(Received for publication, August 2, 1996, and in revised form, November 20, 1996)
From the Department of Biology and the
§ Department of Food Engineering and Biotechnology and the
¶ B. Rappaport Faculty of Medicine, Technion-Israel Institute of
Technology, Haifa 32000, Israel, the
Department of Oncology,
Hadassah University Hospital, Jerusalem 91120, Israel, and the
** Department of Molecular Biology, The Hebrew University-Hadassah
Medical School, Jerusalem 91010, Israel
A vascular endothelial growth factor (VEGF) mRNA species containing exons 1-6 and 8 of the VEGF gene was found to be expressed as a major VEGF mRNA form in several cell lines derived from carcinomas of the female reproductive system. This mRNA is predicted to encode a VEGF form of 145 amino acids (VEGF145). Recombinant VEGF145 induced the proliferation of vascular endothelial cells and promoted angiogenesis in vivo. VEGF145 was compared with previously characterized VEGF species with respect to interaction with heparin-like molecules, cellular distribution, VEGF receptor recognition, and extracellular matrix (ECM) binding ability. VEGF145 shares with VEGF165 the ability to bind to the KDR/flk-1 receptor of endothelial cells. It also binds to heparin with an affinity similar to that of VEGF165. However, VEGF145 does not bind to two additional endothelial cell surface receptors that are recognized by VEGF165 but not by VEGF121. VEGF145 is secreted from producing cells as are VEGF121 and VEGF165. However, VEGF121 and VEGF165 do not bind to the ECM produced by corneal endothelial cells, whereas VEGF145 binds efficiently to this ECM. Basic fibroblast growth factor (bFGF)-depleted ECM containing bound VEGF145 induces proliferation of endothelial cells, indicating that the bound VEGF145 is active. The mechanism by which VEGF145 binds to the ECM differs from that of bFGF. Digestion of the ECM by heparinase inhibited the binding of bFGF to the ECM and released prebound bFGF, whereas the binding of VEGF145 was not affected by heparinase digestion. It therefore seems that VEGF145 possesses a unique combination of biological properties distinct from those of previously characterized VEGF species.
The vascular endothelial growth factor (VEGF)1 isoforms display a limited structural similarity to platelet-derived growth factor and are important regulators of angiogenesis and blood vessel permeability (1-3). The human VEGF isoforms are generated by alternative splicing from a single gene (4-6). The domain encoded by exons 1-5 contains information required for the recognition of the known VEGF receptors KDR/flk-1 and flt-1 (7) and is present in all VEGF isoforms. The amino acids encoded by exon 8 are also present in all the VEGF splice variants. The VEGF isoforms are distinguished by the presence or the absence of the peptides encoded by exons 6 and 7 of the VEGF gene. VEGF121 is 121 amino acids long and lacks both exons. VEGF165 contains the exon 7-encoded peptide, whereas VEGF189 contains both exon 6- and exon 7-encoded peptides (6, 8, 9). VEGF121 and VEGF165 promote angiogenesis, cause permeabilization of blood vessels, and induce proliferation of vascular endothelial cells (10-14). VEGF189 has not yet been purified, but studies with cells expressing VEGF189 indicate that it may induce endothelial cell proliferation (8, 9). Low levels of a mRNA corresponding in size to a mRNA encoding a putative VEGF variant of 145 amino acids (VEGF145) containing exon 6 but lacking exon 7 were detected previously in reverse PCR experiments, but the protein encoded by this mRNA has not yet been characterized (15, 16).
The different VEGF isoforms differ in their heparin binding ability. VEGF121 does not bind to heparin, whereas VEGF165 and VEGF189 do (8, 17-19). The heparin binding affinity of VEGF189 was reported to be higher than that of VEGF165, suggesting that exon 6 contributes to the heparin binding ability of VEGF189 (8). VEGF165 and VEGF121 are secreted efficiently from producing cells and do not bind efficiently to the ECM produced by CEN4 cells (8, 9). In contrast, VEGF189 is retained on the cell surface and in the ECM, from which it can be released by prolonged incubation with heparin. (9). The peptide encoded by exon 7 also seems to affect the receptor recognition patterns of VEGF isoforms. VEGF121 recognizes a single VEGF receptor in endothelial cells that was identified as the KDR/flk-1 VEGF receptor (20, 21). VEGF165 also binds to this receptor but recognizes two additional VEGF receptors of unknown structure that are found in endothelial cells and in several transformed cell types (21, 22).
