down-modulation of tnfsf15 in ovarian cancer by vegf and ......prevails in a normal vasculature...
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ORIGINAL PAPER
Down-modulation of TNFSF15 in ovarian cancer by VEGFand MCP-1 is a pre-requisite for tumor neovascularization
Weimin Deng • Xin Gu • Yi Lu • Chao Gu •
Yangyang Zheng • Zhisong Zhang •
Li Chen • Zhi Yao • Lu-Yuan Li
Received: 28 September 2011 / Accepted: 8 December 2011 / Published online: 31 December 2011
� Springer Science+Business Media B.V. 2011
Abstract Persistent inflammation and neovascularization
are critical to cancer development. In addition to upregu-
lation of positive control mechanisms such as overexpres-
sion of angiogenic and inflammatory factors in the cancer
microenvironment, loss of otherwise normally functioning
negative control mechanisms is likely to be an important
attribute. Insights into the down-modulation of such neg-
ative control mechanisms remain largely unclear, however.
We show here that tumor necrosis factor superfamily-15
(TNFSF15), an endogenous inhibitor of neovasculariza-
tion, is a critical component of the negative control
mechanism that operates in normal ovary but is missing in
ovarian cancer. We show in clinical settings that TNFSF15
is present prominently in the vasculature of normal ovary
but diminishes in ovarian cancer as the disease progresses.
Vascular endothelial growth factor (VEGF) produced by
cancer cells and monocyte chemotactic protein-1 (MCP-1)
produced mainly by tumor-infiltrating macrophages and
regulatory T cells effectively inhibits TNFSF15 production
by endothelial cells in vitro. Using a mouse syngeneic
tumor model, we demonstrate that silencing TNFSF15 by
topical shRNA treatments prior to and following mouse
ovarian cancer ID8 cell inoculation greatly facilitates
angiogenesis and tumor growth, whereas systemic appli-
cation of recombinant TNFSF15 inhibits angiogenesis and
tumor growth. Our findings indicate that downregulation of
TNFSF15 by cancer cells and tumor infiltrating macro-
phages and lymphocytes is a pre-requisite for tumor
neovascularization.
Keywords Inflammation � Ovarian cancer � Angiogenesis
switch � Tumorigenesis � Tumor infiltrating lymphocyte �Macrophage
Introduction
Chronic inflammation, fueled by periodic hypoxic condi-
tions, disrupted vascular integrity, lymphocyte infiltration,
and continual generation of dysfunctional blood vessels, is
a recognized risk factor for malignancies [1–6]. Cancer
cells in hypoxia produce a number of protein factors to
promote angiogenesis, including vascular endothelial cell
growth factor (VEGF) [7, 8]. VEGF, which was known
initially as vascular permeability factor [9], causes an
increase of permeability of the blood vessel. Blood vessel
leakage becomes prominent as a result, which greatly
Electronic supplementary material The online version of thisarticle (doi:10.1007/s10456-011-9244-y) contains supplementarymaterial, which is available to authorized users.
W. Deng � Y. Zheng � Z. Zhang � L.-Y. Li (&)
State Key Laboratory of Medicinal Chemical Biology,
Tianjin, China
e-mail: liluyuan@nankai.edu.cn
W. Deng � X. Gu � Y. Lu � C. Gu � Z. Yao (&)
Key Laboratory of Cellular and Molecular Immunology,
Tianjin Medical University, Tianjin, China
e-mail: yaozhi@tmu.cn
W. Deng � Y. Lu � C. Gu � Y. Zheng � Z. Yao
Key Laboratory of Ministry of Education, Tianjin, China
Y. Lu � L. Chen � L.-Y. Li
Department of Pathology, University of Pittsburgh School
of Medicine, Pittsburgh, PA, USA
Y. Lu � L. Chen � L.-Y. Li
University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA
Y. Zheng � Z. Zhang � L.-Y. Li
Nankai University School of Pharmaceutical Science, Tianjin,
China
123
Angiogenesis (2012) 15:71–85
DOI 10.1007/s10456-011-9244-y
facilitates infiltration of macrophages and T cells [10, 11],
among others. These cells secrete a variety of inflammatory
cytokines, including monocyte chemotactic protein-1
(MCP-1), into the microenvironment, further driving
angiogenesis [12, 13]. MCP-1 is of particular interest to us
because it can be produced by a variety of cell types in the
cancer microenvironment, such as monocytes and macro-
phages in the case of ovarian cancer [14–17], either con-
stitutively or induced under oxidative stress or other
inflammatory conditions [18–21]. Malignant tumors are
thus likened to wounds that will not heal because of this
vicious cycle [22]. It is hypothesized that the maintenance
of vascular integrity is tightly controlled by a balance of
growth factors and cytokines of opposite functions to
impede or accelerate neovascularization and hyperplasia
[23]. Conceivably, while an overall antiangiogenesis effect
prevails in a normal vasculature because of the activities of
endogenous suppressors of angiogenesis, these activities
must be hampered prior to the initiation of a neovascular-
ization process. Insights into how this happens remain,
however, largely unclear.
The ovary is a unique organ where the initiation, for-
mation, and degeneration of blood vessels under physio-
logical conditions occur in a cyclic manner [24–26].
Similar to the rest of the human female reproductive tract,
the ovary is highly dependent on VEGF for normal func-
tions such as endometrial proliferation and corpus luteum
development [27]. The expression and activity of VEGF
influenced by female steroid hormones deems angiogenesis
an important facet of the development of ovarian cancer
when the harmonic balance is compromised [28]. VEGF is
constitutively expressed in both benign and malignant
human ovary tissue [29]. VEGF and its receptors are
present in both primary ovarian carcinomas and ascitic
fluid [30]. Comparison of normal ovarian tissue to ovarian
carcinoma shows that VEGF protein levels are significantly
higher in carcinoma compared with normal tissue [31].
Malignant ovarian cyst fluid has elevated VEGF levels
[32]. Formation of malignant ascites has been directly
linked to VEGF expression in ovarian cancers [33]. VEGF
overexpression is correlated with a shorter disease-free
survival [34]. Similarly, tumor microvessel density in
advanced ovarian carcinoma is associated with a poorer
overall survival [35]. VEGF thus is a main driving force of
ovarian cancer angiogenesis and inflammation.
Tumor necrosis factor superfamily-15 (TNFSF15;
VEGI; TL1A) is a unique endogenous inhibitor of neo-
vascularization [36–38]. TNFSF15 is produced largely by
vascular endothelial cells of established blood vessels and
is a specific inhibitor of endothelial cell proliferation.
TNFSF15 is able to enforce growth arrest on quiescent
endothelial cells but induce apoptosis of proliferating
endothelial cells [39]. TNFSF15 can prevent bone marrow-
derived endothelial progenitor cell (EPC) differentiation
into endothelial cells [40]. Systemically administered
recombinant TNFSF15 exhibits a highly potent inhibitory
effect on tumor angiogenesis and tumor growth in animal
models [41]. It also inhibits the incorporation of bone
marrow-derived EPC into tumor vasculature [42].
TNFSF15 expression is reported to be absent or marginal
in tumor vasculatures in breast cancer [43], prostate cancer
[44, 45], urothelial cancer [46], as well as in wound tissue
[47]. Together these findings support the view that
TNFSF15 is an important component of the negative
control mechanisms of neovascularization.
We report here that TNFSF15 is downregulated in ovarian
cancer by VEGF produced by cancer cells and MCP-1 pro-
duced mainly by tumor-infiltrating macrophage and
CD4?CD25? FOXP3? regulatory T (TReg) cells under the
influence of cancer cells. We show with an ovarian cancer
model that TNFSF15 gene silencing with shRNA leads to
markedly enhanced angiogenesis and tumor growth,
whereas systemic administration of recombinant TNFSF15
inhibits angiogenesis and tumor growth. The findings are
consistent with the view that downmodulation of TNFSF15
is a pre-requisite for tumor neovascularization.
