Paracrine upregulation of VEGF receptor mRNA in endothelial cells by hypoxia-exposed Hep G2 cells

Hidekazu Suzuki1, Koichi Seto1, Yuichi Shinoda2, Mikiji Mori1, Yuzuru Ishimura2, Makoto Suematsu2, and Hiromasa Ishii1

Departments of 1 Internal Medicine and 2 Biochemistry, School of Medicine, Keio University, Shinjuku-ku, Tokyo 160-8582, Japan


    ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

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


    INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

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.


    MATERIALS AND METHODS
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Abstract
Introduction
Materials and methods
Results
Discussion
References

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).

In a separate set of experiments, human umbilical venous endothelial cells (HUVEC) were isolated according to the method of Jaffe et al. (4), plated onto 1.0% gelatin-coated wells, and grown in RPMI 1640 medium (GIBCO BRL, Gaithersburg, MD) with 10% FBS (GIBCO BRL). Before each "indirect" hypoxia experiment, HUVEC were washed twice with PBS and starved overnight in RPMI 1640 plus 1% FBS, to maintain the receptor expression similar to the basal expression levels in the control. HUVEC were treated for 24 h with hypoxia-CM or normoxia-CM (Fig. 1).


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Fig. 1.   Experimental protocols used in the present study. Six different treatment protocols are shown. Control, no treatment with oligodeoxynucleotides. Sense, treatment with a sense oligodeoxynucleotide against vascular endothelial growth factor (VEGF) mRNA. Antisense, treatment with an anti-sense oligodeoxynucleotide against VEGF mRNA. HUVEC, human umbilical venous endothelial cells.

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).

Protected mRNA fragments were loaded on denatured polyacrylamide gel and run at 250 V for ~1 h in Tris borate-EDTA buffer. The gel was transferred to a positively charged nylon membrane (Hybond N+, Amersham) by electroblotting (150 mA, 1.5 h) and cross-linked nucleic acids to the membrane. The transferred membrane was blocked by 30% skim milk, 0.1 M Tris · HCl, and 0.3 M NaCl (pH 7.5) for 30 min and then applied with alkaline phosphatase-labeled anti-digoxigenin antibody (150 mU/ml) for 2 h. Chemiluminescence generated by alkaline phosphatase reaction was detected by CDP-Star detection module (RPN 3510, Amersham Life Science).

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.


    RESULTS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

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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Fig. 2.   Concentrations of VEGF in culture medium of Hep G2 cells. 20% O2, normoxia. 1% O2, hypoxia. Cont, control (no oligodeoxynucleotide treatment). S, treatment with sense oligodeoxynucleotides against VEGF mRNA. AS, treatment with antisense oligodeoxynucleotides against VEGF mRNA. * P < 0.05, ** P < 0.001 compared with control under 20% O2. dagger  P < 0.001 compared with control under 1% O2.

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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Fig. 3.   RT-PCR analysis for VEGF mRNA expression in Hep G2 cells. A: mRNA from Hep G2 cells under normoxic or hypoxic condition gave signals at 486 and 618 bp (corresponding to mRNAs for VEGF121 and VEGF165). Signals for VEGF189 and VEGF206 mRNAs were not detected in Hep G2. B: densitometric analyses of VEGF mRNA species vs. hypoxanthine phosphoribosyltransferase (HPRT) mRNA species as an internal control in Hep G2. DVEGF denotes density calculated as a summation of the signals associated with VEGF121 and VEGF165 (shown in A) (see MATERIALS AND METHODS for formula). * P < 0.05, ** P < 0.05 compared with control under 20% O2. dagger  P < 0.001 compared with control under 1% O2.

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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Fig. 4.   Alterations in fms-like tyrosine kinase 1 (flt-1) mRNA expression in HUVEC treated with culture medium collected from hypoxia-treated Hep G2 cells. Normoxia- or hypoxia-conditioned medium (CM) denotes treatment with culture medium collected from normoxia- or hypoxia-treated Hep G2 cells, respectively. A: RT-PCR analysis showing expression of flt-1 mRNA species in HUVEC treated with normoxia-CM or hypoxia-CM for 24 h. B: densitometric analyses of flt-1 mRNA species on the basis of HPRT (internal control) mRNA species (D'flt-1) in HUVEC treated with normoxia-CM or hypoxia-CM for 24 h. * P < 0.01, ** P < 0.01 compared with control for normoxia-CM.

Figure 5 shows RT-PCR analysis for the expression of another VEGF receptor, KDR, and evaluation by semiquantitative densitometric analysis. HUVEC that were incubated with normoxia-CM derived from Hep G2 cells constitutively displayed 555-bp DNA fragments identical to the KDR mRNA. This signal was attenuated by incubation with normoxia-CM derived from the antisense oligodeoxynucleotide-pretreated Hep G2 cells, suggesting again the involvement of VEGF released from the cells in endothelial expression of this receptor subtype. On the other hand, when HUVEC were incubated with hypoxia-CM from Hep G2 cells, the endothelial expression of KDR was different from that of flt-1. KDR expression under these experimental conditions was only marginally greater and without statistical significance. Treatment with hypoxia-CM from the antisense oligodeoxynucleotide-pretreated Hep G2 cells did not alter these changes.


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Fig. 5.   Alterations in kinase insert domain-containing receptor (KDR) mRNA expression in HUVEC treated with culture medium collected from hypoxia-treated Hep G2 cells. A: RT-PCR analysis showing expression of KDR mRNA species in HUVEC treated with normoxia-CM or hypoxia-CM for 24 h. B: densitometric analyses of KDR mRNA species on the basis of HPRT (internal control) mRNA species (D'KDR) in HUVEC treated with normoxia-CM or hypoxia-CM for 24 h. * P < 0.05 compared with control for normoxia-CM.

The flt-1 mRNA expression was further confirmed by RNase protection assay (Fig. 6). The bands representing protected mRNA fragments (500 bp) were detected. Only a weak activity of protected mRNA fragments was observed in HUVEC with normoxia-CM. On the other hand, a strong activity of protected mRNA fragments was detected in HUVEC treated with hypoxia-CM at 500 bp, having a 5.7-fold greater density of bands compared with that in HUVEC treated with normoxia-CM. Treatment with hypoxia-CM collected from the antisense oligodeoxynucleotide-pretreated Hep G2 cells did not repress these enhanced bands (1:1.1 = hypoxia-CM/hypoxia-CM + antisense), suggesting the presence of an unidentified humoral factor(s) released preferentially from hypoxia-treated Hep G2 cells but not from those treated with normoxia.


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Fig. 6.   RNase protection assay showing expression of flt-1 mRNA species in HUVEC treated with normoxia-CM or hypoxia-CM for 24 h. Arrow indicates 500 bp.


    DISCUSSION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

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.


    ACKNOWLEDGEMENTS

We thank Dr. Kazuyoshi Watanabe, Cytosignal Research for the kind suggestions.


    FOOTNOTES

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.


    REFERENCES
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

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Am J Physiol Gastroint Liver Physiol 276(1):G92-G97
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society