Departments of 1 Internal Medicine and 2 Biochemistry, School of Medicine, Keio University, Shinjuku-ku, Tokyo 160-8582, Japan
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ABSTRACT |
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Although vascular endothelial growth factor (VEGF) plays a role in the growth of hypervascular tumors, mechanisms for paracrine regulation of its receptor expression on vascular endothelial cells remain unknown. This study aimed to investigate whether VEGF released from hypoxia-exposed Hep G2 cells alters expression of the two distinct receptors, kinase insert domain-containing receptor (KDR) and fms-like tyrosine kinase 1 (flt-1), in human umbilical venous endothelial cells (HUVEC). Hep G2 cells were cultured in 20% or 1% O2 for 16 h to examine induction of VEGF mRNA and its protein expression. Conditioned medium from Hep G2 cells (CM) was applied to HUVEC under normoxic conditions, and expression of mRNA for the VEGF receptors was determined by RT-PCR. In response to the hypoxic challenge, Hep G2 cells upregulated VEGF mRNA and the release of VEGF. Hypoxia-CM preferentially stimulated the mRNA expression of flt-1 but not that of KDR in HUVEC. When the VEGF release from hypoxia-exposed Hep G2 cells was blocked by its antisense oligodeoxynucleotide, the endothelial flt-1 mRNA upregulation elicited by the hypoxia-CM was still maintained. These results suggest that hypoxia-exposed Hep G2 cells not only produce VEGF but also evolve paracrine induction of flt-1 through VEGF-independent mechanisms.
reverse transcription-polymerase chain reaction; flt-1; kinase insert domain-containing receptor; antisense oligodeoxynucleotide
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INTRODUCTION |
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CELLS AND TISSUES RESPOND to hypoxia with several compensatory mechanisms to maintain the microenvironments in which they can survive. These compensatory mechanisms include the formation of new blood vessels from preexisting vessels, a process termed angiogenesis. Hypoxia-induced angiogenesis plays an important role not only in physiological situations but also in pathological circumstances involving growth and metastasis of solid tumors (3, 8-10). Increasing evidence suggests that upregulation of expression of vascular endothelial growth factor (VEGF) is a key step in hypoxia-induced angiogenesis, because of its specific and distinct stimulatory action on the growth of vascular endothelial cells. Biological actions of VEGF are thought to depend on expression of its receptors on vascular endothelial cells. At least two distinct receptors are known, that is, fms-like tyrosine kinase 1 (flt-1) and kinase insert domain-containing receptor (KDR).
Among malignancy occurring in humans, hepatocellular carcinoma is one of the representative tumors characterized by hypervascularity. It has recently been shown (5) that hepatocellular carcinoma cells greatly express VEGF, suggesting its role in angiogenic responses. Little information is, however, available as to the detailed molecular mechanisms of interactions between VEGF expression in hypoxia-exposed tumor cells and the receptor expression on endothelial cells. This study was thus designed, using a human hepatoblastoma cell line (Hep G2), to examine the possibility that hepatic tumor cells undergoing hypoxia could overexpress and release VEGF and thereby alter the receptor expression on endothelial cells. The present results provide evidence that Hep G2 cells exposed to hypoxic conditions not only generate VEGF but also release an unidentified factor(s) that evolves expression of flt-1 on vascular endothelial cells, suggesting paracrine upregulation of the specific receptor for VEGF-dependent angiogenesis.
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MATERIALS AND METHODS |
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Cell culture.
Hep G2 cells were obtained from American Type Culture Collection
(Rockville, MD). The cells were cultured in plastic culture dishes and
flasks that were filled with DMEM supplemented with 10% fetal bovine
serum (FBS), L-glutamine (2 mmol/l), pyruvate (1 mmol/l), penicillin (100 U/ml), and streptomycin
(100 µg/ml). Cultures were maintained in a humidified 37°C
incubator with 5% CO2. To expose
Hep G2 cells to hypoxic conditions, we placed the culture dishes in
air-controlled chambers constructed with inflow and outflow valves. A
preanalyzed air mixture (95%
N2-5%
CO2) was infused into the
chambers at a flow rate of 3 l/min (MIC-101 modular
incubator, Asahi Glass, Tokyo, Japan). The
PO2 values measured with an oxygen electrode reached a nadir of 10-15 mmHg in
the culture medium ~6 h after the infusion and were maintained for 16 h without medium change. At the same time, the control Hep G2 cells
were cultured in normoxic conditions and kept for measurements at
desired time points. To examine the cell viability, we counted a
portion of cells using a hemocytometer in the presence of trypan blue.