Materials and methods
Antibodies and reagents
Recombinant VEGF and antibodies against human VEGF,
VEGFR1, VEGFR2, and goat isotype control were pur-
chased from R&D (Minneapolis, MN, USA). Recombinant
human MCP-1, anti-human MCP-1 antibody, and mouse
isotype control were from Sigma (St. Louis, MO, USA).
Anti-mouse CD31 antibody was from BD (Franklin Lakes,
NJ, USA). Antibodies against human CD31, CD4, MAC1
and mouse TNFSF15 were from Santa Cruz (Santa Cruz,
CA, USA). Alexa Fluor-647 anti-human CD25, Alexa
Fluor-488 anti-human CD4 and Pacific Blue anti-human
FOXP3 were from BioLegend (San Diego, CA, USA).
Recombinant human TNFSF15 and anti-human TNFSF15
and mouse monoclonal antibodies 3-12D and 1-8F against
human TNFSF15 were prepared as described (32). Endo-
toxin level in the TNFSF15 preparation was 25 ng/mg.
Cells
Human umbilical vein endothelial cell (HUVEC) and
human ovarian cancer cell line OVCAR3 were purchased
from American Type Culture Collection (Manassas, VA,
USA) and cultured under conditions recommended by the
vendor. Mouse ovarian epithelial papillary serous adeno-
carcinoma cell line ID8 was a gift from Dr. K. F. Roby
72 Angiogenesis (2012) 15:71–85
123
(Center for Reproductive Sciences, University of Kansas
Medical Center). ID8 cells were cultured in Dulbecco’s
modified Eagle’s medium (Invitrogen, CA, USA) supple-
mented with 4% fetal bovine serum, 100 units/ml penicil-
lin, 100 lg/ml streptomycin, 5 lg/ml insulin (which is
known to stimulate VEGF production [48]), 5 lg/ml
transferrin, and 5 ng/ml sodium selenite (Roche, IN, USA).
Mice
Six-week-old female C57BL/6 mice were purchased from
the Jackson Laboratory (Bar Harbor, ME, USA). All pro-
cedures involving experimental animals were performed in
accordance with protocols approved by the University of
Pittsburgh Institutional Animal Care and Use Committee.
Immunohistochemistry
Five-micrometer sections of formalin-fixed, paraffin-
embedded tumors of clinical specimens of human ovarian
cancer or an experimental model of mouse ID8 syngeneic
tumors, as indicated, were deparaffinized, microwaved for
15 min in citric acid solution (pH = 6.0), and then incu-
bated in 3% hydrogen peroxide for 15 min. The sections
were then incubated for 2 h at room temperature with
respective primary antibodies, followed by 50 min incu-
bation with secondary antibody. For the use of fluorescein-
labeled antibodies, the slides were incubated at room
temperature for 2 h in the dark. The sections were sub-
jected to microscopic analysis with a Nikon NIS-Elements
microscope or an Olympus Fluo-view 1000 confocal
microscope. A minimum of 10 fields per section was
analyzed. The intensity of the immunostaining on each
section was assessed by two clinical pathologists inde-
pendently and in blinded manner, using a four-step grading
system (-, ?, ??, ??? for negative, low, high, and very
high, respectively).
CD4? T cell, CD4?CD25? T cell and monocyte
isolation
One hundred milliliters of peripheral blood was collected
from a healthy donor. Procedures involving human donors
were carried out in accordance with a University of Pitts-
burgh Institutional Review Board approved protocol.
Peripheral blood mononuclear cells were prepared by using
density gradient centrifuge (Ficoll, Uppsala, Sweden), and
then used for the isolation of CD4? T cells and
CD4?CD25? T cells with respective immobilized anti-
bodies (Miltenyi, Auburn, CA, USA). The isolation of
monocyte was carried out by depleting non-monocytes
with a Biotin antibody cocktail against CD3, CD7, CD16,
CD19, CD56, CD123 and Glycophorin A (Miltenyi,
Auburn, CA, USA) according to manufacturer’s instruc-
tions. Cells from different donors (three females, one male)
were used for experimental repeats.
Enzyme-linked immunosorbant assay (ELISA)
VEGF, TNFSF15 and MCP-1 concentration was deter-
mined by ELISA respectively. For TNFSF15, mouse
monoclonal antibodies 3-12D and 1-8F were used as the
capturing and detecting reagents, respectively. Recombi-
nant TNFSF15 was used to construct the standard curve.
MCP-1 and VEGF were determined by using the respective
MCP-1 or VEGF Elisa Kit (R&D, Minneapolis, MN,
USA).
Analysis of VEGF, TNFSF15, and MCP-1 expression
OVCAR3 cells (1 9 106 cells in 5 ml DMEM containing
10% FBS in 25-cm2 flasks) were cultured at 37�C in an
atmosphere of 5% CO2 in either 21% O2 (normoxia) or 5%
O2 (hypoxia). The culture media were collected at indicated
time intervals and either analyzed for VEGF protein levels or
used as OVCAR3-conditioned media. Freshly isolated
CD4?CD25? T cells (1 9 106) were suspended in 1.5 ml
RPMI 1640 culture medium (10% FBS) and seeded onto
12-well plates, then treated with 0.5 ml cancer cell condi-
tioned medium that was prepared by culturing OVCAR3
cells (1 9 106) in 5 ml DMEM (10% FBS) in a 25-cm2 flask
for 72 h. The supernatants were subjected to analysis with a
RayBio Human Cytokine Antibody Array G Series-6 panel
that included MCP-1 (Raybiotech, Norcross, GA, USA)
according to the manufacturer’s instructions. Conditioned
media from OVCAR3 cultures or CD4?CD25? T cell
untreated with OVCAR3 were used as controls. HUVECs
(passages 5–7) were plated onto 12-well culture plate with
the density of 2 9 105 cells per well in 2 ml of EGM-2. Once
grown into confluent cultures in about 7 days, the cultures
were treated with VEGF, MCP-1, OVCAR3-CM, or the
conditioned media from OVCAR3-CM treated CD4?
CD25? T cell cultures. Neutralizing antibodies against
VEGF, VEGFR1, VEGFR2, or MCP-1 were added to some
of the culture media as indicated.
Treatment of ID8 tumor-bearing animals
with recombinant TNFSF15
Six-week-old female C57BL/6 mice were injected subcuta-
neously on the flank with ID8 cells (1 9 107) suspended in
PBS (Lonza, Basel, Switzerland). Tumor-bearing animals
were randomized on day 6 into two groups. The experimental
group received intraperitoneal injection of TNFSF15
(125 lg in 0.5 ml PBS) on the same day and every other day
thereafter. The control group received vehicle treatments.
Angiogenesis (2012) 15:71–85 73
123
The animals were observed and weighed daily prior to
treatment. Tumor sizes were measured with a dial caliper in a
blinded manner. Tumor volumes were determined using the
equation: volume = width 9 width 9 length 9 0.52. The
animals were euthanized on the end of the experiment as
indicated according to a University of Pittsburgh IACUC
approved protocol. The tumors were retrieved for pathologic
analysis.
Treatment of ID8 tumor-bearing animals
with TNFSF15 shRNA
Six-week-old female C57BL/6 mice were randomized into
two groups. Animals in the experimental group were
injected subcutaneously on the flank with TNFSF15-
shRNA (1.5 9 105 IFU in 100 ll PBS). The injection sites
were marked with ink. Animals in the control group were
treated with scrambled shRNA in a similar manner. After
3 days, all animals were inoculated subcutaneously with
5 9 106 ID8 cells at the marked sites. The shRNA treat-
ments were repeated on day-6 post-cancer cell inoculation.
The animals were observed, weighed, and the tumor sizes
measured as described above. The animals were euthanized
at the end of the experiment and the tumors were retrieved
for pathologic analysis as described above.
