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Vascular endothelial growth factor receptor-2 inhibitionin experimental murine colitis
Leslie Knod, MD, Eileen C. Donovan, BS, Artur Chernoguz, MD, Kelly M. Crawford, BS,Mary R. Dusing, BS, and Jason S. Frischer, MD*
Division of Pediatric General and Thoracic Surgery, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio
a r t i c l e i n f o
Article history:
Received 4 January 2013
Received in revised form
7 April 2013
Accepted 15 April 2013
Available online 6 May 2013
Keywords:
Inflammatory bowel disease
Angiogenesis
VEGFR2
Colitis
* Corresponding author. Colorectal CenterHospital Medical Center, 3333 Burnett Avenu
E-mail address: [email protected]/$ e see front matter ª 2013 Elsevhttp://dx.doi.org/10.1016/j.jss.2013.04.026
a b s t r a c t
Background: In the setting of inflammatory bowel disease, inflammation is associated with
a simultaneous increase in angiogenesis; moreover, elevated vascular endothelial growth
factor (VEGF) levels implicate angiogenesis as a pathologic contributor to disease severity.We
hypothesize that selectively inhibiting vascular endothelial growth factor receptor-2 (VEGFR2)
in amodel ofmurine colitiswill reduceangiogenesis, resulting indecreased inflammationand
disease severity, providingmechanistic insight into the role of pathologic angiogenesis in IBD.
Materials and methods: In a dextran sodium sulfate model of murine colitis, anti-VEGFR2
monoclonal antibody (DC101) or placebo was administered. Clinical assessments fol-
lowed by histologic and molecular tissue analysis were performed to quantify inflamma-
tion, microvessel density (MVD), VEGF and VEGFR2 gene expression, and phosphorylated
mitogen-activated protein kinase protein expression.
Results: Weight loss began after d 6 with the treatment group demonstrating a more
favorable percent weight change. Inflammation and MVD were similar between cohorts,
both increasing in parallel toward a plateau. VEGF and VEGFR2 messenger RNA expression
were not significantly different, but phosphorylated mitogen-activated protein kinase was
elevated in the DC101 cohort (P ¼ 0.03).
Conclusions: Despite a more favorable weight change profile in the treated group, no differ-
ence was observed between cohorts regarding clinical disease severity. However, a parallel
rise in inflammation and MVD was observed coinciding with weight loss, suggesting their
relationship in IBD. VEGFR2 downstream signaling was significantly elevated in the treated
cohort, possibly by VEGF-independent signal transduction. Early and effective inhibition of
angiogenesis by limiting downstream VEGF signaling or targeting multiple angiogenic
pathways may block angiogenesis, thereby reducing disease severity and provide evidence
toward the mechanism and clinical benefit of antiangiogenics in the setting of IBD.
ª 2013 Elsevier Inc. All rights reserved.
for Children, Division of Pediatric General and Thoracic Surgery, Cincinnati Children’se, MLC-2023, Cincinnati, OH 45229. Tel.: þ1 513 636 3240; fax þ1 513 636 3248.g (J.S. Frischer).ier Inc. All rights reserved.
j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 4 ( 2 0 1 3 ) 1 0 1e1 0 7102
1. Introduction resulting in decreased inflammation and disease severity,
Ulcerative colitis and Crohn’s disease, the two manifestations
of inflammatory bowel disease (IBD), result from a complex
interplay of genetic, immunologic, and environmental factors
[1]. Recent human and animal studies have documented
increased angiogenesis as a pathophysiologic characteristic of
IBD. Elevated vascular endothelial growth factor (VEGF),
a primary angiogenic regulator, in clinical and experimental
IBD is demonstrated in systemic circulation and at the level of
genetic and protein expression, implicating the VEGF pathway
as a regulator of angiogenesis [2e7]. Angiogenesis serves as
a potential venue for molecular pharmacologic control to
decrease disease severity in IBD, similar to the antiangiogenic
benefits demonstrated in cancer management.