We have observed that VEGF145 is one of the main VEGF isoforms expressed by several cell lines derived from carcinomas of the female reproductive system. We have characterized VEGF145 and have compared its biological properties with those of other VEGF forms. Recombinant VEGF145 expressing the exon 6-derived peptide binds to heparin but behaves like VEGF121 with regard to its receptor recognition ability. In contrast to VEGF121 and VEGF165, it binds to a basement membrane like ECM produced by corneal endothelial cells in a biologically active form. VEGF145 therefore represents a VEGF form possessing distinct biological characteristics.
Human recombinant VEGF165 and VEGF121 were purified from Sf-9 insect cells, whereas recombinant human bFGF was produced in bacteria as described previously (19, 23, 24). A rabbit polyclonal antibody directed against VEGF165 (23) and a mouse monoclonal IgM antibody (M-35) directed against full-length VEGF165 were produced in our laboratory using standard techniques. Heparinases type I, II, and III were kindly donated by Dr. J. Zimermann (Ibex Technologies, Montreal, Canada). The bicystronic mammalian expression vector MIRB was kindly provided to us by Dr. Craig MacArthur (Washington University, St. Louis, MO) (25). Anti-VEGF monoclonal antibody clone 26503.11 and peroxidase- and alkaline phosphatase-conjugated anti-rabbit IgG antibodies were from Sigma. Disuccinimidyl suberate was from Pierce. Sodium alginate was from Fluka. Heparin-Sepharose was purchased from Pharmacia Biotech Inc. 125I-Sodium was obtained from New England Nuclear. Anti-mouse IgM antibodies conjugated to alkaline phosphatase were from Southern Biotechnology Associates Inc. (Birmingham, AL). Tissue culture plasticware was from Nunc, and 96-well dishes for enzyme-linked immunosorbent assays were bought from Corning. Grace's medium was obtained from Life Technologies, Inc. All other tissue culture reagents were from Biological Industries Inc. (Kibbutz Beth Haemek, Israel).
Identification of VEGF145 mRNA in Cancer CellsTotal mRNA was prepared from OC-238 human epithelial
ovarian carcinoma cells (26). Complementary DNA was synthesized from 300 ng of total RNA using oligo(dT) as a primer and avian
myeloblastosis virus reverse transcriptase. PCR amplification was
carried out in the presence of a [32P]dCTP tracer (2 µCi in a 100-µl reaction volume), 1 mM of each dNTP,
2.5 mM MgCl2, and 2.5 units of Taq
polymerase. 25 amplification cycles were used, each consisting of a
1-min incubation at 94 °C, a 2-min incubation at 65 °C, and a
3-min incubation at 72 °C. The VEGF-specific oligonucleotides used
were GGAGAGATGAGCTTCCTACAG and TCACCGCCTTGGCTTGTCACA, corresponding to
amino acids 92-98 and to the six carboxyl-terminal amino acids of
VEGF, respectively. A pair of primers from the L19 ribosomal protein
were included in the reactions as an internal control. Amplified
fragments were resolved in a 6% nondenaturing polyacrylamide gel and
were visualized by autoradiography. The band corresponding to the
VEGF145 mRNA (see Fig. 1B) was excised,
reamplified, and sequenced.
A similar procedure using different primers was used to detect
VEGF145 mRNA in A431 and HeLa cells. The VEGF-specific
primers used here were derived from the 5 of the coding region (amino acids 13-20) and from the 3
region encompassed by the last 6 amino
acids of VEGF including the translation stop codon of the VEGF
sequence.
In order to produce recombinant VEGF145, we
prepared a VEGF145 cDNA construct by deleting the
oligonucleotides encoded by exon 7 out of the VEGF189
cDNA. Primers used to amplify exons 1-6 of the VEGF cDNA were
the external primer GCTTCCGGCTCGTATGTTGTGTGG, corresponding to a puc118
sequence, and the internal primer ACGCTCCAGGACTTATACCGGGA, corresponding to a sequence at the 3 end of exon 6. Primers used to
amplify the 3
end of the VEGF cDNA were complementary to the puc118 sequence GGTAACGCCAGGGTTTTCCCAGTC and to the 3
end of the exon
6 sequence (underlined) and to the start of exon 8 (
ATGTGACAAGCCGAGGCGGTGA). Following amplification, the PCR products were precipitated,
and the products were reamplified using only the puc118-derived
external primers. The product was gel purified, subcloned into the
PCR-II vector, and sequenced using the sequenase-II kit from U. S.
