A Novel Alternatively Spliced Form of Murine Vascular Endothelial Growth Factor, VEGF 115*

Takashi SugiharaDagger §, Renu WadhwaDagger , Sunil C. KaulDagger , and Youji MitsuiDagger

From the Dagger  National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, 1-1 Higashi, Tsukuba, Ibaraki 305, Japan and the  Chugai Research Institute for Molecular Medicine, 153-2 Nagai, Nihari-Mura, Nihari-Gun, Ibaraki 300-41, Japan

    ABSTRACT
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Abstract
Introduction
Procedures
Results & Discussion
References

Murine immortal fibroblasts express a form of vascular endothelial growth factor (VEGF) that was cloned, characterized and named VEGF 115. It differs from VEGF 120 by 37 amino acids at the carboxyl terminus. VEGF 115-specific sequence reacted to a single transcript in mouse tissues. Reverse transcription-polymerase chain reaction was performed in mouse tissues and in fibroblasts of normal and immortal divisional phenotypes. The data from mouse tissues suggested that VEGF 115 is not a tissue-specific isoform of VEGF 120, whereas a functional relevance with immortalization is indicated from the latter. The novel cDNA was expressed in Escherichia coli, and the His-tagged VEGF 115 (17.2 kDa) thus obtained was recognized by anti-VEGF antibody. A mammalian expression plasmid, pCMVneo+, encoding for VEGF 115 was transfected to NIH 3T3 cells, and the conditioned medium of stable transfectants was found to have fibroblast growth factor-replacing activity for human umbilical vein endothelial cells. Two independent genomic P1 clonings with primers specific for VEGF 164 and VEGF 115, respectively, resulted in isolation of identical P1 clones. We analyzed these three P1 clones on Southern blots with common and specific probes for VEGF 164 and VEGF 115. The results support the hypothesis that VEGF 115 is a new alternatively spliced form of mouse VEGF.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results & Discussion
References

Vascular endothelial growth factor (VEGF),1 also called vasculotropin or vascular permeability factor, is a potent microvascular permeability-enhancing cytokine and a selective endothelial mitogen that induces angiogenesis in vivo (1). Four types of VEGF mRNA encoding VEGF species (i.e. VEGF 206, VEGF 189, VEGF 165, and VEGF 121) that differ in their molecular mass and other characteristics are transcribed from a single human gene as a result of alternative splicing (2, 3). VEGFs are produced and secreted by several normal human organs and tumors. It is widely thought to be a mediator of neoangiogenesis during tumor development, wound healing, and a variety of other biological events, such as placental growth and retinal vasculature (4-7).

Mouse gene for VEGF has been shown to encode three alternatively spliced forms of VEGF, i.e. VEGF 188, VEGF 164, and VEGF 120 (8). The fourth VEGF isoform, corresponding to human VEGF 206, has not been predicted in mouse. Similar to the corresponding human VEGF forms (VEGF 189, VEGF 165, and VEGF 121), the above three murine forms are glycosylated homodimers with a mass of 34-43 kDa (8, 9). Claffey et al. (10) have shown that VEGF is expressed at relatively low levels in many of the murine adult tissues examined. Tumors frequently show up-regulation of VEGF expression (11, 12). Kim et al. (13) have reported that anti-VEGF antibody treatment inhibits the growth of tumors, although it has no effect on in vitro cell growth. VEGF expression is seen to be dramatically induced during 3T3-adipose differentiation and C2C12 myogenic differentiation (10). On the other hand, it is seen to change from moderate to undetectable levels when transformed undifferentiated PC12 cells differentiate into nonmalignant neuron cells (10). These studies have revealed that VEGF expression is specifically regulated in a transformed cell line and is closely relevant to the process of cellular differentiation. Enomoto et al. (14) have demonstrated VEGF-induced disorganization of actin stress fibers that is accompanied by drastic morphological rounding of Balb/c 3T3 cells. This alteration of cytoskeletal organization has been shown to occur by receptor-mediated tyrosine phosphorylation of cellular proteins. It has been shown that the VEGF receptors, flk-1/KDR and flt-1, are abundantly expressed in endothelial cells and are essential for hematopoietic and vascular development (15, 16). On the other hand, cell types such as bovine corneal endothelial cells, HeLa cells, and human melanoma cells possess cell surface VEGF receptors although these do not proliferate in response to VEGF (2). Whereas mitogenic and angiogenic effects of VEGF have been widely studied for endothelial cells, the significance of VEGF and its receptor expression in some nonendothelial cells remain unknown to date.