Angiogenesis, the formation of new vessels from preexist-
ing vasculature, is essential for physiologic growth and
development, but in disease, angiogenesis becomes pathologic
and a potential therapeutic target, as demonstrated in onco-
logic and chronic inflammatory processes [8e10]. In the field of
cancer therapy, antiangiogenics have been shown to decrease
tumor growth and has become part of many treatment regi-
mens for solid tumors [11,12]. Diseases of chronic inflamma-
tion, including rheumatoid arthritis, atherosclerosis, psoriasis,
and diabetic retinopathy [8,12e14], have upregulated angio-
genesis, which supports a relationship between angiogenesis
and inflammation.
In the VEGF ligandereceptor signaling pathway, the VEGF-
A mitogen is the strongest signal transducer and binds to the
endothelial cell tyrosine kinase receptor, vascular endothelial
growth factor receptor-2 (VEGFR2/Flk-1). VEGFR2 mediates
vascular endothelial cell growth and permeability including
DNA replication, cell proliferation, survival, and migration
[15e17]. Other receptors are less central to angiogenesis,
VEGFR1 exhibits weak tyrosine kinase activity, acting more
like a decoy receptor, and VEGFR3 is predominantly expressed
in lymphatic endothelium [18,19]. The major angiogenic
effects of VEGF are mediated through VEGFR2, making it an
ideal target to inhibit angiogenesis.
Decreased angiogenesis can result from inhibition of
the VEGF receptor or ligand. Selective receptor inhibition
of VEGFR2/Flk-1 by DC101 (anti-Flk-1 monoclonal antibody)
decreases tumor angiogenesis in vitro and in vivo and has
demonstrated tumor growth inhibition [20,21]. Neutralization
of the VEGF ligand with monoclonal antibody in murine IBD
has also shown a reduction in disease severity and inflam-
matory cell infiltrate [22]. Our model of murine colitis
is induced by dextran sodium sulfate (DSS), producing a che-
mically toxic disruption of colonic epithelial cells causing
a mucosal barrier dysfunction that allows bacterial trans-
location leading to an intense inflammatory response [23].
Inflammation and pathologic angiogenesis are likely in-
terrelated processes, possibly with bidirectional cell signaling
from one exacerbating the other [8,24]. Their independent
degree of contribution to disease severity is unknown,making
the angiogenic pathway an attractive target to elucidate the
complex pathophysiology of IBD. We hypothesize that selec-
tive VEGFR2 inhibition by systemic DC101 administration in
a DSS model of murine colitis will reduce angiogenesis,
which will better define the mechanism of pathologic angio-
genesis and its role in IBD.
2. Materials and methods
2.1. Murine colitis model
Experimental colitis was induced in 6- to 8-wk-old male
C57BL/6 mice (Harlan Laboratories, Indianapolis, IN) with a 7-
d course of 1.0%DSS (w/v) supplemented in ad libitum drinking
water. Colitis signs and symptoms were monitored daily as
weights were recorded. Mice were singly housed in the barrier
containment animal facility, and all experiments were per-
formed in accordance with the Institutional Animal Care and
Use Committee (IACUC) of Cincinnati Children’s Research
Foundation.
2.2. Administration of DC101
Subjects were divided into control or DC101-treated cohorts
(n ¼ 18 per group). Commencing on d 3 following the initiation
of DSS, mice in the treated cohort received intraperitoneal
injections of the VEGFR2 monoclonal antibody, DC101 (32
mg/kg in 0.1 mL), every 3 d, whereas control mice received
equivalent injections of phosphate-buffered saline (PBS) until
completion of the experiment on d 14. Optimal dosing was
established to be 400e800 mg per mouse based on published
data that demonstrated a maximal therapeutic response at
800 mg per mouse in C57BL/6 mice [20,25] and in colorectal
tumor xenografts [26]. Animals were killed at 2-d intervals
following the initiation of DSS. At necropsy, dissected colonic
specimens were irrigated with PBS and then longitudinally
subdivided and processed as either fixed or frozen tissues.