Biochemical Corp. This cDNA was further used for protein expression
studies.
The VEGF145 cDNA was subcloned into the BamHI site of the MIRB expression vector (25). Following transfection into BHK-21 cells and selection with 0.6 mg/ml G418, VEGF145-expressing cells were identified using anti-VEGF antibodies. The VEGF145 cDNA was also subcloned into the transfer plasmid pVL-1393 (Invitrogen) downstream from the polyhedrin promoter to yield pVL-1393/v145. This plasmid and baculovirus wild type DNA were co-transfected into Sf9 cells using the calcium-phosphate co-precipitation method, and recombinant baculoviruses were isolated as described (23).
VEGF145 was produced in Sf9 cells as described for VEGF165 (23). The conditioned medium contained approximately 5 mg of VEGF145/liter. The conditioned medium was concentrated by precipitation with 70% ammonium sulfate at 4 °C for 12 h. The precipitate was solubilized in 20 mM Tris, pH 7, and 0.1 M NaCl, dialyzed extensively against this buffer at 4 °C, and applied to a heparin-Sepharose column. The column was washed with the same buffer containing 0.3 M salt, followed by elution with the same buffer containing 0.8 M NaCl. A small residual amount also eluted at 2 M NaCl. The 0.8 M salt eluant was further purified by reverse phase high pressure liquid chromatography on an Applied Biosystems Brownlee C-8 column using a linear gradient of acetonitrile (20-80%) containing 0.1% trifluoroacetic acid. VEGF145 was eluted at 48% acetonitrile. The trifluoroacetic acid in the eluant was neutralized using Tris base, and the acetonitrile was removed using a SpeedVac evaporator at room temperature.
Enzyme-linked Immunosorbent AssaysIncreasing concentrations of VEGF in 50 µl of coating buffer (20 mM K2HPO4, 10 mM KH2PO4, 1 mM EDTA, 0.8% NaCl, pH 7.2) were adsorbed to 96-well dishes for 3 h at 25 °C. Free VEGF was aspirated, and the wells were blocked with coating buffer containing 1% bovine serum albumin for 1 h. The wells were extensively washed with wash buffer (10 mM Tris-HCl, pH 7.2, 150 mM NaCl, and 0.1% Tween 20), incubated with the anti-VEGF M-35 monoclonal antibody for 2 h at 25 °C, washed again, and incubated for 1 h with an alkaline phosphatase-conjugated secondary antibody. After final washing, the amount of bound antibody was determined using para-nitrophenylphosphate as substrate.
Cell Culture and Production of ECM-coated DishesHuman umbilical vein-derived endothelial cells (HUVECs) were prepared from umbilical veins and cultured as described previously in M199 medium supplemented with 20% fetal calf serum, vitamins, 1 ng/ml bFGF, and antibiotics (21, 27). Proliferation assays using HUVECs were done as described previously (11). Bovine corneal endothelial (BCE) cells were isolated from steer eyes and cultured as described previously (28). ECM-coated dishes were prepared from cells grown in the presence or the absence of 30 mM chlorate as described previously (28, 29).
Binding of VEGFs to HUVECs and to ECM-coated DishesThe binding and the cross-linking of 125I-VEGF165 to confluent layers of HUVECs grown in 5-cm dishes in the presence or the absence of various competitors was done essentially as described. VEGF165 was purified from infected Sf9 cells and iodinated as described (19, 21, 23, 30).
Binding of VEGFs to ECM-coated 96- or 24-well dishes was performed at
room temperature. The ECM-coated wells were washed with rinse buffer
(10 mM Tris-HCl, pH 7.2, 150 mM NaCl, and 0.1%
Tween 20). Nonspecific sites were blocked with binding buffer (20 mM K2HPO4, 10 mM
KH2PO4, 1 mM EDTA, 0.8% NaCl, 1 mg/ml bovine serum albumin, pH 7.2) for 1 h at room temperature.
The binding buffer was aspirated and iodinated, or unlabeled growth
factors were incubated with the ECM-coated wells in binding buffer for
2 h at 24 °C. Free growth factors were removed by aspiration,
and the ECM was washed twice with rinse buffer. In binding experiments, 0.2 N NaOH was added to dissociate bound growth factors,
and aliquots were counted in a counter or neutralized using Tris
base and analyzed by SDS-PAGE followed by autoradiography.
