in vivo inhibition of tumor angiogenesis by a soluble vegfr-2 fragment
TRANSCRIPT
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Experimental and Molecular Pathology 76 (2004) 129–137
In vivo inhibition of tumor angiogenesis by a soluble VEGFR-2 fragment$
Kou Baijun,* Li Yulin, Zhang Lihong, Zhu Guibin, Wang Xinrui,Li Yilei, Xia Jianxin, and Shi Yingai
Department of Pathology, College of Basic Medicine, Jilin University, Changchun 130021, China
Received 8 October 2003
Abstract
The interaction of vessel endothelial cell growth factor (VEGF) and its receptors (flt-1, FLK-1/KDR) regulates tumor angiogenesis.
Therefore, blocking the binding of VEGF and the corresponding receptor has become critical for antitumor angiogenesis biological therapy.
Our study extracted sFLK-1 fragment from embryo mouse liver using RT-PCR, recombined it to retrovirus vector, and transfected it to tumor
cell lines (S180 and B16) by the liposome mediated method, then we observed the biological behavior of transgenic cells in vivo.
The results are:
(1) Fragment (1034 bp) was extracted from E9, E11 embryo mouse liver tissue, which was identified by sequence analysis.
(2) This fragment was cloned to retrovirus vector (PLXSN vector), which was further transfected to tumor cells lines (S180 and B16). SDS-
PAGE indicated the suspension of transgenic cells present sVEGFR-2(sFLK-1) fragment; Western blot identified it.
(3) In vivo study showed that the weight and size of tumor in the group of transgenic cells were smaller than in control groups. Microvessel
density (MVD) and FLK-1 expression were obviously different between transgenic and control groups, but there were no differences in
VEGF expression between transgenic and control groups.
In short, the isolated soluble VEGFR2 fragment transfected to tumor cells can be secreted to extracellular suspension and can inhibit
tumor angiogenesis in vivo.
D 2004 Elsevier Inc. All rights reserved.
Keywords: Soluble receptor; VEGF; Angiogenesis; Tumor
Introduction Studies showed that VEGF exists its effects by binding with
Neovascularization including vasculogenesis and angio-
genesis is crucial for embryonal development and the main-
tenance of the vertebrate body (Risau, 1997). Abnormal
angiogenesis is involved in many pathological processes
such as diabetes mellitus, ophthalmological diseases, the
growth, and metastasis of tumors (Folkman and D’Amore,
1996). Accumulating evidence strongly suggests that vessel
endothelial cell growth factor (VEGF) and its receptor system
are very important for the regulation of neovascularization as
well as for pathological angiogenesis (Ferrara and Davis-
Smyth, 1997; Mustonen and Alitalo, 1995; Shibuya, 1995).
0014-4800/$ - see front matter D 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.yexmp.2003.10.010
$ VEGFR: vessel endothelial cell growth factor receptor.
* Corresponding author.
E-mail address: [email protected] (B. Kou).
high affinity to two tyrosine kinase receptors VEGFR-1/Flt-1
(de Vries et al., 1992) and VEGFR2 (KDR/Flk-1) (Terman et
al., 1992) present on endothelial cells. Studies in mouse
embryos have given us some understanding of the early
developmental events mediated by VEGF. Deletion of the
VEGF alleles results in abnormal blood vessel development
and midgestational death (Carmeliet et al., 1996), while null
mutation of KDR/Flk-1 results in defects in differentiation of
hemangioblasts to form angioblastic and hematopoietic cell
lineages. After differentiation, KDR/Flk-1 is downregulated
in hematopoietic but not endothelial cells, indicative of an
early role for VEGF via KDR/Flk-1 in differentiation of the
stem cells of fetoplacental capillaries (Shalaby et al., 1995).
The other receptor of VEGFs, VEGFR-1/Flt-1, appears to
have a later role, as mouse embryos lacking Flt-1 develop
angioblasts but blood vessel assembly and tube formation is
impaired (Fong et al., 1995).