Statistical analysis
At least three repeats were carried for each experiment.
The data were subjected to statistical analysis. The meth-
ods of analysis and P values are indicated in the text.
Results
TNFSF15 gene expression is downmodulated
in ovarian cancer
We determined TNFSF15 protein levels by immunohisto-
chemistry in normal ovary tissues and clinical specimens of
ovarian cancer, using a four-step grading system (-, ?,
??, ??? for negative, low, high, and very high,
respectively). TNFSF15 in normal ovary exhibited vascular
distribution in a pattern similar to that of the endothelial
cell marker CD31 (Fig. 1A). While TNFSF15 expression
was high or very high in most normal ovary specimens, it
was negative or low in the cancer specimens (Table 1).
Remarkably, low TNFSF15 expression (-/?) was found in
nearly all age groups of the patients, and the percentage of
low TNFSF15 expression specimens increased as the
patients aged between 40 and 69 (Table 1). When analyzed
on the basis of Federation of International Gynecology
Organizations (FIGO) stages, TNFSF15 protein levels
decreased markedly in stages I and II, and diminished in
stages III and IV, with the percentages of highly positively
stained specimens (??/???) being 35 and 16%,
respectively (Table 1). TNFSF15 protein levels in ovarian
cancer are thus strongly correlated inversely to the pro-
gression of the disease. No significant differences were
seen among the histological types of the disease or based
on histology staging. Microvessel density (MVD) values
increased significantly as the disease progressed based on
FIGO stages, as expected, with the median MVD values for
normal, stages I/II, and stages III/IV being 8.3, 15.2, and
22.1, respectively. However, of the 84 cancer specimens
that were informative for both TNFSF15 and CD31, MVD
values were significantly higher in low TNFSF15 speci-
mens than in high TNFSF15 specimens, with the median
values being 17.4 and 13.3, respectively (Fig. 1C), indi-
cating that TNFSF15 protein levels are inversely correlated
with the extent of tumor angiogenesis in ovarian cancer.
TNFSF15 downregulation by VEGF
We analyzed VEGF expression in normal ovary and ovarian
cancer specimens by immunohistochemistry. Of the 88
ovarian cancer and 12 normal specimens studied, we found
that, while VEGF staining was basically negative in normal
ovary, an increasing proportion of ovarian cancer cells were
positive for VEGF as the disease progressed (Fig. 2a). About
26% of the cancer cells in stages I and II and as much as 69%
of the cancer cells in stages III and IV were positive for
VEGF (Fig. 2b). Consistently, of the 84 cancer specimens
that were informative for CD31 immunostaining, MVD
values were about 70% higher in cancer specimens that also
exhibited high levels of VEGF (median = 19.8) than that in
those with moderate or no VEGF production (median =
14.1; Fig. 2c). Interestingly, enhanced VEGF production by
cancer cells was accompanied by diminished TNFSF15. Of
the 86 cancer specimens that were informative for both
TNFSF15 and VEGF, the percentage of endothelial cells
with low (-/?) TNFSF15 increased from about 70% ovarian
cancer specimens with moderate or no VEGF staining to
about 90% in specimens with high or very high VEGF levels
(Fig. 2d).
Additionally, we determined the impact of hypoxia on
VEGF production by ovarian cancer cells. OVCAR-3 cells
were cultured under either normoxic (20% O2) or hypoxic
conditions (5% O2). We found that VEGF in the conditioned
media of the cancer cells in hypoxia reached a half-maxi-
mum level in 12 h; in comparison, VEGF production
reached a similar level in about 72 h in cells cultured in
normoxia (Fig. 2e). We then treated HUVEC cultures with
recombinant human VEGF at various concentrations and
found that the treatments substantially inhibited TNFSF15
production (Fig. 2f). To determine whether the inhibition of
74 Angiogenesis (2012) 15:71–85
123
HUVEC production of TNFSF15 by OVCAR-3 cell-condi-
tioned media was specifically caused by VEGF, we treated
HUVEC with OVCAR-3 conditioned media in the presence
or absence of neutralizing antibodies against VEGF, VEG-
FR1, or VEGFR2. We found that ability of OVCAR-3 cell to
inhibit TNFSF15 expression in HUVEC was significantly
hindered by neutralizing antibodies against VEGFR1,
VEGFR2 or VEGF (Fig. 2g). Together these findings
indicate that VEGF produced by the cancer cells is to a large
part responsible for TNFSF15 downregulation.
Correlation between tumor-infiltration of T-cell
and TNFSF15 expression
We determined the correlations between the extent of
T-cell infiltration and TNFSF15 protein levels in clinical
Fig. 1 Relationship between TNFSF15 expression, microvessel den-
sity, and ovarian cancer disease progression in clinical settings.
A Typical images of TNFSF15 and CD31 immunostaining of blood
vessels (brown) of normal ovary and ovarian cancer specimens based
on FIGO staging. Magnification, 9400. B Box plots of MVD (CD31-
positive vessels per 9400 field) of ovarian cancer specimens of
various disease stages. C Box plots of MVD with regard to TNFSF15
protein levels. The number of cases analyzed is indicated for each
group. Horizontal lines indicate median values. **P \ 0.01;
***P \ 0.005; ANOVA (B) or Student t test (C)
Angiogenesis (2012) 15:71–85 75
123
settings. The degree of CD4? T-cell accumulation in
ovarian cancer specimens increased markedly as the dis-
ease progresses based on FIGO staging (Fig. 3A). Of the
86 cancer specimens we analyzed, 75% of stages III and IV
specimens were strongly CD4-positive (??/???),
whereas approximately 41% of FIGO stages I and II
samples were strongly CD4-positive (Fig. 3B). We then
analyzed the relationship between TNFSF15 expression
and T-cell infiltration. Of the specimens in which T-cell
infiltration was negative or marginal (CD4 -/?), 64% was
TNFSF15-negative, whereas of the specimens with high
degree of T-cell infiltration (CD4 ??/???), 83% were
TNFSF15-negative (Fig. 3C). We further determined the
identity of the tumor-infiltrating T-cells by carrying out
CD4-CD25-FOXP3 triple immunostaining of 15 randomly
selected cancer specimens of stage III and IV (Fig. 3D).
We found that only 20% of these specimens were TReg
negative (the ratio of CD4?CD25?FOXP3? cell/CD4? cell
was less than 10%; Fig. 3E). Among the 12 TReg positive
specimens, 30–60% (median = 45%) of the CD4-positive
cells were also CD25?FOXP3? (Fig. 3E). Further analysis
of these specimens indicated that about 95% of the
CD4?CD25? cells were CD4?CD25?FOXP3? (Fig. 3E).
These findings strongly suggest that TNFSF15 protein
levels are inversely correlated with the degree of T-cell
infiltration, especially that of a population of CD4?CD25?
FOXP3? TReg cells, in ovarian cancer with stages III
and IV.
TNFSF15 downregulation by MCP-1
Since a large number of the tumor-infiltrating T-cells in the
stages III&IV ovarian cancer specimens were CD4?CD25?
FOXP3? TReg cells, we determined what cytokine(s) pro-
duced by these cells was responsible for the downregula-
tion of TNFSF15. As more than 95% of tumor-infiltrating
CD4?CD25? cells were CD4?CD25?FOXP3? TReg cells
in the detected specimens, we first treated freshly isolated
CD4?CD25? T cells with OVCAR3 cell conditioned
media. We then treated HUVEC cultures with conditioned
media prepared from these CD4?CD25? T cell cultures.