Alternatively, the wells were further incubated with various anti-VEGF
monoclonal antibodies in binding buffer for 2 h and washed. Bound
antibody was detected with appropriate secondary antibodies coupled to alkaline-phosphatase, using para-nitrophenylphosphate as
substrate. Our M-35 anti-VEGF monoclonal antibody and commercial
anti-VEGF monoclonal antibodies produced identical results in these
assays.
For biological activity experiments, 15,000 HUVECs were seeded on ECM containing various amounts of adsorbed VEGF in a final volume of 1 ml of growth medium. Cells were trypsinized and counted after 3 days in a coulter counter. All experiments were repeated at least twice with similar results.
Reverse PCR analysis of mRNA from OC-238 human epithelial ovarian carcinoma cells (Fig. 1B), HeLa cells, and A431 cells (Fig. 1C) detected a VEGF mRNA containing a coding region smaller than that of VEGF165 but larger than that of VEGF121. The size of the coding region corresponded to the expected size of a mRNA encoding a VEGF form containing exons 1-6 and 8 and should lead to the production of a VEGF form containing 145 amino acids (VEGF145) (15). In all these cell lines the VEGF145 cDNA seemed to be expressed at levels comparable with those of VEGF165. The VEGF145 mRNA was not detected in several other transformed cell lines including C6 glioma cells and U937 cells. Sequence analysis of the PCR product from the OC-238 cells showed that this mRNA was indeed generated by alternative splicing and that it contains exons 6 and 8 but not exon 7 of the VEGF gene.
To study the properties of VEGF145, we have expressed the
VEGF145 cDNA in Sf9 insect cells using the baculovirus
expression system (23). Most of the VEGF145 produced by the
infected Sf9 cells was found in the conditioned medium as a homodimer
of ~41 kDa, with small amounts of monomeric VEGF145 (Fig.
2B). The VEGF145 dimers
dissociated into monomers upon reduction with dithiotreitol (Fig.
2A). VEGF145 was partially purified by
heparin-Sepharose affinity chromatography. VEGF145 was
eluted from heparin-Sepharose columns using a stepwise salt gradient.
Most of the VEGF145 eluted at 0.6-0.7 M NaCl,
indicating that the heparin binding affinity of VEGF145 is
similar to that of VEGF165 (data not shown) (17, 23). The
recombinant VEGF145 was biologically active and induced the
proliferation of HUVECs. The ED50 of VEGF145
was 30 ng/ml, whereas VEGF165 was 6-fold more active than
VEGF145 in this assay (Fig. 3).
VEGF145 Induces Angiogenesis in Vivo
To determine
whether VEGF145 can induce angiogenesis in vivo,
the VEGF145 cDNA was subcloned into the
BamHI site of the mammalian bicystronic expression vector
MIRB (25). The MIRB/VEGF145 plasmid was transfected into
BHK-21 cells (31), and stable cell lines producing VEGF145
were isolated. The VEGF145 produced by the mammalian cells
was biologically active and was secreted into the growth medium. A
stable clone producing 0.1 µg of VEGF145 per
106 cells was isolated. The VEGF145 expressing
cells were embedded in alginate beads, and the beads were implanted
under the skin of BALB/c mice (32). The pellets containing the alginate
beads were removed after 4 days and photographed. Clusters of alginate beads containing VEGF145 expressing cells were dark red
with blood, whereas beads containing cells transfected with vector
alone had a much lower content of blood (Fig. 4). When
examined under higher magnification, pellets containing
VEGF145 producing cells appeared much more vascularized
than pellets containing control cells.
VEGF145 Binds to the KDR/flk-1 Receptor but Not to the Two Smaller VEGF Receptors of HUVECs
125I-VEGF165 forms high molecular
weight complexes with three types of VEGF receptors following
cross-linking to HUVECs (Fig. 5, lane 1),
whereas 125I-VEGF121 only binds to the larger
of these receptors. The common receptor to which both
VEGF121 and VEGF165 bind is the
KDR/flk-1 VEGF receptor (Fig. 5, open arrow)
(21). In order to compare the receptor recognition pattern of
VEGF145 with those of VEGF165, 125I-VEGF165 was bound to HUVECs in the
presence of 1 µg/ml of heparin and increasing concentrations of
VEGF145. Bound 125I-VEGF165 was
subsequently covalently cross-linked to the VEGF receptors.
