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INTRODUCTION |
Both epidemiological and clinical studies demonstrate that alcohol
abuse is directly associated with the increasing incidence of multiple
organ diseases, such as liver injury, cardiovascular diseases,
neurological disorders, and tumor promotion. High consumption of
alcohol has been shown to enhance the invasion and metastasis of breast
cancer, colon cancer, and liver cancer (1-5). Conversely, moderate
alcohol consumption has also been shown to protect against the
incidence of cardiovascular diseases (6). The molecular mechanisms of
its pathogenic and protective effects are not fully established.
Previous studies indicate that ethanol can induce angiogenesis (7, 8).
Angiogenesis, the process of forming new blood vessels out of
pre-existing capillaries (9), is a sequential event that involves
(a) the disintegration of the basement membrane,
(b) the migration of endothelial cells, (c)
endothelial cell proliferation in cords with formation of new vascular
channels, and (d) the formation of a new basement membrane
(10). These processes of angiogenesis are associated with cancer,
cardiovascular diseases, and chronic inflammation. The manipulation of
angiogenesis has great potential as an intervention for a number of
diseases. The stimulation of angiogenesis could prevent and improve
ischemic heart failure, whereas the inhibition of angiogenesis could
potentially cure cancers and chronic inflammation (9).
One of the major steps of angiogenesis is cell migration, which is
pivotally governed by changes in the actin cytoskeleton. Actin is one
of the most abundant proteins in eukaryotic cells. There are two
different forms of actin, mono-actin (G-actin) and actin filaments
(F-actin). Actin filaments are involved in a wide variety of cellular
processes, including cell motility, cell cycle control, cellular
structure, and cell signaling (11). They function in cellular processes
by undergoing dynamic structural reorganization or remodeling, leading
to the formation of discrete structures at the cell periphery for
attachment to the substratum in response to extracellular signals.
These structures include focal adhesions, stress fibers, lamellipodia,
filopodia, and membrane ruffles, which are involved in cell attachment,
cell migration, and signal transduction (12).
Because actin filaments play a critical role in cell migration, the
remodeling of actin filaments may be essential for the process of
angiogenesis. It has been found that insulin-stimulated glucose
transport, gene expression, and alterations of cell morphology are
dependent on the remodeling of actin filaments and that inhibition of
the remodeling of actin filaments abrogates the induction of angiogenesis by insulin (13). The remodeling of actin filaments is
essential for vascular endothelial growth factor-induced formation of
tube-like structures and angiogenesis (14). Actin filaments assemble to
form bundle structures that are oriented along the axis of the tubes at
the periphery of the cells during the formation of vessel tubes.
Inhibition of the actin filament assembly blocks tube formation (13).
The remodeling of actin filaments results in the increases in cell
spreading, nuclear extension, and DNA synthesis (15). The
reorganization of actin filaments is also required for cultured rat
hepatocytes to form three-dimensional structures or spheroids (16).
In this study we demonstrated that ethanol directly remodeled or
reorganized the structure of actin filaments, increased cell migration
and cell invasion, and induced in vitro angiogenesis in
endothelial cells. We further showed that activation of Cdc42 was
important for ethanol-induced in vitro angiogenesis.
Inhibition of Cdc42 activation in endothelial cells abrogated the
effects of ethanol. In addition, we found that ethanol stimulation of endothelial cells induced the production of
H2O2 through activation of Cdc42, and
elimination of H2O2 production abolishes the
effects of ethanol on these cells. Measuring the time course of Cdc42 activation and H2O2 production revealed that
the Cdc42 activation and the increase of H2O2
production lasted more than 3 h, which indicates the mechanisms of
the long duration effect of ethanol on the changes in cell morphology,
cell motility, and in vitro angiogenesis.
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EXPERIMENTAL PROCEDURES |
Materials--
Dulbecco's modified Eagle's medium, catalase,
FITC1-phalloidin,
TRITC-anti-Rabbit antibody were purchased from Sigma. Transwell cell
migration chambers were purchased from Corning Costar (Corning, NY).
Matrigel-coated invasion chambers and Matrigel-coated in vitro angiogenesis plates were purchased from BD Biosciences. Carboxymethyl dichlorofluorescein diacetate was purchased from Molecular Probes (Eugene, OR).
Plasmid Construct and Cell Preparation--
Both dominant
positive Cdc42 (phEFdpCdc42V12) and dominant negative Cdc42
(phEFdnCdc42N14) were the gifts from Dr. Alan Hall (University
College London, UK) and Dr. Wang Lu-Hai (Mount Sinai School of
Medicine). pcDNA3.1/Neo(+)/GPX1 was a gift from Dr. Larry Oberley
(University of Iowa). pGST-WASP-CRIB was a gift from Dr. Pontous
Aspenstrom (Ludwig Institute for Cancer Research, Uppsala, Sweden).