We found that treatments of HUVEC with the T cell
conditioned media that were influenced by OVACR-3 cell
led to a decrease of TNFSF15 in HUVEC culture media by
about 40% (Fig. 4a). To identify any changes in cytokine
production pattern by CD4?CD25? T cells under the
influence of ovarian cancer cells, we carried out microarray
analysis of a panel of 60 cytokines known to be produced
by regulatory T cells (Figure S1). The cytokine profiling
results indicated that, among the 60 cytokines analyzed,
MCP-1 distinguished itself because its production by
CD4?CD25? T cells increased by as much as 4-fold when
these cells were treated with OVACR-3 conditioned media,
compared with that by untreated CD4?CD25? T cells
(Fig. 4b, c; data regarding the production of all cytokines
on the panel is given in supplemental Figure S1). Sus-
pecting that MCP-1 was responsible for downregulating
TNFSF15, we treated HUVEC cultures with recombinant
human MCP-1. We found that MCP-1 treatments led to a
significant decrease of TNFSF15 production by HUVEC in
a dose-dependent manner (Fig. 4d). A neutralizing anti-
body against MCP-1 specifically blocked the inhibitory
effect (Fig. 4e). MCP-1 treatment of HUVEC culture
did not result in discernible changes in the number of
HUVEC cells (Fig. 4f). These findings indicate that MCP-1
produced by tumor-infiltrating lymphocytes in ovarian
cancer is, besides VEGF, responsible for TNFSF15
downregulation.
Correlation between macrophage infiltration
and TNFSF15 expression
Since tumor-associated macrophages are a major source of
MCP-1 [49], we determined the extent of macrophage
infiltration by immunohistochemistry in normal ovary tis-
sue (n = 10) and ovarian cancer specimens (n = 69), using
macrophage marker MAC-1 (Fig. 5A). We also analyzed
Table 1 TNFSF15 protein levels in normal ovary and ovarian cancer
Number of
cases
%a P valueb
-/? ??/???
Normal ovary 12 16.7 (2) 83.3 (10)
Ovary cancer 94 73.4 (69) 26.6 (25) \0.001
Age
B39 9 66.7 (6) 33.3 (3) 0.06
40–49 28 67.9 (19) 32.1 (9) 0.003
50–59 33 75.8 (25) 24.2 (8) 0.01
60–69 13 92.3 (12) 7.7 (1) \0.001
C70 11 63.6 (7) 36.4 (4) 0.06
FIGO stage
I&II 51 64.7 (33) 35.3 (18)
III&IV 43 83.7 (36) 16.3 (7) 0.038c
Histological type
Serous 58 79.3 (46) 20.7 (12)
Mucinous 8 50.0 (4) 50.0 (4) 0.17c
Others 19 63.2 (12) 36.8 (7) 0.267c
Histology staged
Middle-high 63 74.6 (47) 25.4 (16)
Low 22 77.3 (17) 22.7 (5) 0.803c
a Numbers in parentheses are the number of casesb Compared with normal ovary unless otherwise indicated; Chi-
square testc Compared between the sub-categories; Chi-square testd Histology staging method was not applied to all cases studied
76 Angiogenesis (2012) 15:71–85
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Fig. 2 VEGF produced by human ovarian cancer cells inhibits
TNFSF15 production by HUVEC. A Typical images of VEGF
immunostaining (brown) of normal ovary or ovarian cancer speci-
mens grouped on the basis of FIGO staging. B Percentage of VEGF
positive cells in ovarian cancer specimens grouped on the basis of
FIGO staging. Black -/?. White ??/???. C Box plots of MVD of
ovarian cancer specimens grouped on the basis of low (-/?) or high
(??/???) VEGF levels. D Percentage of TNFSF15 positive cells in
cancer specimens grouped on the basis of low (-/?) or high (??/
???) VEGF levels. E VEGF concentrations in conditioned media of
HUVEC cultured under either normoxic (White) or hypoxic (Black)
conditions. F TNFSF15 concentrations determined at different time
intervals in conditioned media of HUVEC cultures treated with VEGF
at indicated concentrations. White bars 24 h. Gray bars 48 h. Blackbars 72 h. G Concentrations of TNFSF15 in conditioned media of
HUVEC cultures treated with OVCAR3 conditioned media in the
absence or presence of various neutralizing antibodies as indicated.
All cell cultures were in triplicate for each experimental condition.
Each experiment was repeated two times. *P \ 0.05; **P \ 0.01;
***P \ 0.005, Chi-square (B, D); Student t test (C, E, F, G)
Angiogenesis (2012) 15:71–85 77
123
MVD in the cancer specimens by staining for CD31
(n = 67). We found that blood vessel density was posi-
tively correlated with the degree of macrophage infiltration
in ovarian cancer (Fig. 5B). We then determined the rela-
tionship between TNFSF15 expression and macrophage
infiltration. We found that, of the cancer specimens in
which macrophage infiltration was negative or marginal
(MAC-1 -/?), 66% were TNFSF15-negative, whereas of
the samples that exhibited high degree of macrophage
infiltration (Mac-1 ??/???), 90% were TNFSF15-neg-
ative (Fig. 5C). These findings indicate that TNFSF15
expression levels are inversely correlated with the degree
of macrophage inflammation in ovarian cancer. To deter-
mine potential influence of ovarian cancer cell on macro-
phage production of MCP-1, we isolated monocytes from
peripheral blood, placed them in cultures, then treated the
cell cultures with OVCAR3 conditioned media and deter-
mined MCP-1 concentration in the culture media
Fig. 3 Tumor infiltration of CD4?CD25? FOXP3? TReg cell in
human ovarian cancer. A Typical images of CD4 immunostaining of
normal ovary and ovarian cancer specimens based on FIGO staging;
magnification, 9400. B Percentage of CD4 positive cells in speci-
mens based on FIGO staging. Black low percentage of CD4? cells
(-/?). White high percentage of CD4 positive cells (??/???).
C Percentage of TNFSF15 positive cells grouped on the basis of the
extent of CD4? T cell infiltration. Black low percentage of TNFSF15
positive cells (-/?). White high percentage of TNFSF15 positive
cells (??/???). D Typical images of CD4-CD25-FOXP3 triple
immunostaining of cancer specimens. Green CD4? (a). Red CD25?
(b). Blue FOXP3? (c). Merged, CD4?CD25?FOXP3? (d). Magnifi-
cation, 9400. E Percentage of TReg-negative and TReg-positive
specimens in ovarian cancer of stages III&IV. Numbers on top ofboxes are the number of cases determined. *P \ 0.05; ***P \ 0.005,
Chi-square (B, C); Student t test (E)
78 Angiogenesis (2012) 15:71–85
123
(Fig. 5D). MCP-1 production by freshly isolated mono-
cytes was negligible. OVCAR3 produced a readily
detectable amount of MCP-1. Remarkably, when treated
with OVCAR3 conditioned media, MCP-1 production by
the monocytes sharply increased by approximately 5-fold
within 72 h. This suggests that, once infiltrated into ovarian
cancer tissues, macrophages could produce a significant
amount of MCP-1 under the influence of the cancer cells.
Changing TNFSF15 levels in tumor microenvironment
markedly impacts on tumor angiogenesis and growth
We used a murine ovarian cancer cell-line ID8 syngeneic
tumor model (44) to determine the effect of changing
TNFSF15 levels in cancer cell microenvironment on
angiogenesis and tumor growth. In order to mimic the high
level of VEGF we observed in ovarian cancer tissues under
Fig. 4 Inhibition of HUVEC
production of TNFSF15 by
MCP-1. A TNFSF15
concentrations in culture media
of HUVEC treated with various
conditioned media (CM) as
indicated. B Protein array
analysis of cytokines secreted
by freshly isolated human
CD4?CD25? T cells treated
with indicated conditioned
media (CM), using a panel of 60
cytokines (refer to
Supplemental Figure S1 for
detail). Circled MCP-1.
C Quantitative analysis of
MCP-1 production shown in
B. RFU, relative fluorescence
unit. D TNFSF15
concentrations in culture media
of HUVEC treated with
recombinant MCP-1.