VEGF145 inhibited the binding of
125I-VEGF165 to the KDR/flk-1
receptor of the HUVECs (Fig. 5). This result was verified in a
cell-free binding experiment in which VEGF145 competed with
125I-VEGF165 for binding to a soluble fusion
protein containing the extracellular domain of the flk-1
receptor (data not shown) (33). In contrast, VEGF145 did
not effectively inhibit the binding of 125I-VEGF165 to the two smaller VEGF receptors
of the HUVECs (Fig. 5, filled arrow), indicating that the
affinity of VEGF145 toward these two receptors is
substantially lower than that of VEGF165. This behavior
resembles the behavior of VEGF121 (21) and indicates that
the presence of exon 6 is not sufficient to enable efficient binding of
VEGF145 to these two receptors, despite the heparin binding
properties that exon 6 confers on VEGF145.
VEGF145 Binds to the ECM Produced by Corneal Endothelial Cells
VEGF189 binds efficiently to the
ECM produced by CEN4 cells, whereas VEGF165 binds to it
very weakly (9). The fact that VEGF189 binds heparin with
high affinity led to the suggestion that the interaction of
VEGF189 with the ECM is mediated by heparan sulfate
proteoglycans (8, 9). The heparin binding affinities of
VEGF145 and VEGF165 are similar and
substantially lower than the heparin binding affinity of
VEGF189 (8). We therefore expected VEGF145 to
bind poorly to ECM. Unexpectedly, experiments in which VEGF145 was bound to an ECM produced by bovine corneal
endothelial cells (28, 34) showed that VEGF145 bound
efficiently, whereas the binding of VEGF165 was marginal
(Fig. 6A). In these experiments the binding
was monitored with anti-VEGF antibodies, but similar results were
obtained when binding to the ECM was assayed directly using
125I-VEGF145 (30 ng/ml) or
125I-VEGF165 (50 ng/ml) (Fig. 6B,
first and third lanes). The binding of
125I-VEGF145 to the ECM was substantially but
not completely inhibited by 10 µg/ml heparin (Fig. 6B,
second lane). The 125I-VEGF145 used
in these experiments contained some impurities (Fig. 6C),
but the major iodinated protein that was recovered from the ECM had a
mass corresponding to that of 125I-VEGF145
(Fig. 6B, first lane). To make sure that
125I-VEGF145 binds to the ECM and not to
exposed plastic surfaces, the ECM was scraped off and washed by
centrifugation, and the amount of adsorbed
125I-VEGF145 in the pellet was determined. The
ECM contained ~70% of the adsorbed
125I-VEGF145. It therefore appears that the
presence of the exon 6-derived peptide in VEGF145 enables
efficient binding to the ECM, whereas the exon 7 derived peptide of
VEGF165 does not suffice to confer this ability on
VEGF165.
VEGF145 Binds to the ECM Using a Mechanism That Is Not Dependent on ECM-associated Heparan Sulfates
The interaction of
bFGF with the ECM is mediated by the heparan sulfate moieties of ECM
associated proteoglycans (35). It was of interest to determine if
VEGF145 uses a similar mechanism. When
125I-VEGF145 was bound to ECM-coated dishes in
the presence of 10 µg/ml heparin, the binding was inhibited by
~60% (Fig. 7A). Under the same conditions
the binding of 125I-bFGF to the ECM was inhibited by 80%
(Fig. 7A). The binding of
125I-VEGF145 to the ECM was also inhibited by
80% in the presence of 0.8 M salt, indicating that the
interaction is probably not hydrophobic (data not shown). These results
are compatible with the expected behavior of proteins that bind to the
ECM via heparin-like molecules. However,
125I-VEGF145 also bound efficiently to an ECM
that was digested with heparinase-II (36). In contrast, there was
almost no binding of 125I-bFGF to heparinase-II-digested
ECM (Fig. 7A) (37, 38).
In order to further investigate the mode of interaction of VEGF145 with the ECM, we measured the heparin- or heparinase-II-induced release of prebound VEGF145 or bFGF from the ECM. When the ECM-coated wells were incubated for 2 h at 37 °C with buffer, only 20% of the bound 125I-bFGF and 13% of the bound 125I-VEGF145 dissociated from the ECM (Fig. 7B). This background release may be attributed in part to a proteolytic activity residing in the ECM (8, 39). When 10 µg/ml heparin were included in the buffer, only 33% of 125I-VEGF145 was released from the matrix, as compared with the release of 78% of the prebound 125I-bFGF. An even sharper difference was observed when heparinase-II was added to the buffer. The enzyme released 72% of the bound 125I-bFGF, but only 17% of the bound 125I-VEGF145 was released (Fig. 7B). Similar results were obtained when the experiment was performed with unlabeled VEGF145, using a commercial monoclonal anti-VEGF antibody to detect VEGF associated with the ECM (data not shown).
