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Blood, Vol. 96 No. 2 (July 15), 2000:
pp. 546-553
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Involvement of vascular endothelial growth factor receptor-3 in
maintenance of integrity of endothelial cell lining during tumor
angiogenesis
Hajime Kubo,
Takashi Fujiwara,
Lotta Jussila,
Hiroyuki Hashi,
Minetaro Ogawa,
Kenji Shimizu,
Masaaki Awane,
Yoshiharu Sakai,
Arimichi Takabayashi,
Kari Alitalo,
Yoshio Yamaoka, and
Shin-Ichi Nishikawa
From the Departments of Gastroenterological Surgery and Molecular
Genetics, Graduate School of Medicine, Kyoto University, Kyoto, and the
Laboratory Animal Center, Ehime University School of Medicine, Ehime,
Japan; and the Molecular/Cancer Biology Laboratory, Haartman Institute,
University of Helsinki, Helsinki, Finland.
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Abstract |
Vascular endothelial growth factor (VEGF) plays a major role in
tumor angiogenesis. VEGF-C, however, is thought to stimulate the growth
of lymphatic vessels because an expression of its specific receptor,
VEGF receptor-3 (VEGFR-3), was demonstrated to be restricted to
lymphatic vessels. Here we demonstrate that the inactivation of VEGFR-3
by a novel blocking monoclonal antibody (mAb) suppresses tumor growth
by inhibiting the neo-angiogenesis of tumor-bearing tissues. Although
VEGFR-3 is not expressed in adult blood vessels, it is induced in
vascular endothelial cells of the tumor-bearing tissues. Hence, VEGFR-3
is another receptor tyrosine kinase involved in tumor-induced
angiogenesis. Micro-hemorrhage in the tumor-bearing tissue was the most
conspicuous histologic finding specific to AFL4 mAb-treated mice.
Scanning microscopy demonstrated disruptions of the endothelial lining
of the postcapillary venule, probably the cause of micro-hemorrhage and
the subsequent collapse of the proximal vessels. These findings suggest
the involvement of VEGFR-3 in maintaining the integrity of the
endothelial lining during angiogenesis. Moreover, our results suggest
that the VEGF-C/VEGFR-3 pathway may serve another candidate target for
cancer therapy.
(Blood. 2000;96:546-553)
© 2000 by The American Society of Hematology.
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Introduction |
Angiogenesis is essential for tumor progression because
it allows oxygenation and nutrient perfusion of the tumor. In the absence of neovascularization, a solid tumor cannot form a large mass.1-3 Angiogenesis is a complex multistep process by
which blood vessels are formed from the preexisting vasculature.
Previous studies of null mutant mice have demonstrated that development of the embryonic vascular system requires the coordinated expression of
various receptor tyrosine kinases (RTKs) and their
ligands.4 Among these ligands, vascular endothelial growth
factor (VEGF) has been shown to play a major role throughout
angiogenesis. VEGF action is mediated by 2 RTKs, VEGFR-1 (FLT-1) and
VEGFR-2 (FLK-1/KDR), expressed primarily by endothelial cells (ECs).
VEGF regulates multiple EC activities such as proliferation, migration,
tube formation, and permeability.5-8 Some molecules, such
as angiopoietins (Ang1 and Ang2)/Tie2, are implicated in the later
stages of vascular development ie, during vascular remodeling
and maturation.9-11 Based on studies using reagents that
neutralize each ligand, it has been suggested that those RTKs involved
in the embryonic process are also involved in tumor
angiogenesis.12-16
Recently, VEGFR-3 was identified as an endothelial-specific
RTK related to VEGF receptors.17 VEGFR-3 is induced in all
endothelial cells during early embryogenesis, though its expression
eventually disappears from the vascular ECs of adult
tissues.18 In contrast to its transient expression in
vascular ECs, VEGFR-3 is expressed constitutively by the adult
lymphatic endothelium.19 VEGF-C, a new member of the
platelet-derived growth factor (PDGF)/VEGF family, was first identified
as a ligand for VEGFR-3.20 Among several distinct forms of
VEGF-C generated by stepwise proteolysis, the maturely
processed VEGF-C could also activate VEGFR-2.21 Moreover,
another ligand, named VEGF-D22 for VEGFR-3, has been identified recently, illustrating the complex ligand-receptor relationship, which poses a problem for understanding the role of
VEGFR-3 during vasculogenesis.
Transgenic overexpression of VEGF-C in the skin has
been shown to induce hyperplasia of the lymphatic vasculature
(lymph-angiogenesis), leaving the vascular structure unaffected.