E TNFSF15 concentrations in
culture media of HUVEC with
recombinant MCP-1 in the
presence or absence of a
neutralizing antibody against
MCP-1. F Analysis of the
number of HUVEC in cultures
treated with recombinant MCP-
1 at indicated concentrations.
All cell cultures were in
triplicate for each experimental
condition. Each experiment was
repeated two times. *P \ 0.05,
Student t test
Angiogenesis (2012) 15:71–85 79
123
clinical conditions, we treated the ID8 cells with insulin
prior to implantation as insulin was known to stimulate
VEGF production in these cells [48]. We first examined the
effect of raising TNFSF15 levels. ID8 cells were subcuta-
neously implanted (5 9 106 cells per injection) on C57BL/
6 mice. The animals were treated by intraperitoneal
injection of recombinant human TNFSF15 (10 mg/Kg;
control group was treated with vehicle) on day 6 post-
inoculation when the tumors were palpable, then repeated
daily. The treatments markedly inhibited the growth of ID8
tumors (Fig. 6A). The tumor volumes of recombinant
TNFSF15-treated group on average were about 40%
smaller than those of the control group.
We then determined the effect of downregulating
TNFSF15 in cancer cell microenvironment on tumor
growth. We subcutaneously injected TNFSF15 shRNA
(1.5 9 105 IFU per injection) 72 h prior the implantation
of ID8 cells on the same site, then repeated shRNA
injection on day 6 post cancer cell inoculation. Monitoring
tumor growth rate, we found that the cancer cells implanted
at TNFSF15 shRNA-treated sites exhibited significantly
facilitated ability to form tumors as compared with the
cells injected at control shRNA-treated sites (Fig. 6B). The
tumor volumes on average were approximately 53% larger
in TNFSF15 shRNA-treated group than those in control
group. Additionally, we determined TNFSF15 protein
levels in the skin of shRNA-treated sites by immunohis-
tochemical analysis. We found that TNFSF15 shRNA-
treatments led to nearly complete elimination of TNFSF15
in the blood vessels (Fig. 6C, a, b). In sharp contrast, the
number of blood vessels identified by CD31-positive
staining increased markedly (Fig. 6C, c, d). Quantitative
analysis of the skin sections revealed that the median MVD
value in TNFSF15 shRNA-treated skin increased by nearly
100% compared with that in the control group (Fig. 6C, e).
We compared the extent of angiogenesis in the tumors
by carrying out CD31 immunostaining of tumor sections
from vehicle- or recombinant TNFSF15-treated animals
Fig. 5 Relationship between
macrophage infiltration, MVD,
and TNFSF15 protein levels in
human ovarian cancer.
A Typical images of tumor-
associated macrophages (BrownMAC-1 positive cells).
Magnification, 9400. B Box
plots of MVD of cancer
specimens grouped on the basis
of low (-/?) or high (??/
???) degree of macrophage
infiltration. The number of cases
in each group is indicated.
C Percentage of TNFSF15
positive cells in cancer
specimens grouped on the basis
of the degree of macrophage
infiltration. White TNFSF15
positive. Black TNFSF15
negative. The number of
informative specimens in each
group is indicated. D MCP-1
concentrations in the culture
media of freshly isolated
monocytes untreated or treated
with OVCAR3 conditioned
media, and OVCAR3
conditioned media alone.
*P \ 0.05; **P \ 0.01;
***P \ 0.005, Chi-square (C);
Student t test (B, D)
80 Angiogenesis (2012) 15:71–85
123
(Fig. 6D, a, b). The median MVD value in tumors of
recombinant TNFSF15-teated group decreased by 75%
compared with that of vehicle-treated group (Fig. 6D, c).
On the other hand, CD31 immunostaining showed that the
degree of angiogenesis was remarkably higher in tumor
sections of TNFSF15 shRNA-treated group than that in the
control group (Fig. 6E, a, b). The median MVD value of
TNFSF15 shRNA-treated group increased by approxi-
mately 120% compared with that of control shRNA-treated
group. Together these findings indicate that increasing
TNFSF15 levels in the cancer cell microenvironment
results in significant inhibition of tumor angiogenesis and
growth, whereas decreasing TNFSF15 levels leads to
greatly facilitated tumor angiogenesis and growth.
Discussion
It is well known that the process of malignant tumor
development resembles abnormal wound healing charac-
terized by uncontrolled inflammation and neovasculariza-
tion driven by highly upregulated pro-angiogenesis growth
factors and cytokines. The initiation of angiogenesis,
however, requires a concomitant downregulation of the
activities of endogenous inhibitors of angiogenesis, as
these inhibitors normally play a critical role to maintain the
stability of an established vasculature. In this study we
focus on how the role of a key endogenous suppressor of
angiogenesis is removed from the normal control mecha-
nism. TNFSF15 is a unique cytokine that functions as an
endogenous inhibitor of neovascularization to maintain
vascular homeostasis [36, 38–41]. It is produced largely by
endothelial cells in an established vasculature and inhibits
the proliferation of endothelial cells themselves. We
hypothesize that downmodulation of TNFSF15 is a pre-
requisite for the initiation of neovascularization such as in
cancers. We determined whether TNFSF15 is downmod-
ulated in ovarian cancer under clinical conditions, what
factors are responsible for the action, and what are the
sources of these factors.
Our data demonstrate in clinical settings that TNFSF15
is present at high levels in the vasculature of normal ovary
but declines sharply at early stages of ovarian cancer
development, and nearly completely diminishes in the
tumor tissues as the disease progresses to later stages. We
found that the downmodulation of TNFSF15 is attributable
to a significant extent to VEGF produced by the cancer
cells. The human female reproductive tract is highly
dependent on VEGF for normal functions such as endo-
metrial proliferation and corpus luteum development [27].
The unique influence of female sex hormones on the
expression and activity of VEGF deems angiogenesis an
important facet of the development of ovarian cancer.
VEGF is highly upregulated under periodical hypoxic
conditions in a malignant tumor. By increasing vascular
permeability in endothelial cells, it facilitates the recruit-
ment of immune cells, mostly noticeably macrophages and
T cells, to the immediate surroundings of the cancer cells,
making tumor formation a process of abnormal wound
healing characterized by uncontrolled inflammation and
angiogenesis. Showing a strong, inverse correlation
between VEGF and TNFSF15 expression in ovarian cancer
in clinical settings, sharp upregulation of ovarian cancer
cell production of VEGF under hypoxic conditions, and
effective downregulation of endothelial cell production of
TNFSF15 by VEGF, our data indicate that TNFSF15 as an
important endogenous inhibitor of angiogenesis is targeted
by VEGF for downmodulation, highly likely as an initial
step of the process of neovascularization.
Our data also reveal a strong, inverse correlation
between the extent of TReg cells and macrophage infiltra-
tion and TNFSF15 protein levels. Additionally, we show
that TNFSF15 expression in human endothelial cells in
culture can be downmodulated by MCP-1 produced by
TReg cell and monocyte (a circulating counterpart of mac-
rophage) under the influence of ovarian cancer cell.
Importantly, the production of MCP-1 by freshly isolated
TReg cells and monocytes is initially insignificant, but is
highly upregulated when treated with ovarian cancer cell
conditioned media. It has been reported MCP-1 under
certain conditions can be produced by a variety of cell
types, including endothelial, fibroblast, epithelial, smooth
muscle, mesangial, astrocytic, monocytic, and microglial
cells, either constitutively or after induction by oxidative
stress, cytokines, or growth factors [18, 20, 21, 50]. MCP-1
has been reported to have an important role in various
disease states, including immunodeficiency, cardiovascu-
lar, and cancer [51–54]. Monocytes and macrophages are
the major source of MCP-1, however, in many cancers,
including ovarian cancer [14–17]. The role of MCP-1 in
cancers appears ambiguous. It stimulates host antitumor
responses by attracting and activating lymphocytes [55],
and is attributed to cancer progression because of its
angiogenic activities [56]. We show that, promoted by
ovarian cancer cells, monocytes and TReg cells produce
high level of MCP-1, and that MCP-1 directly downregu-
lates TNFSF15 expression in endothelial cell. The ability to
downregulate an endogenous suppressor of angiogenesis
suggests that MCP-1 is capable of shifting the equilibrium
between pro- and anti-angiogenesis forces in cancer
microenvironment in favor of inflammation and neovas-
cularization, making MCP-1 a more potent promoter of
cancer progression than simply driving endothelial cell
migration. Further investigation on TNFSF15 downmodu-
lation by tumor infiltrating macrophages and TReg cells is
likely to reveal critical insights into the loss of negative
Angiogenesis (2012) 15:71–85 81
123
control mechanisms in cancers which otherwise would
function to suppress inflammation and angiogenesis.