Fig. 2. The product coming from E9, E11 embryo mouse liver tissues by
RT-PCR is composed of the secretory leader sequence and the N-terminal 3
extracellular immunoglobulin-like domains.
B. Kou et al. / Experimental and Molecular Pathology 76 (2004) 129–137130
VEGF together with its receptors has been shown to play a
significant role in tumor-induced neovascularization (de
Vries et al., 1992; Shalaby et al., 1995; Shibuya, 1995).
Evidence also exists to suggest that inhibition of tumor-
associated angiogenesis can retard tumor growth, prevent
spread, and even cause tumor regression (Bicknell andHarris,
1992; Folkman and Ingber, 1992; Scott and Harris, 1994).
Disruption of the VEGF pathway has been shown in exper-
imental models of tumorigenesis to inhibit angiogenesis and,
consequently, tumor growth. This has been done in a variety
of ways (Kendall and Thomas, 1993; Kim et al., 1993;
Millauer et al., 1996; Strawn et al., 1996), including the
administration of a soluble, truncated receptor that inhibits
VEGF action by a ‘‘dominant-negative’’ mechanism (Lin et
al., 1998).
An antitumor strategy in which the endothelial cells
supporting tumor growth are targeted is appealing because
the endothelial cells are, themselves, normal cells with a low
intrinsic mutation rate and therefore unlikely to acquire a
drug-resistant phenotype. In addition, it is likely that most, if
not all, types of cancer are angiogenesis dependent, provid-
ing a common target for widely heterogeneous tumor types,
thus giving angiogenesis inhibitors broad applicability as
antitumor agents. Millauer et al. reported they transfected
mutant FLK-1 gene to vessel endothelium cell of host tumor
mice by retrovirus vector, and formated heterodimers with
wild FLK-1 gene; the result showed that the neurovascula-
rization was significantly low in the tumor tissue, and the
tumor growth was inhibited by 80–90% (Millauer et al.,
1993, 1994). Then, Lin et al constructed a soluble VEGF
receptor by fusing the entire extracellular domain of murine
flk-1 to a six-histidine tag at the COOH terminus (ExFlk.6
His). In vitro, recombinant ExFlk.6 His protein bound VEGF
with high affinity blocked receptor activation in a dose-
dependent manner and inhibited VEGF-induced endothelial
cell proliferation and migration. ExFlk.6 His bound to
Fig. 1. Soluble VEGFR (sFLK-1) gene fragment was taken from E9, E11
embryo mouse liver tissues by RT-PCR. Lane 1: 1034 bp RT-PCR product,
lane 3: lambda DNA/E co911 marker.
endothelial cells only in the presence of VEGF, and cell
surface cross-linking yielded a high molecular weight com-
plex consistent with the VEGF-mediated formation of a
heterodimer between ExFlk.6 His and the endogenous
VEGF receptor. In vivo, ExFlk.6 His potently inhibited
corneal neovascularization induced by conditioned media
from a rat mammary carcinoma cell line (R3230AC). More-
over, when ExFlk.6 His protein was administered into a
cutaneous tumor window chamber concomitantly with
R3230AC carcinoma transplants, tumor growth was in-
hibited by 75%, and vascular density was reduced by 50%
(Lin et al., 1998). Goldman et al transfected tumor cells with
cDNA encoding the native soluble FLT-1 (sFLT-1) truncated
VEGF receptor that can function both by sequestering VEGF
and, in a dominant-negative fashion, by forming inactive
heterodimers with membrane-spanning VEGF receptors.
Transient transfection of HT-1080 human fibrosarcoma cells
with a gene encoding sFLT-1 significantly inhibited their
implantation and growth in the lungs of nude mice following
intravenous. Injection and their growth as nodules from cells
injected subcutaneous. High sFLT-1 expressing stably trans-
Fig. 3. The construction picture of recombinant vector sFLK-1 fragment-
pMD-18T.