This observation implies a lymphatic-specific role for
VEGF-C/VEGFR-3.23,24 Another report using the cornea
assay,25 however, demonstrated that VEGF-C could induce
angiogenesis of adult tissues. Mice bearing a null mutation
of the VEGFR-3 gene display defects in vascular remodeling, indicating
a role for VEGFR-3 in angiogenesis.26 Nonetheless,
because VEGFR-3 is expressed by vascular ECs in the embryo but
not the adult, the role of VEGFR-3 in adult mice is yet to be
determined. We generated an antagonistic monoclonal antibody
(mAb) against VEGFR-3 to elucidate the role of VEGFR-3 in tumor
angiogenesis. We show that VEGFR-3 is indeed involved in tumor
angiogenesis and is essential for maintaining the integrity of
the endothelial sheet.
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Materials and methods |
Mice
Six- to 8-week-old nude (nu/nu) mice were purchased from SLC
(Shizuoka, Japan). VEGFR-3 null mutant embryos26 were
maintained and mated in our animal facility.
Cell culture
The C6 rat glioblastoma cell line (a gift from Dr H. Kataoka, Kyoto
University, Kyoto, Japan), the F-2 murine endothelial cell
line27 (a gift from Dr K-I Toda, Kyoto University, Kyoto, Japan), and the 293 human embryonic kidney cell line (American Type
Culture Collection, Manassas, VA) were grown in Dulbecco's modified
Eagle's medium (DMEM; GIBCO/BRL, Grand Island, NY) supplemented with
10% fetal calf serum (FCS) (HyClone Laboratories, Logan, UT). PC-3
human prostate adenocarcinoma cell line (a gift from Dr N. Itoh, Kyoto
University, Kyoto, Japan) were maintained in RPMI 1640 medium
(GIBCO/BRL) containing 10% FCS.
Gene transfection
For the preparation of Fc chimeric proteins, cells from the 293 cell
line were plated on a 100-mm tissue culture dish to reach 70% confluence the next day and were transfected with 15 µg
pCDM8-VEGFR-3-Fc (described below), pCDM8-VEGFR-1-Fc,28 or
pCDM8-VEGFR-2-Fc29 plasmid DNA mixed with 25 µg Trans-IT
(Mirus, Madison, WI) according to the manufacturer's instructions.
Transfected cells were grown in DMEM/F12 medium, and culture
supernatants were harvested 5 days after transfection. Fusion protein
was purified using protein G-Sepharose columns (Pharmacia Biotech,
Uppsala, Sweden).
Plasmid vector containing murine VEGFR-3 cDNA (P2B1S
neo/mFLT417) was kindly provided by Dr W. I. Wood
(Genentech, Cambridge, MA). cDNA containing full-length VEGFR-3 with
Not I and Sal I restriction sites at the 5' and
3' ends, respectively, was obtained from P2R1S neo/mFLT4 and
subcloned into the expression vector pCDNA3.
Generation of anti-VEGFR-3 monoclonal antibody
A 2.3-kb fragment of murine VEGFR-3 cDNA (positions 45-2354 in the
GeneBank L07296), encoding the extracellular domain of VEGFR-3, was
subcloned into the expression vector pCDM8-hIgG.29 Rat
monoclonal antibodies against VEGFR-3-Fc protein were produced using
standard methods as described.29 In brief, an 8-week-old Wistar rat was first immunized subcutaneously with 500 µg VEGFR-3-Fc protein in complete Freund's adjuvant (Difco, Detroit, MI) and then
was administered 3 intraperitoneal shots of 250 µg VEGFR-3-Fc protein
in Freund's incomplete adjuvant (Difco) in alternating weeks and
finally was given an intravenous boost of 100 µg
VEGFR-3-Fc protein. Three days after the boost, the spleen cells
were harvested and fused with the murine myeloma X63Ag8. Undiluted
supernatants from hybridoma were screened by enzyme-linked
immunosorbent assay (ELISA) plates coated with 50 ng/mL VEGFR-3-Fc;
VEGFR-2-Fc29 and VEGFR-1-Fc were used as
controls.28 Positive hybridomas were cloned by
the limiting dilution technique and were subcloned twice.
VEGF-C/VEGFR-3 binding inhibition assay by ELISA
The N-terminal signal sequence of mouse stem cell factor (MMU44725;
198-279) and the 5 repeated myc-tag sequences were
inserted to a 5' end of the cDNA fragment corresponding to the
mature VEGF-C (U43142; 705-1052),21 which was generated by
reverse transcription-polymerase chain reaction amplification of mRNA
prepared from PC-3 cells (5-myc-VEGF-C). For the binding inhibition
study, ELISA plates coated with 50 ng/mL VEGFR-3-Fc protein were first
incubated with various dilutions of mAbs and then with the conditioned
medium (CM) of 293 cells transfected by the 5-myc-VEGF-C gene. Binding with 5-myc-VEGF-C was detected by the anti-myc tag antibody (9E10) (Santa Cruz Biotechnology, Santa Cruz, CA) and then by horseradish peroxidase (HRP)-conjugated antimouse IgG (Zymed, San Francisco, CA).
Plate-bound enzymic activity was detected by using
3',3',5',5'-tetramethylbenzidine (Chemo-Sero
Therapeutic Research Institute, Kumamoto, Japan), and absorbance of
each well was measured using the ELISA reader.