SVEC4-10 cells, an immortal mouse endothelial cell line, were
purchased from ATCC (Manassas, VA). Both phEFdpCdc42V12 and
phEFdnCdc42N14 were transfected into SVEC4-10 cell with calphosphate kit (BD Biosciences), and the transfected cells were selected by
G418 to make stable transfected cell lines. pcDNA3.1/Neo(+)/GPX1 was transiently transfected into SVEC4-10 cells with
calphosphate kit (BD Biosciences).
Ethanol Exposure Method--
Because of the volatility of
ethanol, a method utilizing sealed containers (17) was used to maintain
constant ethanol levels in the culture medium. 0.4% of ethanol was
added directly to the culture medium in either tissue culture trays or
dishes. The trays or dishes were then placed in sealed containers
equipped with an ethanol-containing water bath in the bottom. The
concentration of ethanol in the bath was the same as that in the
culture medium. Ethanol from the bath evaporated into the air of the
sealed container and maintained the ethanol concentration in the
culture medium. A small volume of CO2 (60 cc) was injected
into the container before sealing. The ethanol bath was changed daily
to maintain the appropriated ethanol concentration. In control
cultures, the water bath contained no ethanol. All containers were
incubated at 37 °C. Previous studies show that this sealed container
method accurately maintains ethanol concentrations in the culture
medium (17).
Immunofluorescence Assay--
SVEC4-10 cells were grown on
cover slides. After treatment, cells were fixed and permeabilized as
previously described (18) followed by labeling with FITC-phalloidin for
20 min and mounting to the slides with Fluoromount (Fisher). A Zeiss
LSM 510 microscope was used to obtain images. Scale bars were generated
and inserted by LSM software.
Transwell Migration Assay--
Transwell migration assays were
conducted using modifications of the method described by manufacturer
(BD Biosciences). Briefly, the cells were stimulated with 0.4% ethanol
in serum-free media overnight. The Transwells were coated with E-C-L
cell attachment matrix (Upstate Biotechnology) at 20 µg/ml and
incubated for 1 h at 37 °C. The top chambers of the Transwells
were loaded with 4 × 105 cells/ml in 0.4% ethanol in
serum-free media, and the bottom chambers were filled with 5% fetal
calf serum, Dulbecco's modified Eagle's medium media containing 0.4%
ethanol. The 5% fetal calf serum served as an attractant for the
cells. The Transwells were incubated in sealed chambers with 0.4%
ethanol and 0.5% CO2 at 37 °C for 18 h. After the
incubation, the cells that had migrated were fixed with 10% formalin,
stained with Harris modified Fisher hematoxylin (Fisher), and mounted
on slides. These cells were counted using phase contrast microscopy.
Data given for migrating cells represent the average of five typical
fields per each sample.
Wound Healing Assay--
The wound healing assays were performed
according to the methods described by Meng et al.
(4). SVEC4-10 cells were grown on coverslips to 100% confluent
monolayers and then scratched to form a 100-µm "wound" using
sterile pipette tips. The cells were then cultured with 0.4% ethanol
in serum-free media in sealed chambers for 18 h, fixed on
coverslips with formalin, and stained with FITC-phalloidin. A Zeiss LSM
510 microscope was used to obtain images. Scale bars were generated and
inserted by LSM software.
Invasion Assay--
Invasion assays were performed according to
manufacturer's protocol (BD Biosciences). The cells were stimulated
with 0.4% ethanol in serum-free media overnight. Cells (0.5 ml of
1.0 × 105 cells/ml) were loaded on pre-coated
Matrigel 24-well invasion chambers (BD Biosciences). A 0.5-ml aliquot
of 5% fetal calf serum medium containing 0.4% ethanol was added to
the wells of the BD Falcon TC Companion plate to serve as the
chemoattractant for the cells. The Matrigel invasion chambers were
incubated in a sealed chamber with 0.4% ethanol and 0.5%
CO2 at 37 °C for 22 h. After the incubation, the
invading cells were fixed with 10% formalin, stained with Harris
modified hematoxylin (Fisher), and mounted onto slides. The invading
cells were counted and analyzed according to manufacturer's instructions.
Cdc42 Activation Assay--
Cdc42 activation assays were
performed according to the method described by Edlund et al.
(19). Briefly, the cells were washed with 1× phosphate-buffered saline
supplemented with 1 mM MgCl2. After the
washing, the cells were lysed immediately with lysis buffer (50 mM Tris-HCl, pH 7.5, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 500 mM NaCl, 10 mM
MgCl2, 10 µg/ml aprotinin, and 1 mM
phenylmethylsulfonyl fluoride). The lysates were centrifuged at 14,000 rpm for 15 min. A GST-WASP-CRIB fusion protein in
glutathione S-transferase beads was added into the
supernatants to pull-down active Cdc42 proteins, followed by incubation
at 4 °C for 20 min. After the incubation, the beads were washed 2 times with cold wash buffer (50 mM Tris-HCl pH 7.5, 1%
Triton X-100, 150 mM NaCl, 10 mM
MgCl2, 10 µg/ml aprotinin, and 0.1 mM
phenylmethylsulfonyl fluoride). The Cdc42 protein was eluted with
sample buffer and subjected to 15% SDS-PAGE. The Western blot analysis
was performed using anti-Cdc42 polyclonal antibody. Protein bands were
visualized with an enhanced chemiluminescence reagent (Amersham
Biosciences).