Fig. 6. PL(sFLK1 fragment)SN vector was identified with EcoRI and
HindIII enzyme cutting. Lane 1: lambda DNA/Eco911 marker, lane 2:
pLXSN-sFLK1 fragment digested with EcoRI and HindIII, lanes 3 and 4:
pLXSN-sFLK1 fragment.
Fig. 4. sFLK1 fragment-pMD-18T was identified with EcoRI and HindIII
enzyme cutting. Lane 1: lambda DNA/EcoRI + HindIII marker. Lane 2:
pMD-18T-sFLK1 fragment digested with EcoRI and HindIII. Lanes 3 and
4: pMD-18T-sFLK1 fragment.
B. Kou et al. / Experimental and Molecular Pathology 76 (2004) 129–137 131
fected HT-1080 clones grew even slower as subcutaneous
tumors. Finally, survival was significantly prolonged in mice
injected intracranially with human glioblastoma cells stably
transfected with the sflt-1 gene. The ability of sFLT-1 protein
to inhibit tumor growth is presumably attributable to its
paracrine inhibition of tumor angiogenesis in vivo, since it
did not affect tumor cell mitogenesis in vitro (Goldman et al.,
1998). Afterwards, Machein et al. co-removed mouse glioma
cell and retrovirus vector with mutant VEGFR2 to nude
mouse, which specially inhibited the signal conduction of
endothelial cell expressing VEGFR-2 and induced the dele-
tion of signal conduction of cell migration and proliferation
Fig. 5. The construction picture of recombinant vector sFLK-1 fragment-
pLXSN.
between VEGF/VEGFR so that it inhibited glioma angio-
genesis and tumor growth, metastasis. They further explored
the safety of retrovirus-mediated gene transfer. Although
virus sequences were found in different tissues after intra-
cerebral injection of virus-producing cells, no morphological
changes were observed in any tissue after a follow-up time of
6 months (Machein et al., 1999). Recently, a small molecule
tyrosine kinase receptor inhibitor, SU5416, has been applied
in clinical experiment. Study showed that it can reduce
chemical drug-resistance of experimental animal tumor
model by effectively inhibiting signal conduction (Geng et
al., 2001). Another report indicated that it has biologic
activity in patients with refractory acute myeloid leukemia
or myelodysplastic syndromes (Giles et al., 2003). All these
Fig. 7. RT-PCR product of transgenic cells. Lane 1: B�174DNA/BsuRI
marker, lane 2: RT-PCR product.
Table 1
The growth status of three groups of S180 transplanted tumors
Group Number Weight Size (d)
S180 control 10 2.82 F 0.94 2.59 F 0.43
S180-vector 9 2.69 F 0.68 2.71 F 0.33
S180-sFLK1 fragment 10 0.51 F 0.37 0.63 F 0.32
Fig. 8. SDS-PAGE results of transgenic B16 and S180 cell lines’ suspension.
Lane 1: protein marker, lane 2: B16-LN cell suspension, lane 3: B16-sFLK1
fragment cell suspension, lane 4: mixture cell suspension, lane 5: S180-
sFLK1 fragment cell suspension, lane 6: S180-LN cell suspension.
B. Kou et al. / Experimental and Molecular Pathology 76 (2004) 129–137132
events indicated that soluble VEGFR has a considerable
prospect as an antiangiogenesis-dependent tumor antagonist.
In these study, we extracted sFLK-1 fragment from
embryo mouse liver using RT-PCR, recombined it to
retrovirus vector and then transfected it to tumor cells lines
(S180 and B16) through liposome mediated method, and
observed the biological behavior of the transgenic tumor
cells in vivo.
