Activation of VEGF and Ras genes in gastric mucosa during
angiogenic response to ethanol injury
Michael K.
Jones1,
Rabiha M.
Itani1,
Hongtao
Wang2,
Morimasa
Tomikawa2,
I. James
Sarfeh3,
Sandor
Szabo4, and
Andrzej S.
Tarnawski1,2,5
Departments of 5 Medicine,
3 Surgery, and 4 Pathology,
1 Veterans Affairs Medical Center, Long
Beach, 90822; and 2 University of California,
Irvine, California 92697
 |
ABSTRACT |
Our
previous studies demonstrated that ethanol injury triggers the
angiogenic response in gastric mucosa bordering necrosis. The present
study was aimed to determine whether vascular endothelial growth factor
(VEGF) (a potent angiogenic peptide selectively acting on endothelial
cells) and Ras (a mediator of cell proliferation and a putative
regulator of VEGF expression) are involved in gastric angiogenesis
after ethanol injury. We studied the angiogenic response and expression
of VEGF and Ras in gastric mucosa after ethanol injury. Ethanol damage
triggered 1) angiogenesis in the gastric mucosa bordering
necrosis, 2) significant increases in VEGF mRNA and protein
expression, and 3) significant increases in the expression of
Ki-ras mRNA and Ras proteins. Neutralizing anti-VEGF antibody significantly reduced (by greater than threefold) the angiogenic response to ethanol-induced injury. Moreover, mevastatin, an inhibitor of Ras activation, completely blocked the induction of VEGF expression in cultured primary endothelial cells. Because, in other tissues, VEGF
is one of the most potent angiogenic factors and VEGF expression is
dependent on Ras, our data indicate that Ras and VEGF are involved in
gastric mucosal angiogenesis after ethanol injury.
ethanol; oncogenes; neutralizing antibody; competitive reverse
transcriptase-polymerase chain reaction; inhibitor; vascular
endothelial growth factor
 |
INTRODUCTION |
BLOOD FLOW THROUGH MICROVESSELS, i.e.,
capillaries, arterioles, and collecting venules, is essential for
supplying the gastric mucosa with oxygen and nutrients as well as for
the removal of toxic metabolic products (16). Endothelial cells lining
gastric mucosal microvessels are major and early targets of acute
injury by ethanol (34, 37, 41). Injury of the microvascular endothelial cells by ethanol leads to the microvascular stasis and ischemia that
result in focal deep necroses, e.g., mucosal erosions (23, 34, 37, 38,
41). The repair of such injury requires not only restoration of the
surface epithelium, glandular epithelial cells, and connective tissue
but also, most importantly, a reestablishment of the microvascular
network, crucial for delivery of oxygen and nutrients to the area.
Although our knowledge regarding the mechanisms of gastric mucosal
injury and restitution of the surface epithelium has advanced rapidly
in recent years (3, 19, 26, 33, 38), repair of the microvascular
network has not been explored except in our previous experiments. These
studies demonstrated that angiogenesis, i.e., formation of new
microvessels, occurs in the gastric mucosa acutely injured by ethanol.
We have characterized some morphological features of this process (36,
39, 42).
The angiogenic response involved in wound repair results from the
stimulation of endothelial cells by growth factors [including acidic
(aFGF) and basic fibroblast growth factor (bFGF)] to migrate, proliferate, and form the endothelial tubes and capillary structures that undergo transformation into capillary vessels and lead to the
restoration of the microvascular network (1, 13, 21). The importance of
bFGF in promoting dermal wound healing has been established (4). We
have previously demonstrated, in a preliminary study, that
ethanol-induced injury triggers the angiogenic response in the gastric
mucosa bordering necrosis and also activates expression of bFGF and its
receptors in this area (36).
Another growth/permeability factor, vascular endothelial growth factor
(VEGF), which is involved in wound healing (6), has been shown to
stimulate normal angiogenesis as well as the angiogenesis that
underlies tumor metastasis (14, 18, 30). VEGF is the only growth factor
that acts predominantly on endothelial cells upon binding to specific
receptors Flt-1 and Flk-1 (KDR) (9, 12, 24).
Several studies have shown that VEGF expression is upregulated by
oncogenic Ras in transfected cell lines (such as NIH/3T3 fibroblasts
and lines derived from epidermal keratinocytes and intestinal
epithelial cells), suggesting a role for Ras in both VEGF regulation
and angiogenesis in these models (20, 22, 25). However, the expression
and roles of VEGF and Ras have not been extensively studied in the
repair of acute gastric mucosal injury and, to our knowledge, have
never been investigated with regard to ethanol-induced gastric mucosal
injury. Therefore, the aim of this study was to determine whether acute
gastric mucosal injury by ethanol triggers the expression of VEGF and
Ras at both the transcriptional and translational levels and to
establish their relationship to the angiogenic response in the rat
gastric mucosa after ethanol-induced injury.
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MATERIALS AND METHODS |
This study was approved by the Subcommittee for Animal Studies of the
Long Beach Department of Veterans Affairs Medical Center (Long Beach,
CA). Sprague-Dawley rats (weight range 250-300 g) were used in the
experiments. Fifty-four rats fasted for 24 h received,
intragastrically, either 1.5 ml of the indicated concentrations of
ethanol (25, 50, or 100%) or 1.5 ml H2O (controls). At 3, 6, and 24 h after ethanol administration, rats were anesthetized, their
stomachs were excised, and the animals were then euthanized. The
stomachs were then opened along the greater curvature, rinsed with
0.9% NaCl, and examined visually. The macroscopically visible hemorrhagic mucosal erosions were photographed in a standardized manner
and evaluated by image analysis as described earlier (38). The area of
hemorrhagic erosion was expressed as a percentage of total glandular
area (38). Gastric tissue (1.5-2 mm in width) from nonhemorrhagic
areas immediately adjacent to the necrotic lesions was excised and
either immediately frozen in liquid nitrogen and stored at
80°C
for RT-PCR and immunoblotting or fixed in 4% paraformaldehyde for immunohistochemistry.