To determine the efficiency of the heparinase-II digestion, the ECM was metabolically labeled with [35S]sulfate and subsequently digested with heparinase-II. The digestion released 80-85% of the labeled sulfate residues (data not shown). To determine whether VEGF145 can bind to ECM depleted of all types of sulfated glycosaminoglycans, BCE cells were grown in the presence of 30 mM chlorate, an inhibitor of glycosaminoglycan sulfation (29). These ECMs were further digested with a mixture of heparinases I, II, and III (36, 40). Neither of these treatments significantly inhibited the binding of VEGF145 to the ECM, despite a >95% decrease in the content of ECM-associated sulfate moieties (data not shown).
Because endothelial cells do not proliferate when they are seeded on
ECM produced in the presence of chlorate (29), we examined whether
VEGF145 bound to such ECM retains its biological activity. Wells coated with ECM produced in the presence of chlorate were incubated with increasing concentrations of either VEGF145
or VEGF165. The wells were subsequently washed extensively
and HUVECs were seeded in the wells. ECM incubated with
VEGF145 induced proliferation of vascular endothelial
cells, whereas ECM incubated with VEGF165 did not (Fig.
8). We therefore conclude that the ECM-associated VEGF145 is biologically active.
Alternative splicing represents an important mechanism for the generation of diversity in growth factors and in their receptors. The alternative splice forms generated from the VEGF gene share angiogenic properties and are active as mitogens for endothelial cells. The VEGF145 mRNA has been previously detected as a rare VEGF mRNA species in placenta (15, 16). We have found expression of the VEGF145 mRNA in several tumorigenic cell types originating from the female reproductive system at levels comparable with the expression levels of VEGF165. Several transformed cell lines from other sources did not express this mRNA, indicating that the expression of VEGF145 may be more restricted compared with other VEGF forms (6). However, it remains to be seen whether production of VEGF145 is restricted to the female reproductive system.
To study the properties of VEGF145, we have produced recombinant VEGF145 in mammalian and in insect cells. VEGF145 was found to induce endothelial cell proliferation and in vivo angiogenesis, in agreement with previous studies that have indicated that these functions are not dependent on the presence of either exon 6 or exon 7 (10) and seem to be associated with the ability to bind to the KDR/flk-1 VEGF receptor (41, 42). However, VEGF145 seemed to be somewhat less active than VEGF165, and it is possible therefore that the presence of exon 6 in VEGF145 can subtly alter the conformation of the protein at the KDR/flk-1 binding site (7). The VEGF145 protein was secreted into the growth medium of producing cells. VEGF121, VEGF145, and VEGF165 are secreted into the medium by producing cells, whereas VEGF189 and VEGF206 are sequestered by cell surface heparan sulfates (8). It therefore seems that the simultaneous presence of both exon 6 and exon 7 is required for efficient binding of VEGF to cell surfaces, whereas the presence of each of these exons on its own does not confer this property on VEGF.
Unlike VEGF121, VEGF145 was able to bind to heparin-Sepharose columns, indicating that the exon 6 encoded peptide acts as an independent heparin binding domain. The affinity of VEGF145 for heparin was similar to that of VEGF165, even though the structures of the heparin binding domains of the two isoforms differ. All the VEGF isoforms tested to date bind to the KDR/flk-1 receptor of endothelial cells (21), and VEGF145 was no exception. However, unlike VEGF165, VEGF121 and VEGF145 do not bind efficiently to two additional VEGF receptors on HUVECs (21). The fact that VEGF121 and VEGF145 are both mitogenic and angiogenic indicates that these two receptors do not play a central role in the angiogenic and mitogenic response of endothelial cells. The biological function and the molecular structure of these two additional receptors is unknown. They appear to be novel VEGF receptors because they are not recognized by VEGF121, whereas both KDR/flk-1 and flt-1 are recognized by VEGF121.2 In addition these two receptors are not immunoprecipitated by antibodies directed against the intracellular domains of the known VEGF receptors (21), and they can be detected in tumor cells that do not express detectable levels of KDR/flk-1 or flt-1 mRNA (22). Our results suggests that specific exon 7 sequences that are not present in the exon 6-derived peptide are required for the interaction of VEGF165 with these two receptors. The heparin binding ability conferred on VEGF165 by the exon 7 peptide may not play a central role in the recognition of these receptors by VEGF165 because VEGF145 and VEGF165 bind with similar affinities to heparin yet differ in their ability to recognize these two receptors.