Materials and methods
Isolation of sVEGFR2 (sFLK-1) fragment from embryo mice
liver
According to the cDNA sequence of mouse soluble
VEGFR-2, we designed a couple of primers including EcoRI
and HindIII. The upstream sequence: 5VGAC GAA TTC
ATG GAG AGC AAG GCG CTG CTA 3V; the downstreamsequence: 5VCCA CCA AAG ATT TCATCC CAC TAC CG
3V. Total RNA was extracted from E9, E11 embryo mice
liver using RNeasy total RNA system (Promega). The first
strand of cDNA was synthesized using 1 Ag of total RNA
with random hexanucleotide primers (Promega). PCR am-
plification of the cDNA fragments with the above pair of
primers. Cycling times and temperatures were as follows:
denaturation at 95jC for 5 min and 94jC for 45 s, annealing
at 44jC for 1 min, and elongation at 72jC for 4 min 3
recycles; then denaturation at 94jC for 45 s, annealing at
55jC for 1 min, and elongation at 72jC for 2 min 30 recycles
Fig. 9. Western blot evaluation results of expression product.
and 72jC for 10 min. The reaction product was electro-
phoresed through 1.2% agarose gel.
Retroviral vector construction
The reaction product was recovered from agarose gel and
ligated into TA clone vector(pMD-18T) (Takara) to become
sFLK-1 fragment-pMD-18T, which was transformed to E.
coli DH5a and cultured further. The plasmid was extracted
from E. coli DH5a. The recombinant vector was cut with
EcoRI and HindIII and was ligated to retroviral vector
(pLXSN) (Clontech), which has been cut with EcoRI and
HindIII before. Then, recombinant sFLK-1 fragment-pLXSN
was cut with the same enzyme and was electrophoresed with
1.2% agarose gel.
Gene transfection to tumor cell lines
The murine fibrosarcoma cells line S180 and the murine
melanoma cells line B16 were stored by our laboratory,
which were maintained in RPMI1640 medium supple-
mented with 10% bovine serum (GIBCO).
S180 and B16 cell lines were trypsinized and resuspended
at 1 � 105 cells/ml, from which 100 Al were added to each
well of 96-well plate and incubated for overnight. G418
(GIBCO) with different dilutions was added to 96-well plate
incubating for 10 days. The lowest dilution of all cells death
were B16: 600 Ag/ml, S180: 800 Ag/ml.
S180 and B16 (1 � 105) cell lines with complete medium
were added to 6-well plate, respectively, overnight, which
were replaced by serum-free medium with 20 Al liposome
(GIBCO), 20 Al (10 Ag) DNA the next day for 24 h, then
incubated for an additional 48 h, then the cells were cultured
with conditioned medium (with G418 B16: 600 Ag/ml, S180:
800 Ag/ml) for screening. After 3–5 days, the cells were
harvested with conditioned medium of 200–300 Ag/ml
G418. After 2 weeks, the positive clones come into being.
These cells were identified with RT-PCR and agarose gel
electrophoresis after expanded culture.
Table 2
The growth status of three groups of B16 transplanted tumors
Group Number Weight Size (d) Lung metastatic
rate
B16 control 8 1.17 F 0.68 1.04 F 0.42 87.5% (7/8)
B16-vector 7 1.21 F 0.73 1.32 F 0.49 100% (7/7)
B16-sFLK1
fragment
10 0.09 F 0.05 0.06 F 0.05 0
Note. Student’s t test and v2 test.
p of transgenic cells (left) were smaller than that of control group (right).
B. Kou et al. / Experimental and Molecular Pathology 76 (2004) 129–137 133
SDS-PAGE for identification of gene expression
pLXSN is a retroviral expression vector that contains virus
promoter near 5VLTR to regulate gene expression. S180-
sFLK-1 cells, B16-sFLK-1 cells, and control cells were
grown routinely with 10% serum medium to confluence,
then they were changed to serum-free medium for 24 h, from
which suspensions were collected. Samples were added in
12% SDS-PAGE gel for electrophoresis overnight at 80 V.