RNA isolation and RT-PCR.
Frozen tissue specimens were homogenized with a Polytron homogenizer
(Kinematica, Littau, Switzerland) in 4 M guanidinium isothiocyanate,
and total RNA was isolated using the guanidinium isothiocyanate-phenol-chloroform method (8). RT was carried out using a
GeneAmp RNA PCR kit and a DNA thermal cycler (Perkin- Elmer, Norwalk,
CT), which were also used for PCR. An amount of 0.3 µg of total RNA
was used as the template to synthesize cDNA with 2.5 units of Moloney
murine leukemia virus RT in 10 µl of buffer containing 10 mM
Tris · HCl (pH 8.3), 50 mM KCl, 5 mM random hexamer, and
1.4 U/µl of ribonuclease inhibitor. RT was performed at room
temperature for 20 min and then at 42°C for 15 min, at 99°C for 5 min, and at 5°C for 5 min. The resulting cDNA was precipitated and
resuspended at 1.5 µg/µl. PCR amplification of the cDNA was performed using either primers that recognize all four isoforms of VEGF
mRNA (5) for VEGF or Ki-ras (28). Primers that recognize
-actin were used with the same cDNA preparations as an internal control for quantifying mRNA. The primers for VEGF were
5'-CCTGGTGGACATCTTCCAGGAGTACC-3' (sense) and
5'-GAAGCTCATCTCTCCTATGTGCTGGC-3' (antisense).
The primers for Ki-ras were 5'-TGAGTATAAACTTGTGGTAGTTGG-3'
(sense) and 5'-GGTGAATATCTTCAAATGATTTAG-3' (antisense).
The primers for
-actin were 5'-TTGTAACCAACTGGGACGATATGG-3' (sense)
and 5'-GATCTTGATCTTCATGGTGCTAGG-3' (antisense). The primers for
-actin were purchased from Clontech, Palo Alto, CA. PCR was performed with 3 µg of cDNA (2 µl) in 50 µl of buffer containing 10 mM Tris · HCl (pH 8.3), 2 mM
MgCl2, 50 mM KCl, 0.2 mM each of deoxyribonucleoside
triphosphates, 0.4 µM of each primer, and 2 units of Taq DNA
polymerase. For VEGF, the amplification was performed for 33 cycles of
1 min at 94°C for denaturing, 1 min at 55°C for annealing, and 2 min at 72°C for extension. For Ki-ras, the amplification was
performed for 35 cycles of 1 min at 94°C for denaturing, 1 min at
57°C for annealing, and 2 min at 72°C for extension. Aliquots (9 µl) of the products were subjected to electrophoresis on a 1.25%
agarose gel, and the DNA was visualized by ethidium bromide staining.
Location of the products and their sizes were determined by using a
100-bp ladder (GIBCO BRL, Gaithersburg, MD). The gel was then
photographed under ultraviolet transillumination. For the quantitative
assessment of the PCR products, a video analysis system (Image-1/FL,
Universal Imaging, Westchester, PA) was used. The Image-1 system can
distinguish density on a scale of 0-255 units. Each measurement
was standardized by subtracting the background intensity in average.
Competitive RT-PCR.
Competitive RT-PCR was used to quantify the level of VEGF mRNA
expression by employing one set of primers to amplify both the target
cDNA and another DNA fragment, whereby the second DNA fragment competes
with the target DNA for the same primers and thus acts as an internal
standard (15, 31). Serial dilutions of the competitor fragment are
added to PCR amplification reactions containing constant amounts of the
target cDNA samples. By knowing the amount of the competitor added to
the reaction, the mRNA level can be quantitatively determined. The
competitor DNA fragment was constructed using the MIMIC construction
kit (Clontech Laboratories, Palo Alto, CA) according to the
manufacturer's instructions. The composite primers used to construct
the competitor fragment were 5'-
CGCAAGTGAAATCTCCTCCG-3'
(forward) and
5'-
CTCTCCTATGTGCTGGCTCTGTCAATGCAGTTTGTAG-3'
(reverse). The underlined portions of the above primers are the target
gene primer sequences, and the remaining sequences were designed to yield a competitive PCR product approximately twice the size (400 bp)
of the target cDNA PCR product (196 bp). PCR was performed with 3 µg
of total RT cDNA and the indicated concentrations of competitor
fragment. The amplification was performed for 33 cycles of 1 min at
94°C for denaturing, 1 min at 55°C for annealing, and 2 min at
72°C for extension. Aliquots (9 µl) of the products were subjected
to electrophoresis on a 1.25% agarose gel, and the DNA was visualized
by ethidium bromide staining. The gel was then photographed under
ultraviolet transillumination. For the quantitative assessment of the
PCR products, a video image analysis system (Image-1/FL) was used. The
Image-1 system can distinguish density on a scale of 0-255 units.
Each measurement was standardized by subtracting the background
intensity in average.
Immunoblot analysis.