We used a mouse model of ovarian cancer to determine
the impact of raising or eliminating TNFSF15 levels on
tumor angiogenesis. The facts that cancer cells and tumor-
infiltrating immune cells, most prominently macrophages
and TReg cells, cooperate to downmodulate TNFSF15
suggest that the downmodulation of TNFSF15 is a
82 Angiogenesis (2012) 15:71–85
123
prerequisite for the initiation of angiogenesis in tumors.
ID8 is a mouse ovarian cancer cell-line derived from
spontaneous malignant transformation in vitro of C57BL/6
mouse ovarian surface epithelial (MOSE) cell, an equiva-
lent of human epithelial ovarian cancer (EOC) cell
[57–59]. Previously we showed that TNFSF15, delivered
either by various methods of gene-transfer or systemic
administration of a recombinant protein, is able to inhibit
tumor growth in models of breast, prostate, colon cancers
[36, 37, 41]. Our study with the ID8 ovarian cancer model
demonstrates the importance of TNFSF15 as a negative
modulator of to guard against the initiation of angiogenesis.
Our data indicate that systemic application of recombinant
TNFSF15 inhibits angiogenesis and tumor growth, whereas
silencing TNFSF15 with shRNA topically prior to cancer
cell inoculation greatly facilitates angiogenesis and tumor
growth. A well-controlled angiogenesis cycle under phys-
iological conditions is characteristic of normal ovaries,
synchronized with ovulation and corpus luteum formation.
Up- and downregulation of VEGF and other angiogenesis-
promoting factors are implicated in this cycle. However,
insights into the down- and upregulation of endogenous
suppressors of angiogenesis in this cycle are largely
missing. Our findings indicate that the function of
TNFSF15 is likely to be important in the control of angi-
ogenesis cycle in ovary, and that the loss of this negative
control mechanism could be a critical attribute to ovarian
cancer development. It is interesting to notice in this regard
that we found a significant decline of TNFSF15 in ovarian
cancer occurs with patients in age group 40–49 and persists
in older age groups. This is consistent with the observation
that ovarian cancer incidence in Asian populations rises
sharply in age 40–59 and plateaus in older ages [60].
In summary, our data are consistent with the view that
downmodulation of TNFSF15 as an endogenous suppressor
of angiogenesis is a prerequisite for neovascularization in
ovarian cancer. This is achieved by the inhibition of
TNFSF15 expression by VEGF and MCP-1 produced,
respectively, by the cancer cells and tumor-infiltrating
macrophages and TReg cells under the influence of the
cancer cells. Keeping an elevated TNFSF15 level would
thus allow the maintenance of a normal vasculature and
prevention of uncontrolled inflammation and angiogenesis
common to malignant cancers.
Acknowledgments This study is supported in part by grants from
Ministry of Science and Technology of China (2009CB918901 to
L.Y.L), National Institute of Health of the United States (R01CA11
3875 to L.Y.L), and Natural Science Foundation of China (30670801
to W.M.D).
References
1. Rosmorduc O, Housset C (2010) Hypoxia: a link between
fibrogenesis, angiogenesis, and carcinogenesis in liver disease.
Semin Liver Dis 30(3):258–270. doi:10.1055/s-0030-1255355
2. Aggarwal BB, Shishodia S, Sandur SK, Pandey MK, Sethi G
(2006) Inflammation and cancer: how hot is the link? Biochem
Pharmacol 72(11):1605–1621. doi:10.1016/j.bcp.2006.06.029
3. Coussens LM, Werb Z (2002) Inflammation and cancer. Nature
420(6917):860–867. doi:10.1038/nature01322
4. Fiedler U, Augustin HG (2006) Angiopoietins: a link between
angiogenesis and inflammation. Trends Immunol 27(12):552–558.
doi:10.1016/j.it.2006.10.004
5. Coulon S, Heindryckx F, Geerts A, Van Steenkiste C, Colle I, Van
Vlierberghe H (2011) Angiogenesis in chronic liver disease and its
complications. Liver Int 31(2):146–162. doi:10.1111/j.1478-
3231.2010.02369.x
6. Balkwill F, Mantovani A (2001) Inflammation and cancer: back to
Virchow? Lancet 357(9255):539–545. doi:10.1016/S0140-6736
(00)04046-0
7. Folkman J (1995) Angiogenesis in cancer, vascular, rheumatoid
and other disease. Nat Med 1(1):27–31
8. Casanovas O, Hicklin DJ, Bergers G, Hanahan D (2005) Drug
resistance by evasion of antiangiogenic targeting of VEGF sig-
naling in late-stage pancreatic islet tumors. Cancer Cell 8(4):
299–309. doi:10.1016/j.ccr.2005.09.005
9. Dvorak HF, Detmar M, Claffey KP, Nagy JA, van de Water L,
Senger DR (1995) Vascular permeability factor/vascular endo-
thelial growth factor: an important mediator of angiogenesis in
malignancy and inflammation. Int Arch Allergy Immunol
107(1–3):233–235
10. Jeon BH, Jang C, Han J, Kataru RP, Piao L, Jung K, Cha HJ,
Schwendener RA, Jang KY, Kim KS, Alitalo K, Koh GY (2008)
Profound but dysfunctional lymphangiogenesis via vascular
endothelial growth factor ligands from CD11b? macrophages in
advanced ovarian cancer. Cancer Res 68(4):1100–1109. doi:
10.1158/0008-5472.CAN-07-2572
11. Duyndam MC, Hilhorst MC, Schluper HM, Verheul HM, van
Diest PJ, Kraal G, Pinedo HM, Boven E (2002) Vascular endo-
thelial growth factor-165 overexpression stimulates angiogenesis
and induces cyst formation and macrophage infiltration in human
ovarian cancer xenografts. Am J Pathol 160(2):537–548
Fig. 6 Impact of changing TNFSF15 levels in cancer microenviron-
ment on mouse ovarian cancer ID8 syngeneic tumor angiogenesis and
growth. A Growth rates of ID8 tumors on animals treated with
intraperitoneal administration of recombinant TNFSF15 or vehicle.
Closed circles vehicle treated. Open circles TNFSF15 treated. Values
are mean ± SD (number of animal per group, n = 5). B Growth rates
of tumors on animals treated with topical injection of TNFSF15
shRNA or control shRNA. Black bars control shRNA treated. Whitebars TNFSF15 shRNA treated. Values are mean ± SD (number of
animals per group, n = 5). C (a–d) Typical images of TNFSF15 or
CD31 immunostained skin sections collected from the sites of topical
TNFSF15 shRNA or control shRNA treatments, as indicated; (e) Box
plots of MVD values of tumors treated with control or TNFSF15
shRNA, as indicated (number of animals per group, n = 5). D Typical
images of CD31 immunostaining of tumor sections in (a) vehicle
treated or (b) recombinant TNFSF15 treated groups; (c) Box plots of
MVD values of vehicle or TNFSF15 treated tumors (number of
animals per group, n = 5). E Typical images of CD31 immunostain-
ing of tumor sections in (a) control shRNA treated or (b) TNFSF15
shRNA treated groups; (c) Box plots of MVD values of control
shRNA or THFSF15 shRNA treated tumors (number of animals per
group, n = 5). Horizontal bars indicate median values. The exper-
iment was repeated once and the results were reproducible.
