in vivo inhibition of tumor angiogenesis by a soluble vegfr-2 fragment

9
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 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). Studies showed that VEGF exists its effects by binding with 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). 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). www.elsevier.com/locate/yexmp Experimental and Molecular Pathology 76 (2004) 129 – 137

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www.elsevier.com/locate/yexmp

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-sFLK1

fragment

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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