Tumor transplantation
Six- to 8-week-old nude (nu/nu) mice (SLC, Shizuoka, Japan)
underwent subcutaneous transplantation of 2 × 106 C6
rat glioblastoma cells or PC-3 prostate cancer cells in 0.1 mL
phosphate-buffered saline (PBS) on the right flank. Subcutaneous injections of mAbs were given on the left flank of mice. Tumor size was
measured in 2 dimensions, and the volume was calculated using the
formula, width2 × length/2. After 14 days, all mice were humanely killed and autopsied.
Immunohistochemistry
Tissues were fixed in 4% paraformaldehyde in PBS overnight,
embedded in paraffin and sectioned at 5 to 7 µm. The sectioned specimens were incubated first in bleaching solution (methanol, 0.2%
NaN3; 0.6% H2O2) for 30 minutes at
room temperature to block endogenous peroxidase. After rehydration, the
sections were blocked by incubation with 1% bovine serum albumin in
PBS-0.1% Tween 20 (PBS-T) for 20 minutes at room temperature and then
incubated overnight with respective primary antibodies: rat
anti-VEGFR-3 mAb, AFL4 (20 µg/mL); rat anti-VEGFR-2 mAb,
AVAS1229 (10 µg/mL); and rat mAb for murine PECAM-1 (5 µg/mL, Mec13.3; PharMingen, San Diego, CA). After they were washed 3 times in PBS-T for 10 minutes each at room temperature, the sections
were incubated with 1 µg/mL HRP-conjugated anti-rat IgG(H+L)
(Biosource, Camarillo, CA) for 1 hour at room temperature. After
washing with 3 exchanges of PBS-T, the enzymatic reaction with enhanced
DAB substrate kit (TSA-Indirect; NEN Life Science Products, Boston,
MA) was allowed to proceed until the desired color intensity was
reached. For immunohistochemical analysis of AFL4-treated mice, tissues
were incubated with a biotinylated anti-PECAM-1 antibody (1/100) as a
primary antibody, and HRP-conjugated streptavidin (Zymed) (1/1000) was
used as a developing reagent.
The densities of PECAM-1+ and VEGFR-3+ vessels
were calculated according to the method described by Gasparini and
Harris.30 A minimum of 5 fields (×200) was counted
per slide.
Whole-mount immunostaining was performed according to the protocol
previously described.31 In some experiments, stained whole-mount specimens were embedded in polyester wax (BDH, Poole, UK)
and sectioned.
Scanning electron microscopy (SEM)
Tumor masses with surrounding tissues were dissected carefully and
fixed with 3% glutaraldehyde in 0.1 mol/L phosphate buffer (pH 7.4).
The method of SEM observation was as previously reported.32 In brief, the specimens were postfixed with OsO4 and were
hydrolyzed with 8N HCl for 25-60 minutes at 60°C. After
a brief rinse, the specimens were dehydrated through a graded series of
ethanol, immersed in isoamyl acetate, critical-point dried, coated with
platinum, and observed with an SEM (Hitachi S-800, Tokyo, Japan).
Western blot analysis
Western blot analysis was performed as described.29 The
filter was probed with 2 µg/mL anti-VEGFR-3 mAb or a control mAb, followed by treatment with HRP-coupled goat anti-rat IgG, and visualized using the enhanced chemiluminescence reagent (NEN Life Science Products).
Phosphorylation assay
F2 cells were grown to subconfluence in DMEM supplemented with 10%
FCS. The medium was removed and replaced with fresh DMEM containing
10% FCS with or without antibodies (AFL4, rat IgG fraction 50 µg/mL)
for 15 minutes; this was followed by stimulation with one-fifth diluted
VEGF-C CM. Fifteen minutes after VEGF-C stimulation, the cells were
lysed in lysis buffer (10 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% NP-40, 0.2 mmol/L PMSF, 0.5 mmol/L
sodium vanadate, 2 mmol/L sodium fluoride). VEGFR-3 was
immunoprecipitated from cell lysates by protein G-Sepharose after
incubation with the anti-VEGFR-3 mAb. Proteins were resolved with
sodium dodecyl sulfate-polyacrylamide gel electrophoresis under
nonreducing conditions and were probed with HRP-conjugated anti-phosphotyrosine mAb (PY20; Transduction Laboratories, Lexington, KY). Filters were reprobed with anti-VEGFR-3 mAb to measure the amount
of protein loaded on each lane.
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Results |
Production of an antagonistic anti-mouse VEGFR-3 mAb
After screening more than 500 clones, 1 clone, AFL4 (IgG2ak), was
isolated as reacting specifically to VEGFR-3-Fc but not to VEGFR-1-Fc
or VEGFR-2-Fc (Figure 1A). Two polypeptides
were precipitated and immunodetected with AFL4 in cell lysates of
VEGFR-3 gene-transfected 293 cells but not from mock-transfected cells (Figure 1B; lanes 1, 2). Through immunoblot analysis of total extracts
of the F-2 endothelial cell line,27 2 bands of 195- and
125-kd were detected with AFL4 (Figure 1B; lane 3) under nonreducing condition, whereas only the 125-kd band was observed under reducing conditions (Figure 1B; lane 4). These results are consistent with the
previous observation that a 175-kd precursor of VEGFR-3 matures to a
195-kd form, which is then proteolytically cleaved into the 125-kd and
75-kd fragments,33 each linked by disulfide bonds.