*P \ 0.05; **P \ 0.01; ***P \ 0.005, Student t test
b
Angiogenesis (2012) 15:71–85 83
123
12. Ueno T, Toi M, Saji H, Muta M, Bando H, Kuroi K, Koike M,
Inadera H, Matsushima K (2000) Significance of macrophage
chemoattractant protein-1 in macrophage recruitment, angiogen-
esis, and survival in human breast cancer. Clin Cancer Res 6(8):
3282–3289
13. Jin G, Kawsar HI, Hirsch SA, Zeng C, Jia X, Feng Z, Ghosh SK,
Zheng QY, Zhou A, McIntyre TM, Weinberg A (2010) An
antimicrobial peptide regulates tumor-associated macrophage
trafficking via the chemokine receptor CCR2, a model for
tumorigenesis. PLoS One 5(6):e10993. doi:10.1371/journal.
pone.0010993
14. Boelte KC, Gordy LE, Joyce S, Thompson MA, Yang L, Lin PC
(2011) Rgs2 mediates pro-angiogenic function of myeloid derived
suppressor cells in the tumor microenvironment via upregulation of
MCP-1. PLoS One 6(4):e18534. doi:10.1371/journal.pone.0018534
15. Saenz-Lopez P, Carretero R, Cozar JM, Romero JM, Canton J,
Vilchez JR, Tallada M, Garrido F, Ruiz-Cabello F (2008) Genetic
polymorphisms of RANTES, IL1-A, MCP-1 and TNF-A genes in
patients with prostate cancer. BMC Cancer 8:382. doi:10.1186/
1471-2407-8-382
16. Monti P, Leone BE, Marchesi F, Balzano G, Zerbi A, Scaltrini F,
Pasquali C, Calori G, Pessi F, Sperti C, Di Carlo V, Allavena P,
Piemonti L (2003) The CC chemokine MCP-1/CCL2 in pancreatic
cancer progression: regulation of expression and potential mech-
anisms of antimalignant activity. Cancer Res 63(21):7451–7461
17. Negus RP, Stamp GW, Relf MG, Burke F, Malik ST, Bernasconi S,
Allavena P, Sozzani S, Mantovani A, Balkwill FR (1995) The
detection and localization of monocyte chemoattractant protein-1
(MCP-1) in human ovarian cancer. J Clin Invest 95(5):2391–2396.
doi:10.1172/JCI117933
18. Cushing SD, Berliner JA, Valente AJ, Territo MC, Navab M,
Parhami F, Gerrity R, Schwartz CJ, Fogelman AM (1990) Min-
imally modified low density lipoprotein induces monocyte che-
motactic protein 1 in human endothelial cells and smooth muscle
cells. Proc Natl Acad Sci USA 87(13):5134–5138
19. Standiford TJ, Kunkel SL, Phan SH, Rollins BJ, Strieter RM
(1991) Alveolar macrophage-derived cytokines induce monocyte
chemoattractant protein-1 expression from human pulmonary
type II-like epithelial cells. J Biol Chem 266(15):9912–9918
20. Brown Z, Strieter RM, Neild GH, Thompson RC, Kunkel SL,
Westwick J (1992) IL-1 receptor antagonist inhibits monocyte
chemotactic peptide 1 generation by human mesangial cells.
Kidney Int 42(1):95–101
21. Barna BP, Pettay J, Barnett GH, Zhou P, Iwasaki K, Estes ML
(1994) Regulation of monocyte chemoattractant protein-1
expression in adult human non-neoplastic astrocytes is sensitive
to tumor necrosis factor (TNF) or antibody to the 55-kDa TNF
receptor. J Neuroimmunol 50(1):101–107
22. Dvorak HF (1986) Tumors: wounds that do not heal. Similarities
between tumor stroma generation and wound healing. N Engl J
Med 315(26):1650–1659. doi:10.1056/NEJM198612253152606
23. Folkman J, Watson K, Ingber D, Hanahan D (1989) Induction of
angiogenesis during the transition from hyperplasia to neoplasia.
Nature 339(6219):58–61. doi:10.1038/339058a0
24. Fraser HM, Bell J, Wilson H, Taylor PD, Morgan K, Anderson
RA, Duncan WC (2005) Localization and quantification of cyclic
changes in the expression of endocrine gland vascular endothelial
growth factor in the human corpus luteum. J Clin Endocrinol
Metab 90(1):427–434. doi:10.1210/jc.2004-0843
25. Davis JS, Rueda BR, Spanel-Borowski K (2003) Microvascular
endothelial cells of the corpus luteum. Reprod Biol Endocrinol
1:89. doi:10.1186/1477-7827-1-89
26. Fraser HM, Wulff C (2001) Angiogenesis in the primate ovary.
Reprod Fertil Dev 13(7–8):557–566
27. Delli Carpini J, Karam AK, Montgomery L (2010) Vascular
endothelial growth factor and its relationship to the prognosis and
treatment of breast, ovarian, and cervical cancer. Angiogenesis
13(1):43–58. doi:10.1007/s10456-010-9163-3
28. Lebovic DI, Shifren JL, Ryan IP, Mueller MD, Korn AP, Darney
PD, Taylor RN (2000) Ovarian steroid and cytokine modulation of
human endometrial angiogenesis. Hum Reprod 15(Suppl 3):67–77
29. Zhang L, Yang N, Conejo-Garcia JR, Katsaros D, Mohamed-
Hadley A, Fracchioli S, Schlienger K, Toll A, Levine B, Rubin
SC, Coukos G (2003) Expression of endocrine gland-derived
vascular endothelial growth factor in ovarian carcinoma. Clin
Cancer Res 9(1):264–272
30. Boocock CA, Charnock-Jones DS, Sharkey AM, McLaren J, Barker
PJ, Wright KA, Twentyman PR, Smith SK (1995) Expression of
vascular endothelial growth factor and its receptors fit and KDR in
ovarian carcinoma. J Natl Cancer Inst 87(7):506–516
31. Orre M, Rogers PA (1999) VEGF, VEGFR-1, VEGFR-2,
microvessel density and endothelial cell proliferation in tumours
of the ovary. Int J Cancer 84(2):101–108. doi:10.1002/(SICI)1097-
0215(19990420)84:2\101:AID-IJC2[3.0.CO;2-5
32. Hazelton D, Nicosia RF, Nicosia SV (1999) Vascular endothelial
growth factor levels in ovarian cyst fluid correlate with malig-
nancy. Clin Cancer Res 5(4):823–829
33. Kuwahara K, Sasaki T, Kobayashi K, Noma B, Serikawa M,
Iiboshi T, Miyata H, Kuwada Y, Murakami M, Yamasaki S,
Kariya K, Morinaka K, Chayama K (2004) Gemcitabine sup-
presses malignant ascites of human pancreatic cancer: correlation
with VEGF expression in ascites. Oncol Rep 11(1):73–80
34. Bolat F, Gumurdulu D, Erkanli S, Kayaselcuk F, Zeren H, Ali
Vardar M, Kuscu E (2008) Maspin overexpression correlates with
increased expression of vascular endothelial growth factors A, C,
and D in human ovarian carcinoma. Pathol Res Pract 204(6):
379–387. doi:10.1016/j.prp.2008.01.011
35. Juric G, Zarkovic N, Nola M, Tillian M, Jukic S (2001) The value
of cell proliferation and angiogenesis in the prognostic assess-
ment of ovarian granulosa cell tumors. Tumori 87(1):47–53
36. Zhai Y, Ni J, Jiang GW, Lu J, Xing L, Lincoln C, Carter KC,
Janat F, Kozak D, Xu S, Rojas L, Aggarwal BB, Ruben S, Li LY,
Gentz R, Yu GL (1999) VEGI, a novel cytokine of the tumor
necrosis factor family, is an angiogenesis inhibitor that sup-
presses the growth of colon carcinomas in vivo. FASEB J
13(1):181–189
37. Zhai Y, Yu J, Iruela-Arispe L, Huang WQ, Wang Z, Hayes AJ,
Lu J, Jiang G, Rojas L, Lippman ME, Ni J, Yu GL, Li LY (1999)
Inhibition of angiogenesis and breast cancer xenograft tumor
growth by VEGI, a novel cytokine of the TNF superfamily. Int J
Cancer 82(1):131–136. doi:10.1002/(SICI)1097-0215(19990702)
82:1\131:AID-IJC22[3.0.CO;2-O
38. Chew LJ, Pan H, Yu J, Tian S, Huang WQ, Zhang JY, Pang S, Li
LY (2002) A novel secreted splice variant of vascular endothelial
cell growth inhibitor. FASEB J 16(7):742–744. doi:10.1096/
fj.01-0757fje
39. Yu J, Tian S, Metheny-Barlow L, Chew LJ, Hayes AJ, Pan H, Yu
GL, Li LY (2001) Modulation of endothelial cell growth arrest
and apoptosis by vascular endothelial growth inhibitor. Circ Res
89(12):1161–1167
40. Tian F, Liang PH, Li LY (2009) Inhibition of endothelial pro-
genitor cell differentiation by VEGI. Blood 113(21):5352–5360.