Fixed tissues were preserved in 4% paraformaldehyde over-
night followed by serial alcohol dehydrations and then
paraffin embedding for histologic evaluation.
2.3. Histopathologic colitis scoring
Colons were stained with hematoxylin and eosin and then
analyzed under light microscopy at �40 magnification scoring
four consecutive fields, starting at the anorectal junction.
Using an accepted colitis scoring method [27e29] with
amaximumpossible score of 18, two blinded observers graded
the tissues in the following categories: (1) percent area inv-
olved, (2) edema, (3) ulcerations, (4) crypt loss, and (5) immune
cell infiltration to generate a cumulative inflammation score.
2.4. Immunohistochemistry
Paraffin embedded colons were cut into 5 mm sections and
mounted. Following deparaffinization and rehydration,
citrate-based epitope enhancement was performed using
Antigen Retrieval Solution (DAKO, Carpinteria, CA). After a PBS
wash, Protein Blocker Solution (DAKO) was applied by the
manufacturer’s instructions, and slides were incubated with
MECA-32 primary monoclonal antibody (1:10; Developmental
j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 4 ( 2 0 1 3 ) 1 0 1e1 0 7 103
Studies Hybridoma Bank, Iowa City, IA). Alexa 488-tagged
fluorescent secondary antibody (1:200; Invitrogen, Carlsbad,
CA) was applied and followed by mounting with DAPI Vecta-
shield medium (Vector Laboratories, Burlingame, CA).
2.5. Microvessel density
Intestinal sections were immunostained with MECA-32 and
microphotographed for 10 consecutive, �200 fields starting
at the anorectal junction using fluorescent microscopy (Nikon
90i; Nikon Instruments, Inc, Melville, NY). In a blinded
manner, the area (mm2) of mucosal microvessel density (MVD)
per high-powered field was quantitated with NIS-Advanced
Elements software (Nikon Instruments, Inc) to determine the
mean microvascular area for each specimen.
2.6. Colonic VEGFR2 and VEGF-A genetic expressions
VEGF-A and VEGFR2 gene expressions were analyzed from
specimens stored in RNA later (Sigma Life Science, St. Louis,
MO). The tissues were homogenized followed by RNA extrac-
ted using the RNeasyMini Kit (Qiagen, Valencia, CA) according
to the manufacturer’s instruction. RNA was converted to
complementary DNA using the high-capacity complementary
DNA reverse transcription kit (Applied Biosystems, Carlsbad,
CA). Quantitative reverse transcriptionepolymerase chain
reaction was performed by StepOne Plus RT-PCR System
(Applied Biosystems) using SYBR Green detection with b-2-
microglobulin (RealTimePrimers.com; Elkins Park, Phila-
delphia, PA) as the internal reference gene. PCR primers
were used as follows: VEGF-A forward 50-TCTGCTCTCCTT-CTGTCGTG-30 and reverse 50-ACTGGACCCTGGCTTTACTG-30
and VEGFR2 forward 50-CGTTGTACAAATGTGAAGC-30 and
reverse 50-CACAGTAATTTCAGGACCC-30 (Integrated DNA
Technologies, Coralville, IA). Relative quantification of data
by the 2�DDCt method produced results as fold change in
expression of target gene relative to internal control.
Fig. 1 e Percent daily weight change was more favorable in
the DC101 treated cohort compared with controls. Both
groups exhibited a similar chronologic pattern of weight
change, declining after d 6 and reaching a nadir at d 10.