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| Fig 1.
Specificity of AFL4. (A) AFL-4 binding to VEGFR-1-Fc,
VEGFR-2-Fc, or VEGFR-3-Fc was analyzed by ELISA. The absorbance at 450 nm is depicted. Each bar represents the mean ± SEM of triplicate
assays. (B) Cell lysates from the 293 cell line
transiently transfected with murine VEGFR-3 DNA (lane 2) or mock
transfected (lane 1, M) were precipitated with AFL4 and immunoblotted
with the same antibody. (IP, immunoprecipitation.) Two bands of 195- and 125-kd proteins were detected in lane 2 under reducing conditions.
Through immunoblotting of the cell lysates from the murine EC line F2
with anti-VEGFR-3 mAb, 2 bands were detected in nonreducing conditions
(NR), whereas only a 125-kd band was seen under reducing conditions (R)
(lanes 3, 4). Arrows denote the positions of the unprocessed 195-kd
form and the proteolytically processed 125-kd form of VEGFR-3. (C)
Specificity of AFL4 in tissue sections. Sagittal sections were prepared
from embryonic day 9.5 VEGFR-3 / or
VEGFR-3+/+ embryos and stained with AFL4, anti-PECAM-1 mAb,
and anti-VEGFR-2 mAb. Arrows indicate blood vessels in the mesenchymal
region around the neural tube (NT). Note that all PECAM-1+
cells are also VEGFR-2+ and VEGFR-3+ at this
stage of embryonic development.
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To further evaluate the specificity of AFL4, serial transverse sections
were prepared from E9.5 VEGFR-3/ or
VEGFR-3+/+ embryos and immunostained by AFL4. The AFL4
immunostaining was seen in the vascular endothelial lining of the
wild-type embryo but not in that of the VEGFR-3/
embryo, whereas the expression of VEGFR-2 and PECAM-1 was
detected in both groups (Figure 1C). From these results, we concluded
that AFL4 is specific to VEGFR-3 and can be used for various purposes, including immunoprecipitation, immunoblotting, and immunostaining of
fixed tissues.
We next investigated whether AFL4 blocks the function of VEGFR-3. AFL4
could block the binding of myc-tagged VEGF-C to ELISA plates coated
with VEGFR-3-Fc, whereas anti-VEGFR-2 mAb (AVAS12)29 could
not, indicating that AFL4 recognizes the ligand-binding site of VEGFR-3
(Figure 2A). Although these analyses did
not permit a precise determination of the binding affinity of AFL4 for
VEGFR-3, the IC50 for AFL4 inhibition of VEGF-C binding to
VEGFR-3 was estimated to be 0.5 µg/mL. We also examined whether the
blocking of ligand binding leads to the suppression of receptor
signaling. We stimulated F2 cells by VEGF-C in the presence of AFL4 or
AVAS12. Tyrosine phosphorylation of immunoprecipitated VEGFR-3 was
measured by anti-phosphotyrosine mAb. Compared with the control, AFL-4 treatment resulted in 6- and 3-fold reduction of VEGF-C-induced tyrosine phosphorylation at 125-kd and 195-kd bands, respectively (Figure 2B).
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| Fig 2.
AFL4 blocks VEGFR-3 function by inhibiting VEGF-C
binding.
(A) Inhibition of binding of VEGFR-3 to VEGF-C by AFL4. Culture
supernatant of 293 cells transfected with 5 myc-tagged VEGF-C DNA was
incubated with various doses of AFL4 ( ) or anti-VEGFR-2 mAb ( )
and added to microtiter plates coated with mVEGFR-3-Fc. Binding was
quantified by using the anti-myc mAb as a primary antibody, and
absorbance at 450 nm was determined. Data indicate the
background-corrected mean ± SEM from triplicate wells. (B) Tyrosine
phosphorylation of VEGFR-3 in F2 cells was induced by VEGF-C CM in the
presence of control IgG (lanes 1, 3) or AFL4 (lanes 2, 4). Total cell
lysates were immunoprecipitated with anti-VEGFR-3 mAb and subjected to
serial immunoblotting with anti-phosphotyrosine antibody (upper) and
anti-VEGFR-3 mAb (lower). Arrows and arrowheads denote the positions of
195-kd and 125-kd forms of VEGFR-3. Relative density of bands against
125-kd bands of the control lanes (lane 1, upper and lower panels) were
195-kd/125-kd 3.7/1 (lane 1, upper), 0.9/0.2 (lane 2, upper), 0.78/1
(lane 1, lower), and 0.6/1.1 (lane 2, lower). Reduction ratios after
correction by the amount of protein were 0.32 and 0.18 for 195-kd and
125-kd bands, respectively.