VEGF189 is not found in the conditioned medium of producing
cells and is sequestered on cell surface heparan sulfates and in the
ECM. In view of the strong interaction of VEGF189 with heparin, it was suggested that VEGF189 binds tightly to
cell surface and ECM localized heparan sulfates, allowing the
sequestration of VEGF189 in the ECM and on cell surfaces.
This was supported by experiments that showed that heparin can
dissociate bound VEGF189 from the ECM, although long
incubation times and high heparin concentrations were required (9). In
contrast, VEGF165 interacts weakly at best with cell
surface heparan sulfates and with the ECM and is released into the
medium of producing cells (8, 9). We expected VEGF145 to
bind weakly to cell surfaces and to the ECM because VEGF165
and VEGF145 appear to bind to heparin with similar
affinities. Indeed, we have found that VEGF145 is secreted
as expected into the medium of VEGF145 producing cells. However, VEGF145 bound to the ECM produced by corneal
endothelial cells much better than either VEGF165 or
VEGF121. The lowest VEGF145 concentration at
which binding to the ECM was observed was about an order of magnitude
lower than the concentration at which binding of VEGF165 to
the ECM was detected. In addition we have observed that
VEGF145 that is bound to ECM is able to promote
proliferation of endothelial cells. These observations prompted us to
compare the ECM binding behavior of VEGF145 with that of
bFGF, a growth factor that binds specifically to ECM-associated heparan
sulfate moieties (38). Unexpectedly, our observations suggested that VEGF145 and bFGF do not bind to common binding sites on the
BCE cell-derived ECM. Digestion of the ECM with heparinases releases bound bFGF but does not release bound VEGF145. It is
possible that VEGF145 binds to a heparan sulfate
subpopulation that is not recognized by bFGF or by the heparinases used
in the present study. However, VEGF145 was also able to
bind efficiently to an ECM produced in the presence of chlorate, an
inhibitor of glycosaminoglycan sulfation (43) and to ECM digested with
a mixture of three different heparinases resulting in a greater
than 95% depletion of ECM-associated sulfate groups. These experiments
therefore indicate that VEGF145 can bind to ECM components
distinct from the heparan sulfate side chains of proteoglycans. A
similar observation was reported for transforming growth factor-1, a
heparin binding protein (44) that binds to the core protein of the
ECM-associated chondroitin sulfate/dermatan sulfate proteoglycan
decorin (45) rather than to ECM-associated heparan sulfate
moieties.
All the splice variants induce angiogenesis in vivo, so why are five VEGF variants produced? Angiogenesis is often initiated under adverse conditions, such as the conditions encountered during wound healing. Many cell types produce several VEGF forms simultaneously (6, 46), and it is possible that each form offers advantages in different situations. The simultaneous production of several different VEGF forms may therefore ensure a balanced angiogenic response under diverse circumstances. When the properties of the VEGF variants are examined, it is apparent that the differences in their heparin binding abilities may affect their diffusion from a VEGF producing source to target blood vessels. VEGF121 does not bind to either heparan sulfates or to the ECM and should therefore diffuse more readily than the heparin and ECM binding VEGF forms. The ECM may serve as a storage depot for the VEGF forms that bind efficiently to the ECM, and these forms may dissociate slowly from the ECM providing prolonged angiogenic stimulation or be released from the ECM as a result of the activity of proteases (8). The balance may tip toward the production of preferred VEGF isoforms under certain conditions (46). Production of VEGF145 may be such a case because the variety of cell types that produce VEGF145 appears to be limited compared with the range of cell types producing VEGF121 or VEGF165. However, the mechanism that determines what VEGF forms should be produced by a given cell type remains to be elucidated.
We thank Yael Shiffenbauer and Dr. Michal Neeman for help in reverse PCR experiments. We thank Dr. Dina Ron and Dr. Dan Cassel for critically reading this manuscript and for helpful discussions.