After the gel was stained with Coomassie blue R250. Mean-
while, the same samples were added in 12% SDS-PAGE gel
for electrophoresis 2 h at 200 V, then the protein were
transferred onto PVDF membranes for 2 h and blocked
overnight. Detection was performed by incubating the blot
with polyclonal VEGFR2 antibody for 2 h, and subsequently
with the second antibody for another 2 h, DAB was used to
stain it.
Animal tumor models
Specific pathogen-free, age-matched BABL/C and
C57BL/J6 mice were obtained from Medical Animal De-
partment of Jilin University. For generation of murine
subcutaneous tumors, 2 � 106 cells/200 Al of S180-sFLK-1, S180-vector, and S180-control cells were injected sub-
cutaneously to BABL/C mice right flank, respectively. B16-
sFLK-1, B16-vector, and B16-control cells (1 � 107 cells/50
Al) were injected subcutaneously to C57BL/6J mice claw of
right foot, respectively. All the mice were divided into 6
groups: S180-sFLK-1, S180-vector, S180-control, B16-
Fig. 10. After 3 weeks of inoculation, the sizes of tumors in the grou
Fig. 11. After 3 weeks of inoculation, there were three mice occurring obvious blac
tumors were obvious in the group of control cells. The tumors’ sizes in B16-sFLK-1
group.
sFLK-1, B16-vector, B16-control. There were 10 mice in
each group. Tumors were measured twice weekly. The
BABL/c mice were killed and the C57BL/6J mice were
amputated after 3 weeks of injection. The tumors were taken
out and weighed, the diameters were read.
Immunohistochemistry
All the samples were fixed quickly in 4% paraformal-
dehyde for overnight, embedded in paraffin. Sections cut
in 5-Am thick were stained with H.E and immunohisto-
chemical staining with CD34, VEGF, FLK1 as the first
antibody. Anti-CD34 monoclonal antibody, anti-VEGF
polyclonal antibody, and anti-VEGFR2(FLK-1) polyclonal
antibody were purchased in Maixin, China. All the sec-
tions were stained by using a biotin–streptavidin peroxi-
dase system (DAKO), followed by a hematoxylin counter-
stain. Microvessel density (MVD) were manually counted
in four high-powered (200�) fields per slide stained with
anti-CD34 monoclonal antibody. Photo collection of sec-
tions stained with VEGF, and VEGFR2(FLK-1) was car-
ried out using IX70 OLYMPUS microscopy with cold
CCD camera, and photos were analyzed using Image-Pro
Plus analysis software provided by COLD SPRING
HARBER laboratory.
Statistical analysis
For experiments that used the subcutaneous tumor mod-
els, tumor volumes, weight, and MVD were expressed as
k line without mass in B16-sFLK-1 fragment transgenic cells group, but the
fragment transgenic cells group were obviously smaller than that of control
Table 3
The immunohistochemistry results of six groups tissue slides
Group Number MVD (/200�) VEGF FLK1
(A) S180-control 10 165.4 F 36.9 12.5 F 2.2 8.0 F 1.0
(B) S180-vector 9 121.8 F 29.8 9.9 F 3.2 6.5 F 2.1
B. Kou et al. / Experimental and Molecular Pathology 76 (2004) 129–137134
mean F SEM and were compared at the end of the experi-
ments using Student’s t test and v2 test. For that of VEGF and
VEGFR2 (FLK-1), expression were expressed as mean FSEM and were compared at the end of the experiments using
Student’s t test.