2.7. Phosphorylated mitogen-activated protein kinaseprotein expression
Tissues were homogenized in a buffer containing Halt
Protease and Phosphate Inhibitor Cocktail (Thermo Scientific,
Rockford, IL) and Nonidet-P40 substitute in PBS. Protein
concentrations were generated from a standard curve using
the Pierce BCA Protein Assay Kit (Thermo Scientific). Protein
extracts (10 or 30 mg per lane) were loaded with buffer and run
on a 12% TGX gel (Bio-Rad, Hercules, CA) for sodium dodecyl
sulfateepolyacrylamide gel electrophoresis and transferred
onto a nitrocellulose membrane. After application of
nonspecific blocker, the membrane was incubated with
primary antibody, either mitogen-activated protein kinase
(1:1000; MAPK) or phosphorylated MAPK (1:2000; pMAPK)
both from Cell Signaling Technology, Danvers, MA. The
secondary antibody (1:1000, IgG horseradish peroxidase-
linked; Cell Signaling Technology) was applied followed by
LumiGLO application. Densitometric analysis was performed
with ImageJ software (National Institutes of Health, Beth-
esda, MD) and normalized to glyceraldehyde-3-phosphate
dehydrogenase (1:200; Santa Cruz Biotechnology, Santa
Cruz, CA).
2.8. Statistical analysis
Statistical analysis was performed using GraphPad Prism
software (GraphPad Software, Inc, La Jolla, CA). Unpaired t-
test was used to compare bodyweights, inflammation scoring,
MVD, gene expression, and protein levels expressed as mean
� standard error of the mean, in which P � 0.05 represents
statistical significance. The rate of weight change was quan-
tified by linear regression analysis with a 95% confidence
interval (CI).
3. Results
3.1. Clinical assessment
Mice in the treated and control groups developed clinical signs
and symptoms of colitis induced by DSS including weight loss,
ruffled fur, indolence, diarrhea, and bloody stools. After d 6
of DSS administration, both groups began to lose weight. Daily
weights were recorded and percent weight change was cal-
culated based on the initial weight of each mouse. Starting
weights ranged from 20 to 25 g per mouse with amean weight
of 23 g in both cohorts. The weight change of four mice, fol-
lowed daily to d 14, was used to establishweight trends (Fig. 1).
The DC101-treated group exhibited an overall less severe
percent weight loss than controls (control �14.82% � 2.63%
versus treated �11.35% � 2.6%, P ¼ 0.39 at nadir, d 10). The
treated group also had a more clinically favorable rate of
weight loss compared with controls (slope of percent weight
loss in control �4.76 [95% CI �6.35 to �3.16] versus treated
group �2.99 [95% CI �4.17 to �1.8], P ¼ 0.07). A quicker return
toward initial weight in the DC101 group (slope of percent
weight gain in control þ1.23 [95% CI �0.46 to 2.92] versus
treated group þ2.7 [95% CI 1.37 to 4.04], P < 0.16) with a mean
Fig. 2 e Colonic inflammation scoring was similar between
treated and untreated groups, both rising after d 6 and
reaching a plateau at d 8.
j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 4 ( 2 0 1 3 ) 1 0 1e1 0 7104
percent weight change at d 14 of �10.86% � 3.19% control
versus �1.33% � 3.07% treated, P ¼ 0.07.
3.2. Colonic inflammation scoring and MVD
Colonic inflammation markedly increased after d 6 with
a similar trend in both DC101 and control groups, reaching
a plateau that continued through the end of the study (Figs. 2
and 3). Both groups demonstrated peak colonic inflammation
occurring at d 10, with amean inflammation score of 17� 0.01
Fig. 3 e Colons stained with hematoxylin and eosin viewed at
controls (A) and DC101 treated (C) colons, compared with DSS d
inflammatory infiltrate, crypt loss, edema, and ulceration but s
(D) groups. (Color version of figure is available online.)
(n ¼ 2 per group). No statistical difference exists between
treated and untreated groups.
Quantitative MVD increased from baseline after d 6 in
treated and untreated groups (Figs. 4 and 5). Both groups
exhibit elevated MVD between d 8e14 (control 7013 � 599 mm2
versus treated 7865 � 703 mm2, P ¼ 0.39, n ¼ 10 per group). The
apparent MVD difference between groups noted at d 10
(control 2914� 909 mm2 versus treated 8408� 608 mm2, n¼ 2 per
group) did not reach statistical significance (P ¼ 0.09) nor did
this separation in MVD between cohorts persist, as demon-
strated by similar d 14 MVD (control 6620 � 372 mm2 versus
treated 6265 � 358 mm2, P ¼ 0.52, n ¼ 4 per group).
