent angiogenic agent.38 IL-8 binds chemokine receptors
CXCR 1 to CXCR 3 to induce angiogenic response.
HMEC-1s express more CXCR 1 to CXCR 3 and are more
responsive to IL-8 than HUVECs, showing enhanced chemotactic and migratory response.38 Here we showed that contrasting differences also exist in the responses of HMEC-1s
and HUVECs to an applied physiological EF. These include
differences in migration direction, enhancement of migration
speed, and the extent of cell reorientation and elongation.
Each of these cell behaviors is potentially important for
angiogenesis, therefore, clinical application of electrical stimulation should take account of this heterogeneity.
Different types of cells respond to EFs by migration toward
different poles.15 Some cells migrate cathodally, for instance,
neural crest cells, corneal epithelial cells, epidermal keratinocytes, pigmented retinal epithelium, embryonic fibroblasts,
osteoblasts, and bovine aortic endothelial cells.8,34,39 – 42 Other
cells migrate anodally, such as corneal endothelial cells,
fibroblasts, osteoclasts, and peritoneal macrophages.39,43– 45
Intriguingly, lens epithelial cells migrate either cathodally or
anodally, depending on EF strength.46 How cells sense and
transduce electric signals remains largely unknown. Because
both HMEC-1s and HUVECs are endothelial cells and
migrate in opposing directions at the same EF strength, they
offer comparative models to dissect out the mechanisms that
cells use to move directionally in a physiological EF.
The applied EFs of 150 to 400 mV/mm showed significant
effects in directing vascular cell migration, orientation, and
elongation. How does this relate to the EFs these cells will
1238
Arterioscler Thromb Vasc Biol.
July 2004
here that EFs also have significant effects on basic cell
behaviors such as directional migration, alignment, and elongation in these cell types.
In conclusion, small DC EFs of a size equivalent to those
that arise immediately at a wound induced significant directional migration, orientation, and elongation responses of
vascular endothelial cells, fibroblast cells, and SMCs. Distinct heterogeneity in the responses existed among the 4 types
of vascular cells tested. This may have potential physiological
and clinical implications in areas where electrical stimulation
is used to promote angiogenesis or vasculature remodeling.
Acknowledgments
M.Z. is a Wellcome Trust University Award Senior Lecturer
(058551). This work is supported by the British Heart Foundation
(FS/2000056 and PG/99191 to M.Z., C.D.M., J.V.F.). We thank the
anonymous referees for their comments and critiques that helped to
improve this article.
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Figure 4. Orientation and elongation of HMEC-1s, BPAFs,
MASMCs, and HUVECs in EF. Cells exposed to EF at 200
mV/mm showing perpendicular orientation and elongation (b, d,
f, and h; 24 hours in EF). Control cells without EF exposure
showed typical morphology with random orientation (a, c, e, and
g). i, Schematic diagram shows the quantification method of cell
orientation with Oi. Perpendicular alignment to the EF vector
gives an Oi of 1, parallel alignment, an Oi of ⫺1, and randomly
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DC Electric Fields Induce Distinct Preangiogenic Responses in Microvascular and
Macrovascular Cells
Huai Bai, Colin D. McCaig, John V. Forrester and Min Zhao
Arterioscler Thromb Vasc Biol. 2004;24:1234-1239; originally published online May 6, 2004;
doi: 10.1161/01.ATV.0000131265.76828.8a
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Bai et al:
Responses of vascular cells to electric fields
#ATVB/2004/016287R1
Online Supplemental materials
Methods:
Electric Field Stimulation
Cells were seeded in the trough at a density of ~20 × 104 cells/ml and 0.5 ml/trough. A
roof of number 1 cover glass (Chance Propper, Warley, England) was added and sealed
with silicone grease 12-20 h after seeding. The final dimensions of this very shallow
chamber, through which current was passed, were 22 mm × 10 mm × 0.2 mm. Agar-salt
bridges were used to connect silver/silver chloride electrodes in beakers of physiological
saline solution, to reservoirs of culture medium at the ends of the chambers. Field
strengths were measured directly. One hour before EF stimulation, fresh complete
DMEM was used to replace all media including EBM used for HMEC-1 culture. This
kept identical medium conditions for all four types of cells tested.
a
HMEC-1
BPAF
MASMC
HUVEC
HMEC-1
BPAF
MASMC
HUVEC
0.8
0.4
0
4
8
*
**
**
*
1 Orienation
0.8 index
0.6
*
*
**
24
SCos(2q/n)
0.4
0.2
0
#
0h
Orientation index
b
8h
HMEC-1
BPAF
MASMC
HUVEC
1
0.8
0.6
*
*
0.4
*
0.2
#
#
4h
*
*
24h
*
*
*
*
*
*
300
400
*
0
-0.2
0
50
100
150
200
Field strengths ( mV/mm)
Figure I. Time (a) and voltage (b) dependence of the orientation responses of vascular cells in
electric fields. EF of 200mV/mm induced orientation of cells within 4 hours. By 8 hours and 24 hours,
although microvascular cells aligned strongly perpendicular to the EF, each of the other cell types
showed a more robust response (a). The insert shows the response curves of the cells in different timepoints at 200 mV / mm EF strength. *p<0.05 and **p<0.01; # P<0.05-0.000 when compared with
corresponding controls(0 hour) after 4, 8, and 24 hours EF exposure, respectively. n= 65-598 from at
least two independent experiments. b. Voltage dependence of vascular cells at individual field strengths
of 0-400 mV over 5 hours. The number of cells measured for each point are 57~103 , 56~115, 103~116
and 82~114 for HMEC-1, BPAF, MASMC and HUVEC, respectively. *p<0.01-0.0001 when compared
with corresponding controls with no EF (0 mV).
HMEC-1
BPAF
MASMC
HUVEC
Long/short axis ratio
8
**
6
*
4
*
2
0
0 hr
5hr
24hr
Figure II. EF induced elongation of vascular cells. Elongation was quantified as the
long/short axis ratio of individual cells and did not show significant change until 24 hours of
EF exposure. These cell types showed an increased elongation when compared with
corresponding control cells (24hr: 0mV) (*P<0.01- 0.0001). n= 47-343 from at least two
independent experiments. Values are means ± S.E.M.