In Vitro Angiogenesis Assay--
An in vitro
angiogenesis assay was performed with slight modification according to
the methods described by Tang et al. (20). In brief, the
cells were incubated in 0.4% ethanol in serum-free media overnight.
Cells (2 ml of 1-2 × 105 cell/ml) were loaded on
pre-coated Matrigel basement membrane matrix dishes (BD Biosciences)
and cultured in serum-free medium containing 0.4% ethanol at 37 °C
for 5 h followed by staining with Diff-Qick (Fisher) for 20 min.
Images were taken using an Olympus inverted microscope.
Cellular Hydrogen Peroxide (H2O2)
Assay--
H2O2 was monitored using a Biotech
H2O2-560 quantitative
H2O2 assay kit (Oxis International, Inc.,
Portland, OR). The assay is based on the oxidation of ferrous ions
(Fe2+) to ferric (Fe3+) by
H2O2 under acidic conditions. The ferric ion
binds with an indicator dye, xylenol orange, to form a stable colored
complex that can be measured at 560 nm using a PerSeptive Biosystems
Cytofluor multiwell plate reader series 4000 (PerSeptive Biosystems
Inc., Framingham, MA). Measurements were made at 37 °C using 1 × 106 cells suspended in 1 ml of phosphate-buffered
saline. Student's t tests were performed using Sigma Stat programs.
H2O2 Assay in Adherent
Cells--
H2O2 assays in adherent cells were
performed according to the methods described by Moldovan et
al. (21). Briefly, cells were serum-starved overnight and then
stimulated with 0.4% ethanol for the different periods of time. At the
end of the stimulation, carboxymethyl dichlorofluorescein diacetate was
added at a final concentration of 5 µM for 30 min. After
the incubation, the cells were washed two times with phosphate-buffered
saline and mounted on coverslips. A Zeiss LSM 510 microscope was used
to obtain images. Scale bars were generated and inserted by LSM software.
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RESULTS |
Ethanol Stimulation Remodels the Integrity of Actin Filaments in
SVEC4-10 Cells--
Actin filaments mediate signal transduction in
cells and undergo dynamic reorganization or remodeling in response to
stimulation. Several activated forms of kinases remodel the structure
of actin filaments to form the cell motile structures and change the
structures of actin stress fibers. Here we sought to determine whether
stimulation by ethanol has the capability to reorganize the structure
of actin filaments in endothelial cells. SVEC4-10 cells, an immortal
mouse endothelial cell line, were serum-starved overnight and
stimulated with 0.4% ethanol for the different periods of time. After
stimulation, the cells were fixed immediately and stained with
FITC-phalloidin followed by analysis using confocal microscopy. It was
found that the actin stress fibers were well organized in the
unstimulated SVEC4-10 cells, i.e. cells were evenly spread
out, and few cell motile structures and rosette-like dots were found at
the cell leading edges and within cell bodies (Fig.
1A). Upon stimulation with
0.4% ethanol for 3 min, actin filaments were remodeled and protruded
the cell membrane at the leading edging to form cell motile structures
such as lamellipodia and filopodia (Fig. 1B). After
stimulation with ethanol for 10 min, the actin stress fibers dissociated to form the rosette-like structures around the cell body,
and a substantial number of cell motile structures were found at the
cell leading edge, indicating that the integrity of actin filaments was
reorganized upon the stimulation with ethanol (Fig. 1C). The
changes in the structure of actin filaments lasted for about 1 h
and then began to recover (Fig. 1, D-G, and data not
shown). Our data indicate that stimulation with ethanol altered the
structure of actin filaments, which induced the dissociation of actin
stress fiber structures to form actin rosette-like structures in the
cell body and cell motile structures at the leading edges.

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Fig. 1.
Ethanol stimulation induces the
reorganization of actin filaments in endothelial cells. SVEC4-10
cells were grown on coverslips and serum-starved overnight. Ethanol
(0.4%) was used to stimulate the cells for the different periods of
time as indicated. After stimulation, cells were fixed on coverslips
and stained with FITC-phalloidin. Confocal microscopy was used to
analyze the integrity of actin filaments and the changes in cell
morphology.