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Lymphatic specific expression of VEGFR-3 protein in adult mouse
Although a previous study18 and Figure 2 demonstrated
VEGFR-3 expression in the vascular EC of early embryos, AFL4 recognized only lymphatic vessels in later life. Figure
3 showed the whole-mount immunostaining of
the mesentery of E17 embryos, in which lymphatic vessels easily could
be distinguished morphologically from vein and artery running in
parallel (Figure 3A-C). In cross-sections, staining could be found only
in the endothelial cells of the lymphatic vessels that did not contain
blood cells in the lumen (Figure 3D).
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| Fig 3.
Lymphatic-specific expression of VEGFR-3 in embryonic day
17 (E17) embryos and adult dermis.
(A) Whole-mount staining of the mesentery of E17 embryos by AFL4. A
fraction of vascular system is stained. (B) Higher magnification of the
marked area in panel A demonstrates that lymphatic vessels with typical
sac-like structure are stained (arrows). (C) PECAM-1 staining of the
same region shows that blood vessels (arrow) and lymphatic vessels
(arrowhead) are stained. (D) A section of the E17 mesentery
illustrating stained endothelial vessel (arrow) and an unstained blood
vessel (arrowhead) (×200). Note that all stained vessels do not
contain hematopoietic cells. (E) A section of an adult skin illustrates
VEGFR-3+ (arrows) and VEGFR-3 vessels
(×100). Higher magnification (inset, ×200) reveals that
VEGFR-3+ vessels (arrows) do not contain hematopoietic
cells, whereas unstained blood vessels do (arrowheads).
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We next examined VEGFR-3 expression in adult cutaneous tissues. Cells
lining the luminal wall of a vascular structure (indicated by arrows)
were immunostained by AFL4 (Figure 3E). In contrast to the lack of
reactivity to blood vessels containing hematopoietic cells (indicated
by arrowheads), all AFL4-reactive vessels did not contain blood cells,
suggesting lymphatic specific expression of VEGFR-3. This staining
pattern corroborates well with previous in situ analysis showing that
VEGFR-3 is specific to ECs lining the lymphatic vessels.18
To determine the role of VEGFR-3 in the maintenance of the adult
lymphatic system, 1 mg AFL4 was injected subcutaneously into 8-week-old
mice on alternating days for up to 3 weeks. During this 3-week period
of continuous AFL4-injection, we could not detect any gross abnormality
in the treated mice compared with the control mice, which were treated
with non-antagonistic anti-VEGFR-2 mAb or PBS (data not shown). Thus,
VEGFR-3 appeared not to be essential for the maintenance of the adult
lymphatic system.
AFL4 suppresses the growth of xenogenic tumors in nude mice
The antagonistic anti-VEGFR-3 mAb enabled us to examine the
involvement of VEGFR-3 in tumor-induced neo-angiogenesis. For this
purpose, we first used the C6 glioblastoma cell line, which grows
aggressively in the nude mouse.33 C6 cell line has been shown to secrete VEGF34 and VEGF-C.35
To determine the effect of AFL4 treatment on the C6 growth, 200, 600, or 1000 µg purified AFL4 was injected on alternating days for 12 days
into mice grafted with 2 × 106 C6 tumor cells. As
controls, 600 µg AVAS12, nonantagonistic anti-VEGFR-2 mAb was
injected in the same manner. The size of tumor was measured from day 5 to day 14 after the tumor transplantation. As shown in Figure
4A and Table 1,
AFL4 treatment inhibited tumor growth at all doses. Because 200 µg
showed less effect than other doses, we decided to use 600 µg for
subsequent experiments.
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| Fig 4.
Anti-VEGFR-3 suppresses the growth of C6 tumor cells
implanted subcutaneously in nude mice.
(A) Protocol 1: days 0 to 12. Alternate-day treatment with PBS (n = 8),
anti-VEGFR-2 (600 µg/dose; n = 4), or anti-VEGFR-3 (200 µg/dose,
600 µg/dose, 1000 µg/dose; n = 4 each). Tumor size at day 14 was
summarized in Table 1. (B) Protocol 2: days 0 to 6. Injection of
anti-VEGFR-3 treatment (600 µg/dose, closed circle; n = 4), compared
with continuous injection (days 0-12) (open circle; n = 4). (C)
Protocol 3: days 7 to 13. Anti-VEGFR-3 treatment (600 µg/dose, closed
circle; n = 4) and PBS treatment (open circle; n = 4). The growth
curves of tumors in PBS-treated mice were used as reference points
for each figure.