3.3. Tissue genetic and protein analysis
Local colonic VEGF messenger RNA relative expression was
similar between treated and untreated groups at early time
points, before d 6, (8.70� 4.46 control versus 8.13� 2.05 DC101,
P ¼ 0.91, n ¼ 5 per group), but at later time points, after d 6,
VEGF suppression was observed in the DC101 group compared
with controls (7.94 � 5.68 control versus 2.68 � 0.67 DC101, P ¼0.44, n ¼ 4e5 per group), although not statistically significant
(Fig. 6). Between DC101 early and late groups, a decrease in
VEGF gene expression was noted but not significant (8.13 �2.05 early DC101 versus 2.68� 0.67 late DC101, P¼ 0.06, n¼ 4e5
per group). Relative expression of VEGFR2 messenger RNA in
control and DC101 colons was not significantly different at
early time points, before d 6, (3.61 � 0.83 control versus 2.42 �0.67 DC101, P ¼ 0.28, n ¼ 8 per group) or at later time points,
after d 6, (1.39 � 0.17 control versus 1.34 � 0.14 DC101, P ¼ 0.84,
n ¼ 8e10 per group).
340 magnification show limited inflammation in early
10 colons that were more inflamed, exhibiting worse
till appear similar between late control (B) and treated
Fig. 4 e Colonic MVD was similar between both cohorts.
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Downstream signaling of VEGFR2 was measured from DSS
initiation to d 8 bywestern blot analysis expressed as a ratio of
phosphorylated to total MAPK protein (Fig. 7). The percent of
phosphorylated MAPK was higher in the DC101-treated group
(22.5 � 6.4 untreated versus 57.6 � 13.5 treated, P ¼ 0.03, n ¼ 8
per group).
4. Discussion
Murine DSS colitis is a widely used animal model of experi-
mental IBD that produces clinical signs and symptoms similar
to humans including diarrhea, occult blood and weight loss as
Fig. 5 e Colonic MVD visualized with MECA-32 staining at 3200
(A) and DC101 treated (C) groups, but an appreciable increase in
DSS d 10 both control (B) and treated (D) colons. (Color version
well as similar histologic disease exhibiting ulceration,
immune cell infiltrate, and crypt abscesses [30,31]. When
applied to C57BL/6 mice, DSS colitis responds to conventional
IBD pharmacologic intervention demonstrating improve-
ments at the clinical, histologic, and molecular levels, which
demonstrates that this model is translatable into human
disease management [32]. In human subjects with IBD,
increased vascularity is noted in the acutely inflamed bowel,
as such angiogenesis is associated with the pathophysiology
of IBD [2]. The precise role of angiogenesis whether leading to
or exacerbating the inflammatory process remains unknown.
The principal angiogenic factor (VEGF) has its most potent
receptor (VEGFR2) predominantly expressed on vascular
endothelial cells [16,18]. Although no causal relationship is
defined between VEGF and disease activity, expression of
VEGF is elevated in IBD patients [7], and therefore, the VEGF/
VEGFR pathway is a potential target for molecular therapy.
In our model of acute colitis, the VEGFR2 monoclonal
antibodyetreated group exhibited an improved weight
change profile compared with controls, but no difference in
disease severity was present between groups as measured by
inflammation score or MVD. A temporal relationship was
noticed between angiogenesis and inflammation, with both
rising after d 6 of DSS administration, suggesting a linked
association. This parallel rise in vascularity and inflamma-
tion has previously been observed in experimental murine
colitis [33,34]. As weight loss commenced, a simultaneous
rise in histopathologic colitis ensued. Weight loss reached
a nadir by d 10, which corresponds to peak angiogenesis and
inflammation scores, identifying the period of most severe
clinical and histologic colitis. These findings show the
complex interplay that likely exists between angiogenesis
magnification showing similarities between early control
MVD when comparing early to late time points as seen in
of figure is available online.)