Frozen specimens of gastric tissue were homogenized with a Polytron
homogenizer (Kinematica) in a lysis buffer containing 62.5 mM EDTA, 50 mM Tris · HCl (pH 8.0), 0.4% deoxycholic acid, 1% Nonidet
P-40, 0.5 µg/ml leupeptin, 0.5 µg/ml pepstatin, 0.5 µg/ml
aprotinin, 0.2 mM phenylmethylsulfonyl fluoride, and 0.05 mM aminoethyl
benzenesulfonyl fluoride. The homogenates were then centrifuged (14,000 rpm for 10 min at 4°C). The protein concentration of the homogenate
was determined by the bicinchoninic acid protein assay using a
commercial kit (BCA protein assay reagent, Pierce Chemical, Rockford,
IL). Equal amounts of protein from the tissue homogenates were mixed
with 4× sample buffer containing 62.5 mM Tris · HCl (pH
6.8), 10% glycerol, 2% SDS, 5%
-mercaptoethanol, 0.65 mM
dithiothreitol, and 0.06% bromphenol blue. The samples were then
boiled for 2 min and loaded onto 15% acrylamide gels and
electrophoresed. The separated proteins were then transferred onto
nitrocellulose membranes (Hybond ECL, Amersham Life Science, Arlington
Heights, IL), and the membranes were blocked in buffer containing 10 mM
Tris · HCl (pH 7.5), 100 mM NaCl, 0.1% Tween 20, and 5%
milk for 1 h at room temperature before incubation with primary
antibody for either VEGF (rabbit polyclonal, Santa Cruz Biotechnology,
Santa Cruz, CA) or Ras (mouse monoclonal, Oncogene Science, Uniondale,
NY) at a concentration of 1:100 for 1 h at room temperature on a
platform rocker. The membranes were then washed four times in buffer
without milk and incubated with either anti-rabbit IgG peroxidase
conjugate (Sigma Chemical, St. Louis, MO) or anti-mouse IgG peroxidase
conjugate (Transduction Laboratories, Lexington, KY) at room
temperature for 1 h on a platform rocker. The signal was visualized by
the enhanced chemiluminescence method using ECL Western blotting
detection reagents (Amersham). Quantification of the signal was
performed by densitometry scanning using an LKB 2222-020 Ultro Scan XL
laser densitometer (Pharmacia LKB Biotechnology, Uppsala, Sweden).
Assessment for angiogenesis using immunofluorescence staining for
vimentin.
Angiogenesis was assessed using fluorescence staining for vimentin, a
major component of intermediate filaments in mesenchymal cells (41). In
the rat gastric mucosa, vimentin is located predominantly in
endothelial cells lining the mucosal microvascular network (39). When
deep mucosal injury occurs, the microvascular network is destroyed and
vimentin fluorescence is extinct (17). Conversely, the restoration of
mucosal microvessels during the process of angiogenesis is reflected by
the appearance of sprouting tubes of endothelial cells strongly
positive for vimentin (39). The staining for vimentin as an indicator
of endothelial cells was further confirmed in this study by the
staining of similar gastric specimens for factor VIII-related antigen.
Gastric specimens were fixed in 4% paraformaldehyde for 4 h and
subsequently transferred to 0.5 M sucrose in phosphate-buffered saline
for 24 h. They were then frozen at
80°C until cutting. Cryostat
sections (10 µm thick; Jung Cryocut 1800, Leica, Deerfield, IL) were
digested with 0.1% trypsin (Sigma) at 37°C for 10 min and incubated
overnight with an antibody specific for rat vimentin (mouse monoclonal,
Dako, Carpinteria, CA) or factor VIII-related antigen (rabbit
polyclonal, Dako). For control studies, to determine nonspecific
secondary antibody binding, cryostat gastric sections were incubated
overnight with PBS instead of the primary antibody. After washing with
PBS, sections were incubated for 30 min with fluorescein-conjugated anti-mouse or anti-rabbit immunoglobulin (Sigma) diluted 1:100. Immunofluorescence was evaluated using a Nikon Optiphot epifluorescence microscope with B filter composition (Nikon, Garden City, NY). Angiogenesis was assessed quantitatively by counting (under ×400 magnification) the number of mucosal microvessels demonstrating sprouting endothelial tubes. Counting was performed on coded slides in
20 different randomly selected fields of mucosal erosions. At least 100 microvessels per specimen were counted.
Effect of neutralizing anti-VEGF antibody on angiogenesis.
Sprague-Dawley rats (weight range 175-200 g) fasted for 24 h were
injected intravenously with either 200 µg of a goat polyclonal VEGF
neutralizing antibody (R&D Systems, Minneapolis, MN) or 200 µg of
normal polyclonal antibody of the same isotype (controls) in 500 µl
of PBS. Concurrently, the rats received, intragastrically, 1.5 ml 50%
ethanol. Twenty-four hours after ethanol administration, the animals
were anesthetized, their stomachs were excised, and the animals were
euthanized. The stomachs were then opened along the greater curvature,
rinsed with 0.9% NaCl, examined visually, and photographed in a
standardized fashion as described (38). The area of macroscopic
necrosis was measured and expressed as a percentage of total glandular
area (38). Angiogenesis was then assessed quantitatively, as described
above, by counting (under ×400 magnification) the number of mucosal
microvessels demonstrating sprouting endothelial tubes.
Effect of Ras inhibition on VEGF expression in vitro.