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To determine the timing of VEGFR-3 involvement in tumor progression, 3 protocols for antibody injection were tested: (1) day 0 to day 12, (2)
day 0 to day 6, and (3) day 7 to day 13. The continuous injection of
AFL4 suppressed tumor growth by approximately 75%. Because the
discontinuation of treatment on day 7 (protocol 2) resulted in the
prompt recovery of tumor growth, it is likely that VEGFR-3 is
continuously required for tumor growth (Figure 4B). We also attempted
to determine whether growth was suppressed if AFL4 injection was
commenced from day 7 (protocol 3, Figure 4C). Although we did observe a
reduction of tumor size, the effect of this protocol was not
statistically significant (P = .06).
To examine whether AFL4 treatment suppresses the growth of other tumor
types, a human prostatic cancer cell line, PC-3 was used. PC-3 cells
were reported to secrete VEGF-C.20 PC-3 cells grew more
slowly than C6 cells in the subcutaneous region of the nude mice. At
day14, PC-3 tumors reached an average size of 291 ± 90.1 mm3 in control mice (Table 1), whereas we could not detect
PC-3 tumor mass at day 14 in AFL4-treated mice (Table 1).
Induction of VEGFR-3 expression during tumor-induced angiogenesis
Such a dramatic suppression of tumor growth by AFL4 treatment
implicates the role of VEGFR-3 in angiogenesis rather than in the
formation of lymphatic vessels, though VEGFR-3 is not expressed in the
blood vessels of normal tissues. Thus, we hypothesized that VEGFR-3
expression might be induced by tumor transplantation in the surrounding
tissue. To test this possibility, we investigated VEGFR-3 expression in
the tumor-bearing tissues. Sections of tumor and surrounding tissues
were immunostained with AFL4 or anti-PECAM-1 mAb. VEGFR-3 expression
was detected in intratumoral vessels (indicated by arrows) (Figure
5A). Unlike normal tissues (Figure 3E),
VEGFR-3+ vessels in this section contained blood cells,
indicating that VEGFR-3 expression was induced in the tumor blood
vessels. A similar staining pattern was seen for PECAM-1 staining of
serial sections, demonstrating EC-specific expression of VEGFR-3
(Figure 5B). It should be noted, however, that not all EC in the tumor
vessels expressed VEGFR-3. Intratumoral VEGFR-3+ vessel
density, including lymphatics, was 30 ± 1.2 per high-power field,
whereas that of normal skin tissue was 7.4 ± 0.3 per high-power field. Because the intratumoral PECAM-1+ vessel density was
92 ± 2.8, approximately 30% of intratumoral vessels become
activated to express VEGFR-3+, and because
PECAM-1+ vessel density in the normal skin was 38 ±
1.3, the intratumor region was indeed rich in blood vessels. VEGFR-3
staining was also induced in vessels surrounding the tumor (Figure 5C).
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| Fig 5.
Induction of VEGFR-3 expression during tumor-induced
angiogenesis.
Immunostaining for VEGFR-3 (A) and PECAM-1 (B) in adjacent sections of
C6 subcutaneous tumors (day 7) in nude mice (×200). Intratumoral
VEGFR-3+ (A, arrows) containing blood cells are also
positive for PECAM-1 expression (B, arrows). Note that hemorrhages are
not conspicuous at this stage. (C) Immunostaining for VEGFR-3 in a
section of C6 tumors with surrounding tissues (×100). Note the
presence of VEGFR-3+ (black arrows) and VEGFR-3
(white arrows) vessels, both containing hematopoietic cells.
Arrowheads indicate VEGFR-3+ vessels that do not contain
hematopoietic cells. Asterisk indicates the edge of a tumor.
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Histologic basis for tumor suppression in AFL4-treated mice
To gain insight into the VEGFR-3-dependent cellular processes
during tumor-induced angiogenesis, we compared the vascular system
surrounding tumors of AFL4-treated and control mice. In the control
mouse, the size of the vascular trunk supplying branches to tumors was
larger than the corresponding trunk in the tumor-free side of skin,
suggesting an increase of overall blood flow in the tumor-bearing side
(Figure 6A). As expected from the reduced tumor size in AFL4-treated mice, the vascular trunk governing tumor
blood supply was smaller than that of the control mice (Figure 6B). In
control mice, several branches of similar size stemmed from this trunk,
which further divided into smaller branches (Figure 6A,C). In contrast,
though the primary branches were detectable in the AFL4-treated mice,
their sizes were variable, and they did not develop the fan-like
architecture found in the control tumor (Figure 6B,D). Secondary and
tertiary branches appeared to be very thin. Many micro-hemorrhages were
found along the small branches (Figure 6B,D). Although massivebleeding
was frequently found in the necrotic regions of tumors in the control
mice, micro-hemorrhages were rare. This macroscopic observation was
confirmed by microscopic analysis. Control tumor sections stained with
hematoxylin-eosin showed enlarged vessels surrounding tumors (data not
shown), whereas overall vascularity around the tumors was lower in the
AFL4-treated group (data not shown). Moreover, the number of
PECAM-1+ EC within the tumor mass was reduced to 40% in
the control group (Figure 7A,B,E).