Fig. 6 e Local tissue gene expression of VEGF-A was
initially similar between control and DC101, but at later
time points (after d 6), the DC101-treated group
demonstrated decreased VEGF-A expression, but not
significant, when compared with either control groups or
early DC101.
j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 4 ( 2 0 1 3 ) 1 0 1e1 0 7106
and inflammation coinciding with the progression of disease
severity.
By applying DC101 to our DSS model of acute colitis,
we anticipated a decrease in VEGFR2 function by way of
competitive inhibition at the extracellular ligand-binding
domain, which would prevent receptor tyrosine kinasee
mediated signal transduction and thereby dampen the down-
stream angiogenic effect [35,36]. The expected angiogenesis
suppression was not achieved, without significant difference
in MVD between cohorts. Previous studies in our laboratory
demonstrated that VEGFR2 blockade with DC101 caused
elevated VEGF protein expression in wild-type colons
compared with placebo (E.C. Donovan, BS, A. Chernoguz, MD,
K.M. Crawford, BS, M.R. Dusing, BS, J.S. Frischer, MD, unpub-
lished data, 2011). Others have observed that following
Fig. 7 e Receptor tyrosine kinase activity measured by
phosphorylated MAPK (pMAPK) was elevated in the DC101
group (0.57 ± 0.14) compared with controls (0.22 ± 0.06); *P
[ 0.03.
administration of VEGFR2 monoclonal antibody, plasma levels
of VEGF increase suggesting that VEGF levels can serve as
a surrogate marker of effective dosing [26]. However, in this
experiment, gene expression of VEGF was unchanged at early
time points but diminished, though not significantly, in the
DC101 group at later time points. Given these unexpected
results and the clinical similarities between cohorts, additional
angiogenic pathway analysis was required to assess VEGFR2
downstream signaling.
To further evaluate effective receptor blockade, the PLC-
gamma/ERK pathway that leads to endothelial cell prolifera-
tion was assessed [18]. Phosphorylated MAPK expression was
greater in the treated group, giving us a paradoxical response
from the VEGFR2 blockade. This could explain the similarities
between treated and untreated mice regarding colonic angio-
genesis and inflammation. Alternatively, despite blocking
extracellular ligand binding, the downstream signaling of the
VEGF receptor can remain active by VEGF-independent path-
ways [37]. Other plausible explanations include that receptor
blockade may upregulate compensatory processes or alterna-
tive pathways such as platelet-derived growth factor,
epidermal growth factor, or fibroblast growth factor. IBD is
a heterogenous disease with many phenotypic presentations;
certainly, this variability is present among experimental
models of colitis. Angiogenesis upregulation can result from
increased proangiogenic mediators or loss of angiogenic inhi-
bition. Chidlow et al. [5] suggests that the pathologic angio-
genesis in the DSS model of IBD may result from loss of
angiogenic inhibition. Although DC101 was ineffective in
suppressing angiogenesis by means of VEGFR2 blockade, this
receptor remains a potential target given itswell-known role in
physiologic and pathologic angiogenesis. Perhaps combination
treatment targetingmultiple angiogenic pathways or restoring
antiangiogenic activity may be required to decipher the path-
ways contributing to IBD angiogenesis. This is certainly the
case in the oncologic antiangiogenic therapy paradigm [38].
The onset of inflammation and angiogenesis in the initial
disease phase of experimental colitis provides a therapeutic
window in which angiogenic suppression may exude its effect
on inflammation and overall disease severity. Multiple agents
are currently in use for treating the inflammatory components
of IBD [39], but no clinical studies have been designed based
on the contribution of angiogenesis in the disease process.
Treatment with the immunemodulator infliximab, a mono-
clonal antibody to tumornecrosis factor-alpha, has beenshown
to reduce vascularity in IBD but has unfavorable side effects
[39,40]. In future experiments, early and effective inhibition of
angiogenesis or inhibition of downstreamsignalingmay reduce
disease severity and provide evidenced toward the clinical
benefit of antiangiogenics in the setting of IBD.
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