Rat primary aortic endothelial cells were plated in 100-mm tissue
culture dishes and grown until ~80% confluent in DMEM supplemented with 10% fetal bovine serum. For the determination of Ras activation, cells were serum-starved for 16 h and metabolically labeled for an
additional 8 h in serum-free, phosphate-free DMEM containing 200 µCi/ml 32PO4. The cells were incubated during
the final 4 h with vehicle (controls) or 25 µM mevastatin to prevent
Ras localization to the plasma membrane, thus inhibiting Ras
activation. Then 10 ng/ml bFGF was added and the cells were further
incubated for 5 min. Ras activation was determined according to
Downward et al. (10). Briefly, cells were washed with ice-cold PBS and
lysed on ice in 1 ml of lysis buffer [50 mM HEPES (pH 7.5), 500 mM
NaCl, 5 mM MgCl2, 1% Triton X-100, 0.5% deoxycholate,
0.05% SDS, 1 mM EGTA, 10 mM benzamidine, and 10 µg/ml each of
aprotinin, leupeptin, and soybean trypsin inhibitor]. Ras proteins
contained in the cell lysates were immunoprecipitated with rat
monoclonal anti-Ras antibody (Y13-259, Santa Cruz Biotechnology). The
guanine nucleotides bound to the Ras proteins were eluted in 16 µl of
2 mM EDTA, 5 mM dithiothreitol, 1 mM GTP, 1 mM GDP, and 0.2% SDS at
68°C for 20 min and fractionated by thin-layer chromatography.
Quantification was performed using a PhosphorImager
(Molecular Dynamics, Sunnyvale, CA). The percentage of GTP bound to Ras
(as an indicator of Ras activation) was calculated as [counts per
minute (cpm) in GTP]/(cpm in GTP + cpm in GDP) normalized for moles
phosphate in each nucleotide. For determination of VEGF expression,
primary endothelial cells were plated as above until ~80% confluent.
The cells were then serum starved for 24 h and incubated for 4 h with
25 µM mevastatin or vehicle (controls). The cells were further
incubated for 5 min with 10 ng/ml bFGF. The cells were washed with
serum-free DMEM and incubated in this medium for an additional 1 h (for
mRNA expression) and 3 h (for protein expression). The cells were then washed twice with ice-cold PBS and lysed on ice with either 1 ml of 4 M
guanidinium isothiocyanate for total RNA isolation as described under
RNA isolation and RT-PCR or 300 µl of the lysis buffer as
described under Immunoblot analysis. RT-PCR and immunoblot analysis for VEGF expression were performed as described above.
Statistical analysis.
Results are expressed as means ± SD. Student's t-test was
used to determine statistical significance between control and
experimental groups. A P value of <0.05 was considered
statistically significant. Comparisons of data between multiple groups
were performed with ANOVA.
 |
RESULTS |
Assessment of mucosal injury and the angiogenic response of gastric
endothelial cells to ethanol-induced injury.
Administration of 100% ethanol caused severe mucosal injury reflected
as macroscopically visible hemorrhagic necrotic bands, identical to
those described previously (19, 34, 37, 38). The necrosis involved 34 ± 3%, 38 ± 4%, and 42 ± 4% of the total mucosal area at 3, 6, and 24 h, respectively, after ethanol administration. Histological
examination demonstrated that the necrotic bands correspond to deep
hemorrhagic erosions, as described in our previous papers (37, 38).
Within the erosions all mucosal structures, including the gastric
glandular epithelium and the microvascular network, were destroyed as
reflected by immunofluorescence staining for vimentin (an intermediate
filament protein; Fig. 1, A and B), which is strongly expressed in
endothelial cells lining mucosal microvessels of normal gastric mucosa
(Fig. 1, A and B). Immunofluorescence staining of
gastric mucosal sections for vimentin at 24 h after ethanol
administration clearly demonstrated angiogenesis reflected by the
presence of numerous tubes of migrating endothelial cells (Fig.
1C). Staining gastric mucosal sections for both vimentin and
factor VIII-related antigen demonstrated their colocalization in
endothelial cells (data not shown). Quantitative analysis demonstrated that, at 24 h, 9 ± 1% of the microvessels in the mucosa bordering necrosis showed sprouting endothelial tubes, a hallmark of the angiogenic process (Fig. 1C).

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Fig. 1.
Photomicrographs of gastric mucosa showing immunofluorescence staining
for vimentin. A: in normal mucosa, vimentin shows a regular
pattern of distribution in endothelial cells lining microvessels.
Magnification ×400. B: in mucosal erosion resulting from
ethanol-induced injury, there is virtually no vimentin fluorescence,
reflecting destruction of mucosal microvessels (arrows). Magnification
×400. C: gastric mucosa 24 h after ethanol administration.
Migrating tubes of endothelial cells (arrows) are clearly identified,
reflecting angiogenesis. In migrating tubes, vimentin was found to be
colocalized with expression of factor VIII-related antigen (see
RESULTS). Magnification ×1,000.
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Neutralizing anti-VEGF antibody impairs the angiogenic response of
gastric endothelial cells to ethanol-induced injury.
To assess the contribution of endogenous VEGF to the angiogenic
response that occurs in the gastric mucosa within 24 h of ethanol-induced injury, a "neutralizing" anti-VEGF antibody was administered intravenously concurrently with the intragastric administration of 50% ethanol. Twenty-four hours after administration of anti-VEGF and ethanol, macroscopically visible hemorrhagic necrotic
bands were clearly present. The necrosis involved 18 ± 3% of the
total mucosal area. The necrosis resulting 24 h after the concurrent
administration of 50% ethanol with preimmune IgG (controls) involved
significantly (P < 0.025) less of the total mucosal area
(4 ± 3%), indicating that anti-VEGF antibody delays healing.
Angiogenesis, at 24 h after the concurrent administration of anti-VEGF antibody and 50% ethanol, was assessed as described in
MATERIALS AND METHODS. Immunofluorescence staining of
gastric mucosal sections for vimentin clearly demonstrated significant impairment of angiogenesis, as reflected by the severe reduction in
tubes of migrating endothelial cells (Fig.
2B) compared with controls
receiving preimmune IgG concurrent with 50% ethanol (Fig. 2A).