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Ethanol Stimulation Increases Cell Migration and Cell Invasion
through the Activation of Cdc42--
The increase of lamellipodia and
filopodia formation by ethanol stimulation suggests that small Rho
GTPases may be activated in endothelial cells (Fig. 1). Cdc42 is
involved in the formation of filopodia, and Rac is involved in the
formation of lamellipodia (22). We sought to test whether Cdc42 and Rac
were activated. SVEC4-10 cells were treated with 0.4% ethanol for
various periods of time ranging from 3 min to 3 h. Glutathione
S-transferase fusion protein pull-down assays for Cdc42 and
Rac were then performed. The results demonstrate that Cdc42 was
activated upon stimulation with 0.4% ethanol for 3 min, the peak of
Cdc42 activation was detected around 40 min after the stimulation, and
the activation lasted for more than 3 h (Fig.
2A). In contrast, no
activation of Rac was observed (data not shown).

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Fig. 2.
Ethanol stimulation remodels the
structure of actin filaments and increases cell migration and invasion
through the activation of Cdc42 in endothelial cells.
A, ethanol stimulation activates Cdc42. SVEC4-10 cells were
serum-starved overnight and then stimulated with 0.4% ethanol for
different periods of time as indicated. The cells were lysed after
stimulation. Lysates were analyzed for Cdc42 activity. The proteins
were resolved on SDS-PAGE gel, and anti-Cdc42 antibody was used to
detect Cdc42 protein. The densitometries of Cdc42 protein bands were
scanned using EglII software, and the results were plotted by
SigmaPlot. B, ethanol stimulation increases cell migration
through the activation of Cdc42. Both SVEC4-10 cells and SVEC-Cdc42DN
cells were grown on coverslips to 100% confluent monolayers. Sterile
pipette tips were used to scratch the confluent monolayer cells to form
a 100-µm wound, and then the cells were cultured for 18 h with
or without 0.4% ethanol. After the incubation, the cells were fixed on
coverslips and stained with FITC-phalloidin followed by the analysis by
confocal microscopy. C, ethanol stimulation increases the
cell invasion through the activation of Cdc42. Both SVEC4-10 cells and
SVEC-Cdc42DN were cultured on Matrigel-coated Transwells with or
without 0.4% ethanol stimulation for 22 h. The invading cells
were fixed and counted. Five fields were counted randomly. The uncoated
Transwells were used as the controls. The percentage of invading cells
was calculated according to manufacturer's institution. An
asterisk indicates a significant increase. D,
ethanol stimulation reorganizes the structure of actin filaments
through the activation of Cdc42. SVEC-Cdc42DN cells were stimulated
with 0.4% ethanol for different periods of time as indicated. After
stimulation, cells were fixed on coverslips and stained with
FITC-phalloidin followed by analysis with confocal microscopy.
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The increases in cell motile structure at the leading edge of cells
indicate that ethanol may have the capability to increase cell
migration and invasion. To test this possibility, wound healing assays
were performed. SVEC4-10 cell were grown to 100% confluent monolayers
on cover slips and were scratched to form a 100-µm wound with the
sterile pipette tips. The cells were then incubated for 18 h with
or without 0.4% ethanol stimulation. Ethanol-treated SVEC4-10 cells
at the leading edges of the wound migrated and spread to cover the
wound significantly faster than the unstimulated cells (Fig.
2B). We also utilized Transwell migration assays to test the
ability of ethanol to increase the cell migration. The results are
consistent with that of the wound healing assays (data not shown).
Ethanol has been indicated to enhance the metastasis and invasiveness
of several kinds of cancers. We used the Matrigel invasion assays to
examine whether ethanol stimulation can increase the invasion of
SVEC4-10 cells. The results demonstrated that ethanol stimulation
significantly increases the invasiveness of SVEC4-10 cells (Fig.
2C). In the Matrigel assays, 22% of unstimulated SVEC4-10
cells crossed the gel barrier compared with 46% of the ethanol-stimulated cells (p < 0.01, n = 3).
We then investigated whether the activation of Cdc42 was required for
ethanol-induced remodeling of actin filaments and increases in cell
migration and cell invasion. Dominant negative Cdc42 was stably
expressed into SVEC4-10 cells to establish a stable cell line
(SVEC-Cdc42DN) to inhibit the activities of Cdc42. We then treated
SVEC-Cdc42DN cells with 0.4% ethanol to examine changes in the
integrity of actin filaments, cell migration, and cell invasion. Fig.
2D shows that the integrity of actin filaments of
SVEC-Cdc42DN cells exhibited only a minor change upon the stimulation of ethanol. The actin stress fibers were well organized, and there was
no increase in the formation of actin rosette-like structures or cell
motile structures, indicating that the activation of Cdc42 is required
for ethanol-stimulated reorganization of actin filaments. Furthermore,
the expression of SVEC-Cdc42DN decreased the ability of ethanol to
increase the cell migration and cell invasion (Fig. 2, B,
right panels, and C). These data demonstrate that
the activation of small Rho GTPase Cdc42 is essential for
ethanol-stimulated reorganization of actin filaments as well as
increases in cell motility and cell invasion.
Ethanol Induces in Vitro Angiogenesis through the Activation of
Cdc42--
The ability of ethanol to induce in vitro
angiogenesis was tested. Fig.