doi:10.1182/blood-2008-08-173773
41. Hou W, Medynski D, Wu S, Lin X, Li LY (2005) VEGI-192, a
new isoform of TNFSF15, specifically eliminates tumor vascular
endothelial cells and suppresses tumor growth. Clin Cancer Res
11(15):5595–5602. doi:10.1158/1078-0432.CCR-05-0384
42. Liang PH, Tian F, Lu Y, Duan B, Stolz DB, Li LY (2011)
Vascular endothelial growth inhibitor (VEGI; TNFSF15) inhibits
bone marrow-derived endothelial progenitor cell incorporation
into Lewis lung carcinoma tumors. Angiogenesis 14(1):61–68.
doi:10.1007/s10456-010-9195-8
84 Angiogenesis (2012) 15:71–85
123
43. Parr C, Gan CH, Watkins G, Jiang WG (2006) Reduced vascular
endothelial growth inhibitor (VEGI) expression is associated with
poor prognosis in breast cancer patients. Angiogenesis 9(2):
73–81. doi:10.1007/s10456-006-9033-1
44. Zhou J, Yang Z, Tsuji T, Gong J, Xie J, Chen C, Li W, Amar S,
Luo Z (2011) LITAF and TNFSF15, two downstream targets of
AMPK, exert inhibitory effects on tumor growth. Oncogene
30(16):1892–1900. doi:10.1038/onc.2010.575
45. Zhang N, Sanders AJ, Ye L, Jiang WG (2009) Vascular endo-
thelial growth inhibitor in human cancer (review). Int J Mol Med
24(1):3–8
46. Zhang N, Sanders AJ, Ye L, Kynaston HG, Jiang WG (2010)
Expression of vascular endothelial growth inhibitor (VEGI) in
human urothelial cancer of the bladder and its effects on the
adhesion and migration of bladder cancer cells in vitro. Anti-
cancer Res 30(1):87–95
47. Conway KP, Price P, Harding KG, Jiang WG (2007) The role of
vascular endothelial growth inhibitor in wound healing. Int
Wound J 4(1):55–64. doi:10.1111/j.1742-481X.2006.00295.x
48. Bermont L, Lamielle F, Lorchel F, Fauconnet S, Esumi H, Weisz
A, Adessi GL (2001) Insulin up-regulates vascular endothelial
growth factor and stabilizes its messengers in endometrial ade-
nocarcinoma cells. J Clin Endocrinol Metab 86(1):363–368
49. Yoshimura T, Robinson EA, Tanaka S, Appella E, Leonard EJ
(1989) Purification and amino acid analysis of two human
monocyte chemoattractants produced by phytohemagglutinin-
stimulated human blood mononuclear leukocytes. J Immunol
142(6):1956–1962
50. Standiford TJ, Rolfe MR, Kunkel SL, Lynch JP III, Becker FS,
Orringer MB, Phan S, Strieter RM (1993) Altered production and
regulation of monocyte chemoattractant protein-1 from pulmon-
ary fibroblasts isolated from patients with idiopathic pulmonary
fibrosis. Chest 103(2 Suppl):121S
51. Cinque P, Vago L, Mengozzi M, Torri V, Ceresa D, Vicenzi E,
Transidico P, Vagani A, Sozzani S, Mantovani A, Lazzarin A,
Poli G (1998) Elevated cerebrospinal fluid levels of monocyte
chemotactic protein-1 correlate with HIV-1 encephalitis and local
viral replication. AIDS 12(11):1327–1332
52. Marini E, Tiberio L, Caracciolo S, Tosti G, Guzman CA, Schi-
affonati L, Fiorentini S, Caruso A (2008) HIV-1 matrix protein
p17 binds to monocytes and selectively stimulates MCP-1
secretion: role of transcriptional factor AP-1. Cell Microbiol
10(3):655–666. doi:10.1111/j.1462-5822.2007.01073.x
53. Krishnaswamy G, Smith JK, Mukkamala R, Hall K, Joyner W,
Yerra L, Chi DS (1998) Multifunctional cytokine expression by
human coronary endothelium and regulation by monokines and
glucocorticoids. Microvasc Res 55(3):189–200
54. Salcedo R, Ponce ML, Young HA, Wasserman K, Ward JM,
Kleinman HK, Oppenheim JJ, Murphy WJ (2000) Human
endothelial cells express CCR2 and respond to MCP-1: direct role
of MCP-1 in angiogenesis and tumor progression. Blood 96(1):
34–40
55. Krensky AM, Clayberger C (2009) Biology and clinical rele-
vance of granulysin. Tissue Antigens 73(3):193–198. doi:
10.1111/j.1399-0039.2008.01218.x
56. Soria G, Ben-Baruch A (2008) The inflammatory chemokines
CCL2 and CCL5 in breast cancer. Cancer Lett 267(2):271–285.
doi:10.1016/j.canlet.2008.03.018
57. Janat-Amsbury MM, Yockman JW, Anderson ML, Kieback DG,
Kim SW (2006) Comparison of ID8 MOSE and VEGF-modified
ID8 cell lines in an immunocompetent animal model for human
ovarian cancer. Anticancer Res 26(4B):2785–2789
58. Holtz DO, Krafty RT, Mohamed-Hadley A, Zhang L, Ala-
gkiozidis I, Leiby B, Guo W, Gimotty PA, Coukos G (2008)
Should tumor VEGF expression influence decisions on combin-
ing low-dose chemotherapy with antiangiogenic therapy? Pre-
clinical modeling in ovarian cancer. J Trans Med 6:2. doi:
10.1186/1479-5876-6-2
59. Li Z, Huang H, Boland P, Dominguez MG, Burfeind P, Lai KM,
Lin HC, Gale NW, Daly C, Auerbach W, Valenzuela D, Yanco-
poulos GD, Thurston G (2009) Embryonic stem cell tumor model
reveals role of vascular endothelial receptor tyrosine phosphatase
in regulating Tie2 pathway in tumor angiogenesis. Proc Natl Acad
Sci USA 106(52):22399–22404. doi:10.1073/pnas.0911189106
60. Kim K, Zang R, Choi SC, Ryu SY, Kim JW (2009) Current status
of gynecological cancer in China. J Gynecol Oncol 20(2):72–76.
doi:10.3802/jgo.2009.20.2.72
Angiogenesis (2012) 15:71–85 85
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