Quantitative analysis demonstrated that administration of the
neutralizing anti-VEGF antibody resulted in a threefold reduction vs.
controls (without neutralizing antibody) in the percentage of the
microvessels showing sprouting endothelial tubes in the mucosa
bordering necrosis (2.9 ± 1.2% vs. 9 ± 1.3%,
P < 0.0001).

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Fig. 2.
Effect of "neutralizing" anti-vascular endothelial growth factor
(VEGF) antibody on angiogenic response to ethanol-induced injury to the
gastric mucosa. Photomicrographs of gastric mucosa showing
immunofluorescence staining for vimentin. A: representative
staining of gastric mucosa from controls receiving normal (preimmune)
goat IgG concurrent with administration of 50% ethanol. Similarly to
Fig. 1C, migrating tubes of endothelial cells (arrows) are
clearly identified, reflecting angiogenesis 24 h after ethanol
administration. Magnification, ×1,000. B: representative
staining of gastric mucosa from animals receiving neutralizing
anti-VEGF antibody concurrent with administration of 50% ethanol.
Significant impairment of angiogenesis is clearly demonstrated by
dramatic reduction in tubes of migrating endothelial cells.
Magnification, ×1,000.
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Expression of VEGF mRNA is increased following ethanol-induced
gastric mucosal injury.
VEGF mRNA expression after ethanol-induced gastric mucosal injury was
determined quantitatively. Competitive RT-PCR, in which a DNA fragment
of known concentration competes for the same PCR primers as the cDNA
resulting from the reverse transcription reaction, thus allowing
quantification of the amount of cDNA from a given RNA sample (15, 31),
was used to quantify VEGF mRNA expression (Fig. 3,
A-D). With this method, we
determined the level of VEGF mRNA expression in ethanol-treated gastric
mucosa to be 0.667 ± 0.139, 0.586 ± 0.125, and 0.408 ± 0.046 amol/µg total RNA at 3, 6, and 24 h, respectively, compared with
0.106 ± 0.021 amol/µg total RNA in control gastric mucosa.
Ethanol-induced injury, therefore, significantly increased VEGF mRNA
expression in the gastric mucosa bordering necrosis to 629%
(P < 0.0003), 553% (P < 0.0003), and 385%
(P < 0.0001) at 3, 6, and 24 h, respectively, compared with controls (Fig. 3E).

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Fig. 3.
Quantification of VEGF mRNA expression in gastric mucosa at 3, 6, and
24 h after ethanol (EtOH) treatment by competitive RT-PCR. Competitive
RT-PCR was performed as described in MATERIALS AND METHODS
to provide quantification of VEGF mRNA expression in gastric mucosa of
control animals (A) and of animals at 3 h (B), 6 h
(C), and 24 h (D) after intragastric administration of
100% ethanol. Known amounts of a competitor DNA fragment were used in
PCR amplification reactions containing a constant amount of total
target cDNA (3 µg). Amounts of competitor DNA fragment used in PCR
reactions: lane 1, 2 × 10 1 amol; lane
2, 2 × 10 2 amol; lane 3, 2 × 10 3 amol; and lane 4, 2 × 10 4
amol. E: quantitative data of competitive RT-PCR shown in
A-D. Log of ratio of VEGF target intensity to competitor
intensity was plotted against log of competitor amounts used. Amount of
VEGF target cDNA was calculated by determining the x-intercept
for point on curve at which ratio of target to competitor equaled 1. Total mRNA content of each sample was normalized using -actin as
internal control (data not shown). Values are means ± SD. For each
time point, n = 6.
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Expression of VEGF protein is increased after ethanol-induced injury
to the gastric mucosa.
Immunoblot analysis demonstrated that ethanol-induced injury of the
gastric mucosa also results in increased expression of the secreted
isoform of VEGF protein, VEGF165, at 3, 6, and 24 h in the
mucosa bordering necrosis. A protein band of ~20 kDa, corresponding
to VEGF165, was found to be significantly increased at each
of the three time points over the controls, with the greatest increase
being at 3 h [367% (P < 0.001) compared with 318%
(P < 0.001) at 6 h and 185% (P < 0.002) at 24 h] as shown in Fig. 4A. Expression
of the larger, nonsecreted form of VEGF was not significantly different
compared with controls (data not shown). Quantitative data for VEGF
protein expression are presented in Fig. 4B.

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Fig. 4.
Western blot analysis of VEGF protein expression in gastric mucosa
after ethanol injury compared with gastric mucosa of
controls. A: analysis of VEGF protein expression was
performed as described in MATERIALS AND METHODS. Level of
VEGF protein from gastric mucosa samples of control animals is shown
together with that of gastric mucosa samples from animals at each time
point after intragastric administration of 100% ethanol. B:
quantitative data of VEGF protein expression (shown in A)
obtained by densitometric scanning using values of peak area. Values
are means ± SD. For each time point, n = 6.
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Expression of Ki-ras mRNA is increased after ethanol-induced injury
to the gastric mucosa.
Because VEGF has been shown to be upregulated by oncogenic Ras in vitro
(20, 22, 25), we studied the level of gastric Ki-ras mRNA
expression in the mucosa bordering necrosis in response to
ethanol-induced injury. RT-PCR demonstrated that the gastric mucosa
(bordering necrosis) of rats that received ethanol had increased
expression of Ki-ras mRNA to 163% (P < 0.05) and
190% (P < 0.02) at 3 and 6 h, respectively, compared with
the controls (Fig. 5A, top).
Although this analysis indicated that the increased expression of
gastric Ki-ras mRNA resulting from ethanol-induced injury
remains twofold greater at 24 h compared with the controls, upon
normalizing for
-actin mRNA as shown in Fig. 5A, bottom, the
P value was outside the range of statistical significance (P = 0.097). Quantitative data for Ki-ras mRNA
expression are presented in Fig. 5B.