3A shows the control without
ethanol treatment. Untreated SVEC4-10 cells failed to form the
tube-like structures and remained as individual cells on Matrigel up
to 5 h of incubation (Fig. 3A). In contrast,
ethanol-treated SVEC4-10 cells formed extensive tube-like structures
on Matrigel within the same incubation time (Fig. 3B).
Development of tube-like structures is characteristic of in
vitro angiogenesis. The results indicate that ethanol stimulation induced in vitro angiogenesis in SVEC4-10 cells. Next, we
tested whether the overexpression of dominant negative Cdc42 would
affect the ability of ethanol to induce in vitro
angiogenesis in SVEC4-10 cells. The results show that the expression
of Cdc42 dominant negative protein in SVEC4-10 cells was sufficient to
inhibit in vitro angiogenesis stimulated by ethanol (Fig.
3D), indicating that the activation of Cdc42 is involved in
ethanol-induced in vitro angiogenesis. These results lead us
to test whether the overexpression of dominant positive Cdc42 alone
would induce in vitro angiogenesis in endothelial cells.
Dominant positive Cdc42 was stably expressed into SVEC4-10 cells to
create a stable cell line (SVEC-Cdc42DP). The results show that the
expression of dominant positive Cdc42 was sufficient to induce in
vitro angiogenesis in SVEC4-10 cells within 5 h of
incubation (Fig. 3E), whereas neither SVEC4-10 cells nor
SVEC-Cdc42DN cells were able to induce in vitro angiogenesis
within the same period of time (Fig. 3, A and C).
To the best of our knowledge, this is the first time that activation of
Cdc42 alone has been demonstrated to be sufficient to induce in
vitro angiogenesis. To investigate whether actin filaments are
involved in ethanol-induced in vitro angiogenesis, we
treated SVEC4-10 cells with an actin filament inhibitor,
cytochalasin D, and examined its effects on ethanol-induced in
vitro angiogenesis. Fig. 3F shows that cytochalasin D
preincubation completely blocked the process of ethanol-stimulated
in vitro angiogenesis, implying that the remodeling of actin
filaments is essential for ethanol-stimulated in vitro
angiogenesis. Taken together, the data demonstrate that the activation
of Cdc42 is not only necessary to induce ethanol-stimulated in
vitro angiogenesis but is sufficient to induce in vitro
angiogenesis.

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Fig. 3.
Ethanol stimulation induced in
vitro angiogenesis through the activation of Cdc42 in
SVEC4-10 cells. SVEC4-10 cells, SVEC-Cdc42, and SVEC-Cdc42DP
were grown on Matrigel with the different treatments as indicated for
5 h followed by staining with Diff-Qick. Images are representative
of typical fields seen in three experiments. The magnification is
5×.
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Ethanol Stimulation Induces the Formation of
H2O2 through the Activation of Cdc42 in
SVEC4-10 Cells--
It has been shown that reactive oxygen species
play a major role in ethanol-induced tissue damage (23).
H2O2 is generated via dismuation of the
superoxide radical (O
). It can be converted to the hydroxyl
radical (·OH) via Fenton-type reactions in the presence of metal
ions. Thus, H2O2 is a key intermediate of
reactive oxygen species. To investigate the role of
H2O2 in ethanol-induced angiogenesis, we
detected H2O2 formation from SVEC4-10 cells
stimulated by ethanol. The results demonstrated that stimulation of the
cells by ethanol increased H2O2 production (Fig. 4A). Treatment with
catalase, a specific scavenger of H2O2, reduced
the H2O2 production to the control level (Fig.
4A). To further confirm the functional roles of ethanol in
H2O2 production, glutathione peroxidase was
transiently overexpressed in SVEC4-10 cells to determine whether it
could inhibit ethanol-induced H2O2 production.
Glutathione peroxidase is an important antioxidant enzyme that removes
H2O2 from cells and protects against oxidant damage (24). Our results showed that overexpression of glutathione peroxidase substantial decreased the production of
H2O2 upon stimulation with ethanol (Fig.
4A). These results demonstrate that stimulation with ethanol
induces the production of H2O2 in endothelial
cells.

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Fig. 4.
Ethanol stimulation produces the
reactive oxygen species, H2O2, through the
activation of Cdc42. A, ethanol stimulation induces the
production of H2O2 in SVEC4-10 cells.