(C) S180-sFLK1fragment
10 56.9 F 9.4 10.4 F 1.8 13.9 F 2.1
(D) B16-control 8 8.4 F 2.1 10.2 F 1.6 5.7 F 0.7
(E) B16-vector 7 8.6 F 1.7 9.4 F 1.7 6.1 F 1.3
(F) B16-sFLK1
fragment
5 1.4 F 0.6 9.0 F 1.9 11.2 F 1.9
Results
Identification and structure of sVEGFR2 (sFLK-1) fragment
We isolated a fragment from E9, E11 embryo mouse liver
tissue by RT-PCR, and analyzed it with 1.2% agarose gel
electrophoresis. The size of the fragment is between 702 and
1264 bp compared with lambda DNA/E co911 marker (Fig.
1). The murine sFLK-1 mRNA contains the entire 2.3 kb,
including the secretory leader sequence and extracellular
immunoglobulin-like domain (Matthews et al., 1991).
According to the primers we designed, it should be a
1034 bp size fragment of murine FLK-1 cDNA extracellular
domains. The product is composed of the secretory leader
sequence and the N-terminal 3 extracellular immunoglobu-
lin-like domains. The receptor is missing the membrane-
proximal 4–7th immunoglobulin-like domain, the trans-
membrane-spanning sequence, and the kinase domains.
Thus, it is a soluble form of FLK (Fig. 2). Then, we
approved it with sequence analysis.
Restriction mapping of recombinant vector
Because the product of RT-PCR is A-viscosity terminal,
and TA clone vector is T-viscosity terminal, we cloned the
RT-PCR product to TA clone vector (pMD-18T) and made
a recombinant vector sFLK-1 fragment-pMD-18T(Fig. 3).
The size is 3.7 kb. Then, the recombinant vector was cut
by EcoRI and HindIII (Fig. 4). Meanwhile, we cut retro-
viral vector (pLXSN) by EcoRI and HindIII and ligated to
the linear sFLK-1 fragment with EcoRI and HindIII termi-
nal (Fig. 5). After transforming to and extracting from E.
coli DH5a, the recombinant vector PL (sFLK1 fragment)
SN vector was cut by EcoRI and HindIII for identification
(Fig. 6).
Fig. 12. H.E staining of transgenic cells tumor tissue. The left is S180-trans
(400�).
Identification of transgenic cells and expression
After stable transfection, the cells lines of S180 and B16
were expand cultured, respectively. Cells (2 � 107) were
taken to extract the total RNA and RT-PCR, respectively. The
method is the same as before. The product was electro-
phoresed through 1.2% agarose gel and compared with
B�174DNA/BsuRI marker, the size is between 872 and
1087 bp (Fig. 7). Thus, it approved that the transfection
was a success.
Because retroviral vector (pLXSN) is an expression,
vector and the secretory leader sequence was contained in
the fragment sequence. When the fragment gene has been
transfected to cells, it can be secreted to extracellular
suspension. Thus, we collected the cells’ suspension and
analyzed it with SDS-PAGE (Fig. 8). The positive bands
were between 31.0 and 43.0 kDa. The molecular weight is
in accordant with the base pair. Western blot with polyclonal
VEGFR2 antibody identified it (Fig. 9).
Histopathological observation of tumor models
The weight and size of tumors in the group of trans-
genic cells were smaller than that of control group. After 3
weeks, there were three mice occurring obvious black line
without mass in B16-sFLK1 fragment transgenic cell
group, but the tumors were obvious in the group of control
cells. The tumors’ weights and sizes of B16-sFLK1
fragment transgenic cells group were obviously smaller
than that of control group. The ratio of lung metastasis was
Note. Student’s t test.
genic cells tumor tissue, the right is B16-transgenic cells tumor tissue
Fig. 13. The immunohistochemical staining of CD34 in S180-sFLK-1 fragment (left) and B16-sFLK-1 fragment (right) transgenic mice (400�).
B. Kou et al. / Experimental and Molecular Pathology 76 (2004) 129–137 135
decreased in the group of transgenic cells than that of
control group (Tables 1 and 2).