Pooled media were then spun (at 3,200 g for 10 min at 4°C), sequentially
passed through 0.45- and 0.2-µm sterile filters, and used immediately
or stored at 80°C for up to 1 wk. Under such conditions, the
pH values between normoxia- and hypoxia-conditioned medium (CM) did not
remarkably differ (7.6 vs. 7.5, respectively), presumably because of
the presence of HEPES buffer. On the other hand, as one might expect,
levels of lactic acid were remarkably different in hypoxia-CM (72.4 ± 2.8 vs. 133.0 ± 13.8 mg/dl for normoxia-CM vs. hypoxia-CM, respectively).
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Secretion of VEGF from Hep G2 cells. The VEGF levels, especially VEGF121 and VEGF165, in the culture medium were measured by sandwich enzyme immunoassay using affinity-purified anti-human VEGF mouse IgG (monoclonal antibodies, 16F1+2E1; IBL, Fujioka, Japan). The total cell number was evaluated by the modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (Promega, Madison, WI). The levels of VEGF were normalized by the total cell number in each culture dish.
Isolation of total RNA. Total cellular RNA was isolated from near confluent Hep G2 cells at 16 h after normoxic or hypoxic incubation by a one-step phenol-chloroform extraction method. Cells were lysed in RNAzol B (Cinna/Biotex Laboratories, Houston, TX) solution and homogenized. The homogenates were transferred to RNase-free centrifuge tubes, and 0.1 vol of chloroform was added to the tubes. Phase separation was achieved by centrifugation at 1,200 g at 4°C for 15 min. The aqueous phase was transferred to clean RNase-free tubes, and RNA was precipitated with an equal volume of isopropanol at 4°C. The RNA pellet was washed once with 75% ethanol and resuspended in 1 mM EDTA. First-strand cDNA synthesis was performed by reverse transcription of 0.5 µg of total RNA using avian myeloblastosis virus RT (Promega).
Antisense and sense oligodeoxynucleotides. A 22-mer antisense oligodeoxynucleotide complement of the 5' region of human VEGF mRNA containing the initiator AUG codon and the corresponding sense oligodeoxynucleotide was synthesized by the phosphorothioate approach using tetraethylthiuram disulfide. Sequences of antisense and sense oligodeoxynucleotides were 5'-CCCAAGACAGCAGAAAGTTCAT-3' and 5'-ATGAACTTTCTGCTGTCTTGGG-3', respectively (7). Sense as well as antisense oligodeoxynucleotides were incubated with the liposome composed of N-[1-(2,3-dioleyloxy)propyl]N,N,N-trimethylammonium chloride and dioleoyl phosphatidylethanolamine (lipofectin reagent, GIBCO BRL) for 30 min, and the colloidal suspension was added to cells 6 h before the exposure of hypoxia.
PCR. Hypoxanthine phosphoribosyltransferase (HPRT) was used as positive internal control. Primer sequences for detecting HPRT mRNA were 5'-AATTATGGACAGGACTGAACGTC-3' and 5'-CGTGGGGTCCTTTTCACCAGCAAG-3'. Primer sequences for detecting VEGF mRNA were 5'-GAGAATTCGGCCTCCGAAACCA TGAACTTTCTGCT-3' and 5'-GAGCATGCCCTCCTGCCCGGCTCACCGC-3'. It has been known that four alternatively spliced products can be generated from the single VEGF gene, yielding different protein products composed of 121, 165, 189, and 206 amino acids, designated as VEGF121, VEGF165, VEGF189, and VEGF206, respectively. Among them, only VEGF121 and VEGF165 are secreted and induce mitogenesis of endothelial cells; VEGF189 and VEGF206 are membrane anchored and act as vascular permeability factors (1, 2). Nomura et al. (7) previously employed such a sensitive RT-PCR technique to determine which VEGF forms are expressed in Hep G2 cells, by using the primers against the regions common to the four alternatively spliced products. With these primers, 486-, 618-, 690-, and 741-bp-long cDNA fragments would be amplified from VEGF121, VEGF165, VEGF189, and VEGF206 mRNA templates, respectively.