SVEC4-10 cells (1 × 105/ml) were stimulated with
0.4% ethanol for 3 min and then monitored for the production of
H2O2 using a Biotch
H2O2 quantitative H2O2
assay kit (1) and confocal microcopy (2).
a, without stimulation; b, with stimulation by
0.4% ethanol; c, same as b but with 5000 units/ml catalase added; d, same as b but with
overexpression of glutathione peroxidase. The asterisk
indicates a significant increase, and the pound sign (#)
indicates a significant decrease (p < 0.05, n = 3). A square region of interest, 500 × 500 µm, was centered on each fluorescent image, and the average gray
value of the entire field (range 0-255, black = 0, white = 255) was extracted using Optimas image analysis software. The values of
average gray level: 2a, 8.09; 2b, 59.81;
2c, 1.30; 2d, 5.05. B, ethanol-induced
production of H2O2 is through the activation of
Cdc42. SVEC4-10 cells (a) and SVEC-Cdc42DN (b)
cells were monitored for the production of H2O2 before and after
stimulation by 0.4% ethanol using a Biotech
H2O2 quanitative H2O2
assay kit (top panel) and confocal microscopy (bottom
panels). An asterisk indicates a significant decrease
for that of SVEC4-10 cells (p < 0.01, n = 3). A square region of interest, 500 × 500 µm, was centered on each fluorescent image, and the average gray
value of the entire field (range 0-255, black = 0, white = 255) was extracted using Optimas image analysis software. The values of
average gray level: 2a, 54.78; 2b, 6.09. C, the overexpression of a constitutively dominant positive
Cdc42 is sufficient to induce the production of
H2O2. Both SVEC4-10 cells and SVEC-Cdc42DP
cells were measured for the production of
H2O2.
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To determine whether the production of H2O2
upon stimulation with ethanol is regulated by Cdc42, SVEC4-10 cells
expressing a dominant negative Cdc42 protein were stimulated with
ethanol. The results showed that expression of dominant negative Cdc42 inhibited ethanol-induced H2O2 production (Fig.
4B). Interestingly, expression of dominant positive Cdc42
itself is sufficient to enhance H2O2 production
(Fig. 4C). These results indicate that Cdc42 is important in
ethanol-induced H2O2 production.
The Production of H2O2 Is Essential for
Ethanol-induced Effects on SVEC4-10 Cells--
To determine whether
H2O2 mediated the effects of ethanol on
SVEC4-10 cells, cells were pretreated with the
H2O2 scavenger catalase for 2 h and
subsequently stimulated with 0.4% ethanol. Immunofluorescence assays
show that pretreatment with catalase blocked the ability of ethanol to
induce remodeling of the structure of actin filaments in SVEC4-10
cells (Fig. 5A). To further
confirm the role of H2O2 in mediating
ethanol-induced actin filament remodeling, SVEC4-10 cells were
incubated with 1 mM peroxide for 5 min followed by the
immunofluorescence analysis. The results showed that the addition of
peroxide partially mimicked the effects of ethanol on the changes of
actin filament integrity (Fig. 5A, 4), indicating that H2O2 production is important in
ethanol-induced actin filament remodeling.

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Fig. 5.
Elimination of H2O2
abrogates the effects of ethanol on SVEC4-10 cells. A,
the free radical scavenger, catalase, inhibits ethanol-induced
reorganization of actin filaments. SVEC4-10 cells were grown on
coverslips and serum-starved overnight. The cells in panel 1 had no stimulation, and the cells in panel 2 were stimulated
with 0.4% ethanol for 3 min. The cells in panel 3 were
preincubated with 5000 units/ml catalase for 2 h followed
by stimulation with 0.4% ethanol for 3 min. The cells in panel
4 were stimulated with 1 mM exogenous hydrogen
peroxide for 5 min. After stimulation, cells were fixed and stained
with FITC-phalloidin followed by analysis with confocal microscopy.
B, the free radical scavenger catalase inhibits
ethanol-stimulated cell migration. SVEC4-10 cells were stimulated with
0.4% ethanol in serum-free media overnight followed by incubation in
the Transwells for 18 h. The cells in panel 2 were
preincubated with 5000 units/ml catalase for 2 h. After
incubation, the cells were fixed, and the images were taken using an
Olympus inverted microscope (panel 1 and panel
2). Five fields of the migrating cells were also counted using
phase contrast microscopy. Cell migration data were analyzed and
plotted using Microsoft Excel (panel 3). The
asterisk indicates a significant decrease from the positive
control (p < 0.01, n = 3).
C, the free radical scavenger, catalase, inhibits
ethanol-stimulated cell invasiveness. SVEC4-10 cells were preincubated
with 5000 units/ml catalase for 2 h and then cultured on a
Matrigel-coated Transwell with 0.4% ethanol stimulation for 22 h.
The cells without preincubation of catalase served as controls. The
invading cells were fixed, and five fields were counted randomly. The
uncoated Transwells were used as controls. The percentage of invading
cells was calculated according to manufacture's instruction. The data
were analyzed and plotted by Microsoft Excel. The asterisk
indicates a significant decrease from the positive control
(p < 0.01, n = 3). D,
inhibition of free radical formation inhibits in vitro
angiogenesis-induced by ethanol. SVEC4-10 cells were either
preincubated with 5,000 units/ml catalase for 2 h (panel
3) or were transiently overexpressed with glutathione peroxidase
(panel 4). SVEC4-10 cells without the pretreatment were
used as a positive control (panel 2). Cells were incubated
on Matrigel with 0.4% ethanol for 5 h. Panel 1 is a
negative control in the absence of 0.4% ethanol. Images are
representative of typical fields seen in three experiments. The
magnification is 5×.