Theweight and size in the groups of S180-sFLK1 fragment
transgenic cells and B16-sFLK1 fragment transgenic cells
were significantly lower and smaller than that of the control
groups (P < 0.05). The lung metastatic rate in the group of
B16-sFLK1 fragment transgenic cells was lower than that of
the control groups (P < 0.01) (Figs. 10–12 and Table 3).
Microvessel density (MVD) and VEGF, VEGFR2(FLK-1)
expression analysis
After counting microvessel density (MVD), we found
that MVD in transgenic cells groups were lower than in
control groups. There were significant difference between
transgenic cells groups and control groups ( pAC =0.0106,
pBC = 0.0443; pDF = 0.0268, pEF = 0.0064).
Meanwhile, FLK-1 expression in the transgenic cells
groups was obviously higher than in control groups ( pAC =
0.0207, pBC = 0.0237; pDF = 0.0084, pEF = 0.0441).
Because the binding side of polyclonal antibody located
on the extracellular domain of cells and the protein expres-
sion of FLK1 included the entire FLK1 and soluble FLK1
fragment, the FLK1 protein expression in the groups of
transgenic were higher than that of control. This indicated
that the sFLK1 fragment peptide has secreted to the
extracellular matrix and have a possibility to combine to
VEGF to play a role, but there was no obvious difference of
VEGF expression between transgenic cells groups and the
control groups (P > 0.05) (Figs. 13–15).
Fig. 14. The immunohistochemical staining of VEGF in S180-sFLK-1 fragment (l
tumor cells’ cytoplasm (400�).
Discussion
Angiogenesis is a predetermined factor to tumor growth
and metastasis. Antiangiogenesis treatment is based on the
theory that solid tumor’s growth and metastasis depend on
angiogenesis (Folkman, 1972). Therefore, the treatment
mechanism is restricting neovascularization through block-
ing the integration of vascular growth factor and vascular
endothelial cell in tumor tissues, which induce tumor tissues
of continual growth to be broad necrosis for ischemia and
inhibit tumor’s growth and metastasis.
One of the key molecules promoting angiogenesis is the
endothelial cell-specific mitogen, vascular endothelial cell
growth factor (VEGF), which acts as a marker of tumor’s
metabolism and metastasis. Its biological activity is mediated
by two receptor tyrosine kinases, VEGFR-1 (Flt-1) and
VEGFR-2 (KDR/Flk-1). Study showed (de Vries et al.,
1992; Dougher-Vermazen et al., 1994; Terman et al., 1991)
that the interaction of VEGF and its receptor flt-1, KDR/Flk-1
is very important to regulate tumor angiogenesis. Tumor cells
secrete VEGF, which reacts to the corresponding receptor on
endothelial cells to induce tumor proliferation, angiogenesis,
and metastasis. VEGFR-2 (KDR/Flk-1), which forms a high-
affinity complexes with VEGF, only exists in vascular
endothelial cell and some of tumor cells. Its expression level
is so low that it is difficult to detect it in normal tissue. Many
tumor cells express and secrete VEGF, which combines to the
high-affinity receptor that existed on endothelial cells and
some of tumor cells and induces endothelial cell proliferation,
neovascularization, tumor growth through autocrine and
eft) and B16-sFLK-1 fragment (right) transgenic mice. VEGF expressed on
Fig. 15. The immunohistochemical staining of FLK-1 in S180-sFLK-1 fragment (left) and B16-sFLK-1 fragment (right) transgenic mice (400�).
B. Kou et al. / Experimental and Molecular Pathology 76 (2004) 129–137136
paracrine function. Study showed (Boocock et al., 1995;
Brown et al., 1995; Kolch et al., 1995; Yoshiji et al., 1996)
that the high KDR/Flk-1 expression of tumor vessel endo-
thelial cell was related to many of tumors’ angiogenesis,
proliferation, and metastasis. Therefore, blocking the binding
of VEGF and the corresponding receptor is important and
most direct target to antitumor angiogenesis therapy.