Primer sequences for flt-1 mRNA were 5'-GAGAATTCACTATGGAAGATCTGATTTCTTACAGT-3' and 5'-GAGCATGCGGTATAAATA CACAT-3' and those for detecting KDR mRNA were 5'-TATAGATGGTGTAACCCGGA-3' and 5'-TTTGTCACTGAGACAGCTTGG-3'. The amplification was carried out through a cycle program for 32 cycles at 94°C for 30 s, 60°C for 1 min, and 68°C for 2 min, followed by an additional 7 min of extension at 68°C. RT-PCR products were analyzed on 1% agarose gel using appropriate molecular weight markers and then its images were transferred to a fluorescent image analyzer (FluoroImager, Molecular Dynamics, Tokyo, Japan). The level of total VEGF PCR products was semiquantitatively evaluated by using the densitometric analysis program of the fluorescent image analyzer in four different experiments. To compute the density of the total VEGF PCR product (DVEGF), we divided the density of VEGF121 (DVEGF121) and VEGF165 (DVEGF165) by the density of HPRT (internal control) (DHPRT), according to the equation VEGF = (DVEGF121 + DVEGF165)/DHPRT. The levels of flt-1 or KDR PCR products were also semiquantitatively evaluated by the densitometry derived from three different experiments. To compute the density of the flt-1 PCR product (Dflt-1) or the KDR PCR product (DKDR), we divided each density by the density of HPRT (internal control) (DHPRT), according to the equation D'flt-1 = Dflt-1/DHPRT or D'KDR = DKDR/DHPRT, respectively.RNase protection assay for flt-1.
To generate a cRNA probe that allowed us to detect
flt-1
mRNA, we amplified a 500-bp fragment of
flt-1
mRNA by nested RT-PCR. A combination of first 5' primer for
nested PCR
"flt-1(1)A"
(5'-CGCGCTGCTCAGCTGTCTGCTTCTC-3'), first 3' primer
for nested PCR
"flt-1(1)B"
(5'-GGTGTGCTTATTTGGACATCTATGA-3'), second 5' primer
"flt-1(2)A"
(5'-CACATCATGCAAGCAGGCCAGACAC-3'), and second 3'
primer
"flt-1(2)B"
(5'-TTGACTGTTGCTTCACAGGTCAGAA-3') was chosen for nested
PCR. For reverse transcription, 1 µg of total RNA from the lung was
reverse transcribed with 200 U of Moloney murine leukemia virus
transcriptase (GIBCO BRL), using standard protocols and oligo(dT)
(GIBCO BRL) for priming the reverse transcriptase reaction. From the
total volume of 20 µl, 3 µl of the cDNA were used for the PCR. The
reaction was performed using a final volume of 20 µl containing 2 µl 10× concentrated buffer (supplied with enzyme), 2 µl of
2.5 mM dNTPs, and 1 U of Taq
polymerase (Boehringer Mannheim). RNA was used and performed as
described above. PCR conditions were 32 cycles with denaturation at
94°C (1 min), annealing at 60°C (1 min), and extension at
72°C (1 min) for
flt-1
cDNA. Purified fragments were cloned in the polylinker site of pGEM-T
(Promega) after EcoR V digestion.
Sequencing of the inserts performed from both SP6 and T7 by ABI-PRISM
(model 310, version 3.0) confirmed the identity of the cloned insert and the orientation in the polylinker site. Plasmids were linearized and yielded antisense probes of 500 bp after in vitro transcription with digoxigenin-labeled GTP (GIBCO BRL) and SP6 as well as T7 polymerase. Transcripts were labeled with digoxigenin and purified on a
Sephadex G50 spin column. For hybridization, total RNA was dissolved in
a buffer containing 80% formamide, 40 mM PIPES, 400 mM NaCl, and 1 mM
EDTA (pH 8). Total RNA (10 µg) was hybridized in a volume of 50 µl
at 45°C overnight with digoxigenin-labeled probe (600 pg). After
hybridization overnight, 350 µl of RNase T1 were added (final concn,
0.1 U/µl). The RNase digestion with RNase T1 was carried out at
37°C for 30 min and terminated with RNase
inactivation/precipitation mixture (RPA II, Ambion) for 30 min at
37°C, and then the tubes were transferred to a 20°C freezer for 30 min. The tubes were centrifuged at 12,000 g at 4°C for 20 min and then the
precipitate was loaded with gel-loading buffer (RPA II, Ambion).
Statistical analysis. Statistical comparison among groups was determined by one-way ANOVA and Fishers multiple comparison test. All values are reported as means ± SD. P < 0.05 was considered to be statistically significant.
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RESULTS |
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Hypoxia-induced VEGF release and its blockade by antisense
oligodeoxynucleotide.