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Both the cell invasion and the cell migration assays demonstrated that
pretreatment with catalase inhibited the ability of ethanol to increase
the migration and invasiveness in SVEC4-10 cell (Figs. 5, B
and C). In vitro angiogenesis assays demonstrated that pretreatment with catalase abrogated the process of angiogenesis induced by ethanol (Fig. 5D). Fig. 5D also shows
that overexpression of glutathione peroxidase had the same inhibitory
effect as catalase did in ethanol-induced in vitro
angiogenesis. These results indicate that the production of
H2O2 mediates the ethanol-induced
reorganization of actin filaments, the increase in cell migration and
cell invasion, and in vitro angiogenesis in SVEC4-10 cells.
Ethanol Stimulation Induces a Long Duration of
H2O2 Production--
To further explore how
the transient activation of Cdc42 can induce the reorganization of
actin filaments in a relatively long period of time, the time course of
H2O2 production upon ethanol stimulation were
measured. SVEC4-10 cells were stimulated with ethanol for different
periods of time ranging from 3 min to 3 h followed by the
detection of H2O2 production using a confocal microscope. It was found that the increase of
H2O2 production began in 3 min after ethanol
stimulation and lasted for 3 h (Fig. 6). Further experiments revealed that the
pattern of increase of H2O2 production lasted
about 24 h (data not shown). The results indicate that the
activation of Cdc42 upon ethanol stimulation induces a profound effect
on the production of H2O2, which keeps actin
filament remodeling to lead to the increases in cell motility and
in vitro angiogenesis.

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Fig. 6.
Time course of H2O2
production upon ethanol stimulation. SVEC4-10 cells were
stimulated with 0.4% ethanol for different periods of time ranging
from 3 min to 3 h as indicated. After the stimulation, the cells
were stained with carboxymethyl dichlorofluorescein diacetate. A Zeiss
LSM 510 microscope was used to detect the production of
H2O2. Images are representative of typical
fields seen in three experiments. A square region of interest, 500 × 500 µm, was centered on each fluorescent image, and the average
gray value of the entire field (range 0-255, black = 0, white = 255) was extracted using Optimas image analysis software.
The values of average gray level: A, 2.3; B, 58;
C, 38; D, 71; E, 72; F,
93.
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DISCUSSION |
The ethanol concentration (400 mg/dl) applied here is relatively
high but is physiologically relevant to the blood alcohol concentration
of heavy alcoholics (25). In general, the concentrations of ethanol
used for in vitro studies are higher than those required to
produce similar effects in vivo. One example relates to the effects of ethanol exposure on cell proliferation. The duration of the
cell cycle is increased by 29% in the telencephalic ventricular zone
of rats with a blood alcohol concentration of ~180 mg/dl (26).
Similarly, the length of the cell cycle of cultured neuroblastoma cells
(used as a model for proliferating neuronal cells) is prolonged (+37%)
by treatment with 400 mg/dl of ethanol (27).
In this report, we demonstrate that ethanol stimulation in endothelial
cells could induce the remodeling of actin filaments, disrupt actin
stress fibers, and induce the formation of cell motile structures. More
importantly, the remodeling of the structure of actin filaments is
concomitant with the increases in the cell motility and cell invasion.
The reorganization of actin filaments is essential for cell signal
transduction. It is a highly coordinated process that is regulated by
cell signaling cascades. It has been shown that the reorganization of
the structure of actin filaments is one of driving forces that cause
the cell membrane to protrude forward to form the cell motile
structures, which are involved in the increases in both actin filament
polymerization and cross-linking (28-30). The reorganization of actin
filaments plays a major role in the cell migration and invasion. It is
essential in cell extension, attachment, contraction, and release
during cell migration and cell invasion (31). We
demonstrate here that ethanol stimulation could promptly reorganize the
structure of actin filaments to induce the formation of cell motile
structures and the dissociation of actin stress fibers within several
minutes. Interestingly, similar results are found in insulin-mediated
signal transduction, in which the reorganization of actin filaments
occurs within several minutes upon the stimulation with insulin (13,
32). It is likely that the fast response of actin filaments to
stimulation with ethanol enables the cells to adapt to changes in the
environment properly and to transfer and propagate the signals.