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Fig. 5.
RT-PCR analysis of Ki-ras mRNA expression in gastric mucosa
after ethanol injury compared with gastric mucosa of controls. A,
top: RT-PCR using specific primers for Ki-ras was performed
as described in MATERIALS AND METHODS. Samples from control
animals are shown together with samples from each time point after
intragastric administration of 100% ethanol. A, bottom: RT-PCR
of same reverse transcription products in A, top, using
specific primers for -actin as internal control for total mRNA.
B: quantitative data for Ki-ras mRNA expression (shown
in A) using a computerized video analysis of amplified PCR
products. Each signal was normalized against corresponding -actin
signal, and results are expressed as Ki-ras/ -actin. Values
are means ± SD. For each time point, n = 6.
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Expression of Ras protein is increased after ethanol-induced injury
to the gastric mucosa.
Immunoblot analysis demonstrated that expression of gastric Ras protein
is also increased as a result of ethanol-induced injury. A protein band
of ~21 kDa was detected by a monoclonal anti-Ras antibody, which
recognizes all four mammalian isoforms of Ras and was found to be
increased to 840% (P < 0.05) and 241%
(P < 0.005) at 3 and 6 h, respectively, over the controls
as shown in Fig. 6A. By 24 h, the
level of gastric Ras protein expression had normalized in gastric
mucosal tissue of rats treated with ethanol to that of the controls
(Fig. 6A). Quantitative data for Ras protein expression are
presented in Fig. 6B.

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Fig. 6.
Western blot analysis of Ras protein expression in gastric mucosa after
ethanol injury compared with gastric mucosa of controls. A:
analysis of Ras protein expression was performed as described in
MATERIALS AND METHODS. Gastric mucosa samples of control
animals are shown together with gastric mucosa samples from animals at
each time point after intragastric administration of 100% ethanol as
described in Fig. 4. Because antibody used to detect Ras proteins
recognizes all 4 isoforms of mammalian Ras, signal obtained by this
analysis represents level of all Ras proteins in each sample.
B: quantitative data for Ras protein expression (shown in
A) obtained by densitometric scanning using values of peak
area. Values are means ± SD. For each time point, n = 6.
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The increased expression of both VEGF and Ki-ras mRNA and VEGF and
Ras protein resulting from ethanol-induced injury to gastric mucosa is
dependent on ethanol concentration.
To determine whether the increased expression of VEGF and
Ras, at both the transcriptional and translational levels,
resulted from ethanol-induced injury to the gastric mucosa in a
dose-dependent manner, the effect of three concentrations of ethanol on
VEGF and Ras expression was investigated. Although VEGF
mRNA expression increased to 153% in the gastric mucosa at 3 h after
administration of 25% ethanol (Fig.
7A), this increase was not
statistically significant compared with controls (P = 0.09).
However, administration of 50% ethanol resulted in deep necrosis in
the gastric mucosa at 3 h and a significant increase to 359%
(P < 0.03) in VEGF mRNA expression in the gastric mucosa
bordering the necrosis compared with controls (Fig. 7A).
Administration of 100% ethanol resulted in an increase to 556%
(P < 0.005) in VEGF mRNA expression in the mucosa bordering
the necrosis compared with controls (Fig. 7A). VEGF protein
expression was also not significantly altered in the gastric mucosa at
3 h after administration of 25% ethanol (Fig. 7B). However,
administration of 50% and 100% ethanol did result in significant
increases in expression of VEGF165 in the mucosa bordering
necrosis to 286% (P < 0.005) and 380%
(P < 0.001), respectively, at 3 h (Fig. 7B).
Expression of the larger, nonsecreted form of VEGF was not
significantly altered at 3 h after administration of either 50% or
100% ethanol compared with controls (data not shown). Administration
of 25% ethanol did result in a slight but significant
(P < 0.04) increase in Ki-ras mRNA expression in
the gastric mucosa to 131% at 3 h compared with controls (Fig.
8A). Expression of Ki-ras
mRNA was increased in the gastric mucosa bordering necrosis at 3 h
after administration of 50% and 100% ethanol to 174%
(P < 0.01) and 229% (P < 0.001), respectively (Fig. 8A). The expression of Ras protein was not significantly different in the gastric mucosa at 3 h after administration of 25%
ethanol compared with controls (Fig. 8B). Ras protein
expression was, however, significantly increased in the gastric mucosa
bordering necrosis at 3 h after administration of 50% and 100%
ethanol to 373% (P < 0.001) and 680%
(P < 0.001), respectively (Fig. 8B).

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Fig. 7.
Dose dependency of ethanol concentration on VEGF expression. A,
top: RT-PCR using specific primers for VEGF was performed as
described in MATERIALS AND METHODS. A representative sample
from control animals is shown together with representative samples
obtained at 3 h after intragastric administration of 25%, 50%, and
100% ethanol. A, middle: RT-PCR of same reverse transcription
products using specific primers for -actin as an internal control
for total mRNA. In bottom, values are means ± SD
(n = 6). B: analysis of VEGF protein expression was
performed as described in MATERIALS AND METHODS. Level of
VEGF protein from a representative gastric mucosa sample of control
animals is shown together with that of representative gastric mucosa
samples from animals at 3 h after intragastric administration of 25%,
50%, and 100% ethanol. Quantitative data of VEGF protein expression
were obtained by densitometric scanning, by using values of peak area.
Values are means ± SD. For each concentration of ethanol, n = 6.
|
|

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Fig. 8.