Figure 2 depicts differences in excretion
of VEGF from Hep G2 cells cultured under normoxic and hypoxic
conditions. Under normoxic conditions, there was a detectable level of
VEGF in the culture medium, indicating that VEGF was constitutively
secreted from these cells. Pretreatment with the antisense
oligodeoxynucleotide against VEGF mRNA, but not with the sense
oligodeoxynucleotide, significantly attenuated the level of VEGF
released from the cells compared with the control. In response to
hypoxic insults, the level of VEGF release was markedly elevated,
increasing by approximately twofold. The hypoxia-elicited elevation of
the VEGF release was abolished and further decreased by pretreatment
with the antisense oligodeoxynucleotide against VEGF mRNA to a level
equivalent to that observed in the normoxic cells undergoing the same
pretreatment but not by that with the sense oligodeoxynucleotide.
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Expression of distinct VEGF mRNAs in hypoxia-exposed Hep G2 cells.
We attempted to examine whether hypoxia alters VEGF expression at mRNA
levels in cultured Hep G2 cells. Because Northern blot analysis could
not clearly discriminate the four mRNA species, we employed a sensitive
RT-PCR technique to determine which VEGF forms are expressed under
normoxic and hypoxic conditions. Figure 3
illustrates the effects of hypoxia on expression of VEGF mRNA assessed
by RT-PCR. As shown in Fig. 3A, mRNA
from Hep G2 cells gave signals at 486 and 618 bp that corresponded to
mRNAs for VEGF121 and VEGF165, respectively. These signals were
markedly inhibited by incubation with the antisense
oligodeoxynucleotide against VEGF mRNA (Fig.
3A,
left). On the other hand, signals for VEGF189 and VEGF206 mRNAs were not detected in Hep G2 cells throughout these experiments. Under hypoxic conditions, the signals of
mRNAs for VEGF121 and VEGF165 were significantly enhanced, while those
for VEGF189 and VEGF206 mRNAs were not evident (Fig. 3A,
right). Pretreatment with the
antisense oligodeoxynucleotide against VEGF mRNA significantly
attenuated the hypoxia-evolved increase in the mRNA expression for
VEGF121 and VEGF165 (Fig. 3A,
right). Figure
3B shows semiquantitative
densitometric analyses of VEGF mRNA level based on four repeated
experiments. DVEGF
was 2.3 in the control and 2.4 in the sense
oligodeoxynucleotide-treated cells under 20%
O2 (Fig.
3B). When treated with
the antisense oligodeoxynucleotide, the level decreased significantly
to 1.79 (Fig. 3B). Under hypoxic
conditions, the DVEGF values
increased compared with those in the control and sense
oligodeoxynucleotide-treated cells under normoxia (Fig.
3B). This elevation was small
compared with the increase in the VEGF protein expression under hypoxia shown in Fig. 2 but was reproducible and statistically
significant. The
DVEGF value in the sense
oligodeoxynucleotide treatment was similar to that of the hypoxic
control (Fig. 3B). On the other hand, pretreatment with the antisense oligodeoxynucleotides against VEGF mRNA again attenuated the
DVEGF values (Fig.
3B). These findings provide evidence
that hypoxic insult under the current experimental conditions evolves
upregulation of VEGF at mRNA levels and its protein release into the
medium in cultured Hep G2 cells.
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VEGF receptor subtypes in HUVEC incubated with hypoxia-CM of Hep G2
cells.
Evidence for the extracellular release of VEGF in hypoxia-exposed Hep
G2 cells led us to examine whether the culture medium containing VEGF
could alter the expression of its receptors,
flt-1 and KDR, at the side of
HUVEC. Figure 4 illustrates
the expression of flt-1 in HUVEC
assessed by RT-PCR. HUVEC that were incubated with normoxia-CM from Hep
G2 cells constitutively displayed 1,098-bp DNA fragments, corresponding
to flt-1 mRNA. This signal was
attenuated by incubation with normoxia-CM derived from the antisense
oligodeoxynucleotide-pretreated Hep G2 cells. As seen in Fig.
4A
(right), HUVEC undergoing the treatment with hypoxia-CM for 24 h exhibited a marked enhancement of
the flt-1 mRNA expression compared
with those treated with normoxia-CM. However, the hypoxia-CM-induced
enhancement of the flt-1 expression
was reproduced even when HUVEC were incubated with hypoxia-CM derived
from the antisense oligodeoxynucleotide-treated Hep G2 cells. As seen
in densitometric analyses for
D'flt-1 in Fig.
4B, treatment with normoxia-CM
collected from the antisense oligonucleotide-treated Hep G2 cells
induced an ~25% reduction of the
flt-1 expression in HUVEC, suggesting
that VEGF released from Hep G2 cells plays a role in the constitutive
expression of flt-1 in HUVEC. On the
other hand, treatment with hypoxia-CM collected from Hep G2 cells
significantly upregulated the
D'flt-1 values, indicating
a ~20% elevation compared with the control. However, treatment with
the hypoxia-CM collected from the antisense oligodeoxynucleotide-pretreated Hep G2 cells displayed no significant reduction of the flt-1 receptor
expression in HUVEC.