Rho GTPases Cdc42, Rac, and Rho play a central role in the
reorganization of the structure of actin filaments. It has been demonstrated that Cdc42 regulates the formation of filopodia, Rac
regulates the formation of lamellipodia, and Rho regulates the
formation of stress fibers (22). The morphological changes in the
structure of actin filaments, primarily the increase in the formation
of filopodia and lamellipodia, upon stimulation by ethanol implies that
Cdc42 and Rac could be activated. Indeed, our results demonstrate that
ethanol stimulation activated Cdc42 in SVEC4-10 cells. However, no
activation of Rac was detected upon stimulation with ethanol in
SVEC4-10 cells (data not shown). A literature search found that it was
not unprecedented that Cdc42 remodeled actin filaments to form both
filopodia and lamellipodia structures independent of the activation of
Rac (19, 33). The observation that expression of dominant negative
Cdc42 blocked the effects of ethanol indicates that the activation of
Cdc42 is necessary to reorganize the structure of actin filaments and to increase the cell migration and cell invasion upon stimulation with
ethanol. Cdc42 has been found to control signaling pathways, such as
apoptosis, cell cycle progression, and cell transformation (34).
Recently, Cdc42 was found to be essential in
V
3 integrin-mediated angiogenesis.
Inhibition of
V
3
integrin-dependent activation of Cdc42 results in the
suppression of angiogenesis in vivo (35). Cdc42 was also
found to mediate factor VIIa/tissue factor-induced angiogenesis (36).
Mutation of its downstream kinase p21-activated kinase is
sufficient to block angiogenesis (37). In this study, we demonstrated
that Cdc42 is essential for ethanol-induced in vitro
angiogenesis in SVEC4-10 cells. More importantly, we found that the
overexpression of dominant positive Cdc42 is sufficient to induce
in vitro angiogenesis in mouse endothelial cells. This is
the first time that the overexpression of a constitutively dominant
active Cdc42 alone has been demonstrated to induce in vitro
angiogenesis. Our results also indicate that the in vitro angiogenesis observed in this study may be mediated through the reorganization of actin filaments. Cytochalasin D is an actin filaments
inhibitor (38), and preincubation of SVEC4-10 cells with cytochalasin
D blocked both the dominant positive Cdc42 (data not shown) and
ethanol-induced in vitro angiogenesis.
Ethanol has been shown to induce in vitro angiogenesis
through the activation of protein kinase C and mitogen-activated
protein kinase signaling pathways (39). Recently, ethanol has been
found to activate the angiogenic stimulator vascular endothelial growth factor through HIF-1
and Ras (7, 40). It is unclear at this time whether the activation of Cdc42 upon stimulation by ethanol is
related to those pathways. Further studies are needed to identify the
connections among these signaling pathways in ethanol-induced in
vitro angiogenesis.
The results obtained from the present study show that
H2O2 generated by ethanol-stimulated cells may
play a key role in ethanol-induced angiogenesis and that Cdc42 is
responsible for the H2O2 generation. The
following experimental observations support these conclusions. (a) The generation of H2O2 upon
stimulation by ethanol was visualized using confocal microscopy and
detected by the change in fluorescence of scopoletin in the presence of
horseradish peroxidase; (b) the increase of
H2O2 production lasts a relatively long period
of time; (c) catalase, a scavenger of
H2O2, decreased its generation; (d)
dominant negative Cdc42 cells generated a low level of
H2O2 compared with wild type cells;
(e) catalase prevented ethanol-induced remodeling of actin
filaments, decreased in cell migration and cell invasion, and reduced
angiogenesis; (f) the addition of peroxide extracellularly
induced actin filament remodeling; and (g) overexpression of
glutathione peroxidase decreased the H2O2
formation inside the cells and inhibited ethanol-induced angiogenesis.
It should be noted that H2O2, a key member of
the reactive oxygen species family, plays a pivoted role in various
cellular processes. Recently, it has been reported that chronic alcohol
toxicity is mediated primarily through oxidative stress in liver,
central nervous system, and other tissues (23). Electron spin resonance
and the analysis of indirect makers of oxidative stress, such as lipid
peroxidation protein modification, and alternation in the levels of
endogenous tissue antioxidants, lead to the hypothesis that oxidative
stress is an important mediator of alcohol-induced cellular injury
(23). Based on this hypothesis, therapeutic strategies have been
developed for the treatment of alcohol toxicity by administration of
antioxidants (23). The present study shows that
H2O2 is an important mediator in
ethanol-induced remodeling of actin filament integrity and in
vitro angiogenesis, and Cdc42 is an upstream mediator of
H2O2 generation. This study provides a
molecular basis for the role of H2O2 in the
ethanol-induced cell responses. Currently, we are actively identifying
the downstream signals of H2O2 in mediating the
effects of ethanol on SVEC4-10 cells.
Taken together, our data demonstrate that ethanol stimulation of
endothelial cells has the ability to produce
H2O2 through the activation of Cdc42, which
induces the reorganization of actin filaments to lead to the increases
in cell migration, cell invasion, and in vitro angiogenesis.
Data also demonstrate that the overexpression of a constitutively
dominant active mutant Cdc42 alone is sufficient to induce the
formation of H2O2 and to cause in
vitro angiogenesis. Our study identifies a novel signaling pathway
that links the ethanol-induced changes in Cdc42,
H2O2, actin filaments, and cell motility to
in vitro angiogenesis. These observations may have implications for understanding the molecular mechanisms of angiogenesis in general.