Dose dependency of ethanol concentration on Ras expression. A,
top: RT-PCR using specific primers for Ki-ras was performed
as described in MATERIALS AND METHODS. A representative
sample from control animals is shown together with representative
samples obtained at 3 h after intragastric administration of 25%,
50%, and 100% ethanol. A, middle: RT-PCR of same reverse
transcription products using specific primers for -actin as an
internal control for total mRNA. In bottom, values are means ± SD (n = 6). B: analysis of Ras protein expression
was performed as described in MATERIALS AND METHODS. Level
of Ras protein from a representative gastric mucosa sample of control
animals is shown together with that of representative gastric mucosa
samples from animals at 3 h after intragastric administration of 25%,
50%, and 100% ethanol. Quantitative data of Ras protein expression
were obtained by densitometric scanning, by using values of peak area.
Values are means ± SD. For each concentration of ethanol, n = 6.
|
|
VEGF expression is mediated through Ras.
To determine whether nononcogenic Ras is required for VEGF expression,
the effect of an inhibitor of Ras activation on growth factor-induced
VEGF expression was investigated in vitro. For this study, rat primary
endothelial cells were used, because they undergo angiogenesis and form
microvessels. bFGF was used to induce VEGF expression because bFGF has
been shown to induce expression of VEGF in endothelial cells undergoing
angiogenesis (29). In our study, VEGF mRNA expression was increased in
rat primary endothelial cells to 469% in response to bFGF (Fig.
9B). Mevastatin, which inhibits
posttranslational farnesylation of Ras and thus Ras activation (11),
inhibited the expression of VEGF mRNA induced by bFGF in these cells by
184% (Fig. 9B). Furthermore, although incubation of the
primary endothelial cells with bFGF resulted in an increase in protein
expression of the VEGF165 isoform to 526% (Fig.
9C), this increase was also inhibited 295% by mevastatin (Fig.
9C). Under the same conditions, mevastatin completely blocked
bFGF-induced Ras activation in primary endothelial cells, indicating
that the inhibition of growth factor-induced VEGF expression by
mevastatin results from the inhibition of Ras activation (Fig.
9A).

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Fig. 9.
Effect of Ras inhibition on VEGF expression. Effect of mevastatin, an
inhibitor of Ras activation, on expression of VEGF mRNA and protein was
investigated in rat primary endothelial cell culture. For this study,
basic fibroblast growth factor (bFGF) was used to induce Ras activation
and VEGF expression (29). A: percentage of GTP bound to Ras
protein (as an indicator of percentage of Ras activation) was
determined as described in MATERIALS AND METHODS. Lane
1, percentage of GTP (12.4 ± 2.1) bound to Ras
immunoprecipitated from serum-starved rat primary endothelial cells
treated for 4 h with vehicle alone (control). Lane 2, percentage of GTP (67.0 ± 8.4) bound to Ras immunoprecipitated from
serum-starved rat primary endothelial cells treated for 4 h with
vehicle, followed by treatment for 5 min with 10 ng/ml bFGF. Lane
3, percentage of GTP (16.8 ± 4.0) bound to Ras
immunoprecipitated from serum-starved rat primary endothelial cells
treated for 4 h with 25 µM mevastatin, followed by treatment for 5 min with 10 ng/ml bFGF. Values are means ± SD of 3 independent
experiments. B, top: RT-PCR using specific primers for VEGF was
performed as described in MATERIALS AND METHODS. Lanes show
VEGF mRNA expression from serum-starved rat primary endothelial cells
treated for 4 h with vehicle alone (control; lane 1), treated
for 4 h with vehicle followed by treatment for 5 min with 10 ng/ml bFGF
(lane 2), or treated for 4 h with 25 µM mevastatin followed
by treatment for 5 min with 10 ng/ml bFGF (lane 3). B,
middle: RT-PCR of same reverse transcription products in B,
top, using specific primers for -actin as internal control for
total mRNA. In bottom, values are means ± SD of 3 independent
experiments. C: analysis of VEGF protein expression was
performed as described in MATERIALS AND METHODS. Lanes show
VEGF protein expression from serum-starved rat primary endothelial
cells treated for 4 h with vehicle alone (control; lane 1),
treated for 4 h with vehicle followed by treatment for 5 min with 10 ng/ml bFGF (lane 2), or treated for 4 h with 25 µM mevastatin
followed by treatment for 5 min with 10 ng/ml bFGF (lane 3).
Values are means ± SD of 3 independent experiments.
|
|
 |
DISCUSSION |
Ethanol, after intragastric administration, penetrates deeply into the
gastric mucosa because of high lipid solubility and, at concentrations
of 50-100%, causes microvascular damage and hemorrhagic lesions
(23, 34, 37, 38, 41). Ethanol-induced injury to the gastric mucosa is a
time-related process in which disruption or exfoliation of the gastric
surface epithelium is followed by necrosis of deeper mucosal layers,
including the mucosal proliferative zone and the microvasculature (23,
37, 38, 41). Angiogenesis is a prerequisite for the healing of
ethanol-induced deep gastric mucosal damage. Our previous study has
shown that ethanol-induced injury to gastric mucosa triggers an
angiogenic response as well as an increase in bFGF, a known angiogenic
factor, in the mucosa bordering necrosis (36, 39).