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DISCUSSION |
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To exert action on mitosis and proliferation of vascular endothelial cells, locally produced VEGF requires its receptors to be specifically expressed on the endothelial cells. The present study provided evidence that tumor cells, such as Hep G2 cells, undergoing hypoxia could alter the mRNA expression of the specific VEGF receptor, i.e., flt-1, on cultured endothelial cells. The enhanced expression of flt-1 mRNA in HUVEC with hypoxia-CM was demonstrated by RNase protection assay as well as by RT-PCR. Another important finding in the present study is that, among four different alternative splicing products of VEGF, only the secretory forms with mitogenic activities (VEGF121 and VEGF165) were detectable in the medium collected from the tumor cells.
As seen in the current study, the mechanisms through which Hep G2 cells can change phenotypes of the receptor expression on endothelial cells appear to depend greatly on humoral factors released from these cells, suggesting an important role of paracrine interactions between tumor and endothelial cells. Among such factors, VEGF released from the tumor cells obviously plays a pivotal role in the receptor expression on the endothelial cells, inasmuch as normoxia-CM collected from the tumor cells pretreated with the antisense oligodeoxynucleotide significantly lowered the basal expression of two distinct VEGF receptors, flt-1 and KDR. This finding thus supports the concept that malignant cells can utilize VEGF released from themselves to help upregulate the VEGF receptor expression for proliferation of the regional endothelial cells. In contrast, our study has demonstrated that hypoxia-CM from the tumor cells can further upregulate the endothelial expression of the VEGF receptors but through mechanisms distinct from those observed in normoxia-CM. First, hypoxia-CM can preferentially induce the expression of flt-1 but not of KDR in endothelial cells. Second, the humoral factor(s) responsible for the flt-1 upregulation does not involve VEGF released from the hypoxia-treated tumor cells, since the medium collected from the tumor cells pretreated with the antisense oligodeoxynucleotide did not attenuate the mRNA expression of the receptor on endothelial cells. These results collectively suggest that, under hypoxic conditions, tumor cell-dependent and VEGF-independent paracrine mechanisms play an active role in regulation of VEGF receptor expression on endothelial cells.
Considering that both the increasing release of VEGF and upregulation of the receptor on vascular endothelial cells are required for angiogenesis, there are several putative pathways that contribute to stimulation of the neovascularization in tumor tissues. First, when the tumor exhibits a rapid growth and enlargement in size, endothelial cells occurring at the hypoxic core of the tissue are known to upregulate VEGF and stimulate themselves to be proliferated (6, 7), serving as autocrine mechanisms. Second, tumor cells enhance the VEGF generation in response to hypoxia (8) and can thereby trigger the angiogenic responses in the adjacent endothelial cells, that is, a paracrine interaction. At least under the current experimental conditions, involvement of homologous upregulation of the VEGF receptor appears to be little if any, since the blockade of VEGF production by antisense deoxynucleotides in hypoxia-exposed tumor cells did not alter the endothelial receptor expression. In addition to these mechanisms for tumor angiogenesis, the present study provided circumstantial evidence for the presence of another paracrine mechanism in the tumor cells involving the release of humoral factors besides VEGF that are specifically operated under hypoxic conditions to regulate the receptor expression at the side of endothelium. Such tumor-derived factors would involve those capable of increasing the endothelial receptor expression or those that can downregulate or block an inhibitor(s) of the flt-1 mRNA expression. In this context, hypoxic tumor cells could not only produce VEGF as well documented by other investigators previously (8, 9) but also produce soluble factors with a potential to upregulate the receptor expression. Further investigation is clearly needed to define such unidentified factors for tumor angiogenesis as well as to examine subsequent protein expression of flt-1 and its functional consequence on proliferation of the endothelial cells.
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ACKNOWLEDGEMENTS |
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We thank Dr. Kazuyoshi Watanabe, Cytosignal Research for the kind suggestions.
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FOOTNOTES |
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This study is supported by Grant-in-Aid No. 09670575 for Scientific Research from the Ministry of Education, Science, and Culture of Japan and by a grant from the Kanae New Drug Research Foundation.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: H. Ishii, Dept. of Internal Medicine, School of Medicine, Keio Univ., 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582 Japan.
Received 3 February 1998; accepted in final form 18 September 1998.
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