Conversely, 4 times more micro-hemorrhagic regions were detected in the
AFL4-treated group (Figure 7C,D,F).
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| Fig 6.
AFL4 treatment inhibits tumor angiogenesis.
Tumor-bearing regions were photographed on day 14 after tumor
transplantation. Gross appearance of representative vascularization of
control (A) and AFL4-treated (B) mice. Arrows and arrowheads indicate
the vascular trunks governing tumor blood supply and those of the
tumor-free side, respectively. Note that the size of the trunk is
larger on the tumor-bearing side than on the other side. Such a
dilatation is not clearly seen in the AFL4-treated mouse. (C, D) Higher
magnification than that in A and B, respectively. Secondary and
tertiary branches are poorly developed in the AFL4-treated mouse. Note
the many micro-hemorrhages in this AFL4-treated tumor (arrows).
Asterisks indicate tumors.
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| Fig 7.
Histology of intratumor vasculatures.
(A, B) Tumor vasculatures are visualized by anti-PECAM-1
immunostaining. Micrographs are of representative sections prepared
from the control (A) and AFL4-treated mice (B) (×200). (C, D)
Representative H&E-stained tumor sections from controls (C) and
anti-VEGFR-3-treated mice (D) (×200). In anti-VEGFR-3-treated
tumors, many micro-hemorrhages are observed (arrows). (E) Vessel counts
per field were determined from at least 5 different vision fields of
sections from control (n = 4; 65 ± 5.2/high-power field) and
AFL4-treated mice (n = 4; 27.5 ± 5.3/high-power field)
(×200). Data are plotted as mean ± SEM. *P < .01. (F) The number of micro-hemorrhages was scored in high-power fields
(×200) of H&E-stained tumor sections (at least 5 different vision
fields each of 4 tumors). Control, 0.73 ± 0.13/high-power field;
AFL4, 3.9 ± 0.46/high-power field. Data are plotted as mean ± SEM. *P < .01. Statistical differences between groups were
computed using the Student t test. (G) Representative SEM of a
postcapillary venule in control tumors (×3800). (H) SEM of a
postcapillary venule in anti-VEGFR-3-treated tumors reveals disruption
of the endothelial sheet, exposing red blood cells (arrow)
(×2200).
|
|
To determine the morphologic basis of the micro-hemorrhage in the
AFL4-treated tissues, the vessels connecting to the tumor were analyzed
systemically by SEM. We could not detect any abnormalities in the
morphology of the proximal vessels of AFL4-treated mice, suggesting
that these vessels developed normally (data not shown). However, at the
level of postcapillary venules, disruption of the endothelial lining
was observed frequently in AFL4-treated mice (Figure 7G,H).
Erythrocytes could be seen through the cleft. The formation of such
clefts was barely detectable in the postcapillary venule surrounding
the tumors in control mice.
| |
Discussion |
In this study we established an antagonistic mAb to VEGFR-3 (AFL4)
and used AFL4 to evaluate the role of this RTK in tumor angiogenesis.
Although VEGFR-3 is not expressed in the vascular EC of adult mice, our
result showed that VEGFR-3 expression is induced in the vascular EC
upon the implantation of tumor cells. Furthermore, the growth of C6
glioma cells and PC-3 prostate carcinoma cells was suppressed by the
injection of AFL4, presumably because of the inhibition of the
establishment of the vascular architecture in tumor-bearing tissues.
Taking the previous study on VEGFR-3/
embryos26 into account, besides embryonic angiogenesis,
VEGFR-3 is involved in the neo-angiogenesis of adult tissue.
Sustained AFL4 treatment resulted in no gross anomalies in normal mice.
Thus, during the 3 weeks of mAb injection, the architecture of the
lymphatics and the vascular system remained unaffected in the absence
of VEGFR-3 function; however, the effect over a longer period of time
remains to be investigated. Recently, it has been demonstrated that the
prolonged suppression of VEGF activity in the adult mouse has no effect
on the maintenance of the vascular system, though it suppresses
angiogenesis in the newborn mouse.36 The fully established
lymphatic and vascular systems are basically resistant to treatment
with various reagents that suppress neo-angiogenesis. Because of the
neo-angiogenesis-specific effect of these reagents, this approach has
been expected to be chosen for cancer therapy. Our current results
demonstrate clearly that VEGFR-3 is a potentially useful molecule for
targeting in future cancer therapy.
Compared with the phenotype of VEGFR-2/ mice,
in which formation of the primitive vascular plexus is
impaired,37 it has been indicated that VEGFR-3 is involved
at a later stage of vascular development, particularly in the
remodeling of the primitive plexus to a higher-order
architecture.26 The absence of secondary and tertiary
branches in AFL4-treated mice suggests a VEGFR-3 role in the remodeling
of tumor-induced neo-angiogenesis. Angiopoietins/Tie-2 has also been
implicated in the remodeling process of embryonic and tumor-induced
angiogenesis. It is likely that the molecular requirements for vascular
development in the embryo and for tumor-induced neo-angiogenesis are
essentially the same and involve an ordered expression of multiple
tyrosine kinase receptors.