The present study demonstrates for the first time that ethanol-induced
injury to the gastric mucosa activates VEGF gene expression as
reflected by increases in VEGF at both the transcriptional and
translational levels. VEGF is the only known growth factor to act
predominantly on endothelial cells, which are one of the few cell types
to express the receptors for VEGF (9, 12, 24). Although not yet
characterized for gastric mucosa, in various other tissues VEGF induces
angiogenesis by acting both as an endothelial mitogen and by enhancing
microvascular hyperpermeability, which stimulates the formation of a
fibrin-rich extracellular matrix promoting endothelial cell migration
(5). VEGF has been implicated in epidermal wound healing (6) and in the
accelerated healing of experimental duodenal ulcers and ulcerative
colitis (27, 32). VEGF may also play a role in the angiogenesis
involved in the healing of chronic gastric ulcers, which represent a
distinctly different type of injury from acute ethanol-induced necrosis
(35). In that study, VEGF expression, which was found predominantly in
epithelial cells of normal gastric mucosa, was shown to be induced in
gastric fibroblasts of ulcer margins and ulcer beds (35). Our finding
that VEGF expression is increased in the gastric mucosa in response to
ethanol injury strongly suggests the importance of VEGF as a mediator
of the angiogenesis crucial for the repair of gastric mucosal erosion.
This rapid increase in the expression of VEGF was associated with an
angiogenic response to ethanol-induced injury as demonstrated in the
present study, 24 h after ethanol administration, by the endothelial
cell migration and tube formation, in the mucosa bordering necrosis.
We have previously demonstrated that administration of exogenous VEGF
enhances angiogenesis and accelerates the healing of chemically induced
lesions in the upper and lower gastrointestinal tract (27, 32, 40). The
importance of VEGF in the angiogenic response to ethanol-induced
gastric mucosal injury is further supported by demonstration in this
study that angiogenesis was significantly inhibited by administration
of a neutralizing anti-VEGF antibody. Moreover, the extent of
macroscopic injury remained greater than fourfold that of controls,
indicating that neutralization of endogenous VEGF significantly delays
healing of mucosal erosions. The finding that administration of the
neutralizing anti-VEGF antibody led to a threefold reduction in the
angiogenic response (compared with controls receiving normal antibody),
but did not completely inhibit it, can be explained by the presence of
other angiogenic factors such as bFGF, which is increased in response to ethanol-induced injury (36). Our data show that gastric VEGF expression at both the transcriptional and translational levels increases early in response to ethanol-induced injury, becoming maximal
within 3 h after intragastric ethanol administration. These increases
in VEGF mRNA and protein expression were also dose dependent on ethanol concentration.
The finding that intragastric administration of 25% ethanol did not
result in increased VEGF expression is not surprising. It has long been
known that ethanol at this concentration can produce exfoliation of the
surface epithelial cells but does not result in necrotic injury of the
deeper mucosa or of microvessels. In fact, this concentration of
ethanol has a cytoprotective action against the necrotizing effect of
higher concentrations of ethanol administered subsequently (7). The
finding that intragastric administration of 50% ethanol results in
significant increases in both VEGF mRNA and protein in the gastric
mucosa bordering necrosis is clinically relevant because alcoholic
beverages produced for human consumption can reach this concentration.
The present study also demonstrates for the first time that
ethanol-induced injury to the gastric mucosa activates ras gene expression, increasing Ras at both the transcriptional and
translational levels. As with VEGF expression, the increase in Ras mRNA
and protein expression in response to ethanol-induced injury was dose dependent on ethanol concentration. Several studies have shown that
VEGF expression is upregulated by oncogenic Ras in transfected cell
lines, suggesting that one of the roles of oncogenic Ras in the growth
of solid tumors is the induction of angiogenesis (20, 22, 25). In the
present study, we have demonstrated in primary rat endothelial cell
culture that the expression of VEGF induced by bFGF is mediated through
Ras activation. In addition, the induction of VEGF by hypoxia was shown
to be blocked by the Ras inhibitor, RasN17, in untransformed NIH/3T3
cells, suggesting that nononcogenic Ras may play a role in mediating
this induction (22). Furthermore, upregulation of VEGF expression has
been shown to be induced by myocardial ischemia in vivo, suggesting a
link between tissue hypoxia-induced VEGF expression and the angiogenic
response to ischemia resulting in coronary neovascularization (2).
Because ethanol-induced injury to the gastric mucosa is associated with
microvascular damage [e.g., rupture of the endothelium, thrombi
formation, and endothelial and capillary necrosis (37, 41)] leading to
cessation of oxygen delivery and thus ischemia, it is very likely that
the observed increase in VEGF occurs through a similar regulatory
mechanism. Expression of Ras in the gastric mucosa, in response to
ethanol-induced injury, has not been previously investigated. Our data
demonstrating that the increase in Ras protein expression in response
to ethanol-induced injury is maximal within 3 h after ethanol
administration and is normalized to control levels by 24 h are
consistent with an early involvement of Ras in the repair of mucosal
erosion, possibly through angiogenesis. We currently have no
explanation for why Ki-ras mRNA expression continues to
increase at 6 and 24 h after ethanol administration. The decreased
expression of total Ras protein (which includes H-Ras and N-Ras, as
well as Ki-Ras) at these time points, compared with the expression at 3 h after ethanol administration, implies differential regulation at the
transcriptional and translational levels.
Because the induction of VEGF by hypoxia has been shown to be blocked
by Ras inhibition, and is thus dependent on Ras, and because data in
the present study indicate that, in rat endothelial cells, the
expression of VEGF induced by bFGF is mediated through the Ras pathway,
the increase in Ras expression resulting from ethanol-induced gastric
mucosal injury is likely to cause upregulation of VEGF expression and
thus the observed angiogenic response.
 |
ACKNOWLEDGEMENTS |
This study was supported by Merit Review Awards to A. S. Tarnawski
and I. J. Sarfeh from the Medical Research Service of the Department of
Veterans Affairs.
 |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: A. S. Tarnawski,
VA Medical Center, 5901 E. Seventh St., Long Beach, CA 90822 (Email:
atarnawski{at}pop.long-beach.va.gov).
Received 9 March 1998; accepted in final form 22 February 1999.
 |
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