VEGFR-2 (KDR/Flk-1) is a membrane-spanning recep-
tors, which is composed of seven extracellular immunoglob-
ulin-like (Ig-like) domains, a transmembrane region, and an
intracellular region with the tyrosine kinase domain (Strawn
et al., 1996). Soluble VEGFR-2 (sKDR/Flk-1) is a series of
different size fragment of extracellular immunoglobulin-like
(Ig-like) domains. This kind of soluble form retains its high-
affinity binding to VEGF, but cannot work to the receptor
tyrosine transphosphorylation and activation of downstream
signal transduction to induce endothelial proliferation for the
lack of the tyrosine kinase domain. Study has approved
(Millauer et al., 1996) that Soluble VEGFR-2 (sKDR/Flk-1)
forms a kind of heterodimers with wild VEGFR-2 or
dominant-negative to block the activity of entire VEGFR-2
by competitive suppression principle. Thereby, it inhibits
tumor angiogenesis and tumor growth. Therefore, soluble
VEGFR-2 (sKDR/Flk-1) is a potent and selective endoge-
nous inhibitor of VEGF-mediated angiogenesis. The present
study took it as a theoretical base that tumor growth depends
on angiogenesis, and the KDR/flk-1 expression is different
between normal tissue and tumor tissue. The aim is to find a
product to inhibit angiogenesis-dependent tumor’s growth
and metastasis.
Because molecular weight of KDR/Flk-1 is so big that
there is a certain difficulty to express functional study of the
entire extracellular domain, selecting the main functional
domain to study is easier and can get good result. Some
researchers selected the Ig-like 5–7 domain as a target to
study the antiangiogenesis effect. The reason is that the Ig-
like 5–7 domain has a typical combination structure, which
is related to signal transduction and the combination site of
heparin, and heparin can improve the combination efficiency
of VEGF165 and KDR/Flk-1 (Soker et al., 1997). Other
researchers took the Ig-like 2–4 domain as a target to study
antitumor angiogenesis. The reason is that this part can
combine to different type of VEGF directly and does not
depend to heparin. Study proved that the extracellular Ig-like
2 domain of KDR/flk-1 contains VEGF combination site,
and Ig-like 1 domain and Ig-like 3 domain near Ig-like 2
domain have a promoting effect for the combination (Davis
et al., 1996). In this study, we selected the fragment from
ATG of the beginning, which is about 1000 bp including
secretory leader sequence. We took the fragment as a target
to study the antitumor angiogenesis function.
Retrovirus vector is a kind of expression vector having
been applied widely in mammalian cells. pLXSN as a
vector possesses much of excellence. It can transfect the
proliferative cells efficiently and integrate extrinsic gene to
receptor’s genome exactly. The recombinant virus vector
can be made into different sizes and different functional
genomes. It can also be reverse transcripted to cDNA clone,
which was integrated to cell’s chromosome and get copy
with the cell’s cleavage. Moreover, extrinsic gene will be
integrated at specific site; thus, the vector gene structure
can be protected, not be impaired. Until now, there is no
report about wild virus product of pLXSN, so it is safe. In
this study, we took retrovirus vector (pLXSN) as vector to
carry out gene recombination and transfect it to tumor cells.
Because there is secretory leader sequence after ATG in the
fragment, the fragment after being integrated to tumor cell’s
chromosome can be secreted to extracellular matrix. Be-
cause tumor cell grows fast, its proliferation is fast with the
fast amplification of the fragment in tumor cell’s chromo-
some. Thus, the fragment protein secreted to extracellular
matrix increased rapidly. This increased fragment protein
combines to VEGF secreted by the same tumor cells to
inhibit the combination of VEGF and functional VEGFR so
that it inhibits vessel endothelial cell proliferation and
tumor angiogenesis.
Acknowledgments
Work from our laboratory referred to in this paper has
been supported by Chinese National grants for PhD. student.
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