Which process of angiogenesis is affected by the inhibition of VEGFR-3?
Although the role of VEGFR-3 in the remodeling of vascular formation
has been implicated, these reports did not specify the
process beyond the word remodeling.26 This may be, in part, because of an inherent difficulty in studying embryonic angiogenesis in which angiogenesis proceeds asynchronously according to
region-specific timetables. In other words, various intermediate steps
of angiogenesis are mixed within an embryo. In contrast, tumor-induced
angiogenesis is a synchronous process that can be induced in a
relatively homogeneous microenvironment. Moreover, the progression of
angiogenesis during tumor growth has been described in detail. With
these considerations in mind, we attempted to obtain insight into the
histologic basis of the phenotype induced by AFL4-injection. Although
AFL4 treatment appeared to be identical to other anti-angiogenic
reagents in that it inhibited the supply of vascular branches to the
tumor, we demonstrated that micro-hemorrhage, presumably because of the
disruption of the endothelial lining at the postcapillary venule level,
is a characteristic feature of AFL4-treated tissues. It is difficult to
rule out the possibility that this effect is caused by the cytotoxic
reaction of AFL4 to VEGFR-3+ EC, but we prefer to think
that this disruption is derived from the blockage of VEGFR-3 function.
This histologic sign has not been described in previous experiments in
which other RTKs are blocked. Because micro-hemorrhages are too
conspicuous to be overlooked, frequent micro-hemorrhages may be
specific to VEGFR-3 inhibition.
How VEGFR-3-block caused the disruption of endothelial structure is
difficult to specify. According to previous studies, sprouting of EC
occurs only at the levels of capillaries, postcapillary venules, and
precapillary arterioles, where no smooth muscle is present.38 By the repeated sprouting, splitting, and
anastomosis of blood vessels at this level, the overall peripheral
vascular bed in the tumor-bearing tissues increases. This increase in
the vascular bed contributes to the reduction of regional vascular resistance, thereby resulting in the increased blood supply. The change
of blood supply induces restructuring of the more proximal vessels
connecting to tumor, as observed in the current study. Because the
angiopoietins/Tie2 signal was shown to regulate interactions between
ECs and smooth muscle cells,9-11 it is conceivable that this signal is required for vascular remodeling in which the distal blood vessel is restructured to the more proximal form associated with
smooth muscles. In the Tie2-block experiment, however,
micro-hemorrhages in tumor-bearing tissues has not been indicated.
Hence, failure in interaction between ECs and smooth muscle cells may
not lead to disruption of the endothelial structure.
The frequency of micro-hemorrhages found in AFL4 treated-mice suggests
that AFL4 treatment inhibits maintenance of the integrity of the
endothelial sheet during angiogenesis. Although further cell biology
studies are required for an understanding of the underlying mechanisms,
the presence of mural cells at the level of precapillary arterioles and
postcapillary venules of AFL4-treated mice suggests that it may not
result from inhibition of the interaction between EC and smooth muscle
cells. It has been suggested that vascular permeability is increased at
the site of tumor-induced angiogenesis. Thus, it is likely that the
endothelial lining is agitated during neo-angiogenesis. Indeed,
sprouting and pruning would imperil the integrity of the endothelial
lining. Yet, micro-hemorrhage is not a frequent outcome of
neo-angiogenesis, indicating that regulatory mechanisms
maintain the integrity of the endothelial sheet even
during dynamic restructuring of the vascular system. Therefore, we
speculate that in the process of sprouting and pruning, during which
integrity of the EC layer is disturbed, additional signals such as
VEGFR-3 may be required for quick restoration of the EC sheet that
otherwise leads to the formation of irreparable clefts. If such clefts
causing micro-hemorrhage are generated during neo-angiogenesis, the
rheologic resistance of the vascular system should increase, thereby
resulting in the collapse of more proximal vessels as observed in the
AFL4-treated mouse.
| |
Acknowledgments |
We thank Dr W. I. Wood (Genentech) for VEGFR-3 cDNA, Dr K. I. Toda for the F-2 cell line, and Dr N. Itoh for the PC-3 cell line. We also thank Drs H. Kataoka, M. Hirashima, and H. Yoshida for
their helpful advice, and we thank Dr S. Fraser for critical reading of
the manuscript.
| |
Footnotes |
Submitted September 13, 1999; accepted March 6, 2000.
Supported by grants from the Japanese Ministry of Education,
Science and Culture (07CE2005, 07457085, and 06277102), from the
Japanese Ministry of Health and Welfare, and from the Japan Society for
the Promotion of Science Research.
Reprints: Hajime Kubo, Department of
Molecular Genetics, Graduate School of Medicine, Kyoto
University, Shogoin Kawaharacho 53, Sakyo-ku, Kyoto 606-8507, Japan;
e-mail: kuboflt{at}kuhp.kyoto-u.ac.jp.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Top
Abstract
Introduction