While characterizing the spontaneously immortalized mouse fibroblast RS-4, an immortalized clone from CD1-ICR mouse embryonic fibroblasts (CMEFs) (17), we initially concluded that the conditioned medium from RS-4 cells has growth-supporting activity for human umbilical vein endothelial cells (HUVECs). The activity factor was purified and characterized as VEGF by amino acid analysis (18). By using VEGF 164 primers, we detected an up-regulation of VEGF expression in RS-4 cells as compared with CMEFs. In addition to the three known alternatively spliced forms (VEGF 120, VEGF 164, and VEGF 188), a fourth form of VEGF was detected by RT-PCR as a 304-bp fragment (18). In the present study, we report a cloning and characterization of a cDNA that encodes a protein of 141 amino acid residues containing 26 amino acids residues of signal sequence (thus named VEGF 115). Functional analysis determined it to be a specific endothelial mitogen, and it corresponds to the FGF-replacing factor in the conditioned medium of RS-4 cells, identified in our previous study. Genomic characterization revealed that VEGF 115 is the fourth alternatively spliced form of murine VEGF gene.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results & Discussion
References

Cell Culture-- Embryonic fibroblasts (CMEFs) were isolated from CD1-ICR strain of mice as described earlier (19). These were regularly maintained in Dulbecco's modified Eagle's minimal essential medium (Sigma) supplemented with 10% fetal calf serum (FCS) (Bio Cell), penicillin, streptomycin (Sigma), and fungizone (Life Technologies, Inc.) at 37 °C in a humidified atmosphere of 95% air and 5% CO2. Spontaneously immortalized clones arising from CMEFs during the end phase of their life span were isolated and cloned (17). RS-4 cells that represent stage II of immortalization were used for the present study. These cells could grow on serum-free medium and were also detected to harbor a growth-supporting factor(s) for fibroblasts and endothelial cells (HUVECs) in their conditioned medium. HUVECs were isolated and maintained as described (20). They were cultured in MCDB 151 medium (Sigma) supplemented with 15% FCS, 5 µg/ml heparin (Sigma), and 10 ng/ml FGF-1 in culture flasks precoated with bovine fibronectin (1 µg/cm2).

Cloning of VEGF 115-- Total cellular RNA (1 µg) from RS-4 cells was annealed with random hexanucleotides and reverse transcribed for 75 min at 37 °C using Superscript II reverse transcriptase (Life Technologies, Inc.) as described earlier (18). The reaction mixture was then heated at 95 °C for 5 min and chilled on ice. PCR was performed on <FR><NU>1</NU><DE>18</DE></FR>th of the RT mixture by using AmpliTaq DNA polymerase (Perkin Elmer Corp.), VEGF 164 sense (bp 248-267, 5'-TGAGACCCTGGTGGACATCT-3') and antisense (bp 640-658, 5'-CACCGCCTCGGCTTGTCAC-3') primers (18) under the conditions of 94 °C for 1 min, 58 °C for 2 min, and 72 °C for 3 min, for a total of 30 cycles. The PCR product was purified (Geneclean II kit, BIO 101, Inc.) and cloned in pT7-blue TA cloning vector (Novagen, Inc., Madison, WI) from which the insert could be released by EcoRI and PstI digestion. The cloned DNA was sequenced on automated DNA sequencer (ABI model 370A) following the manufacturer's protocols.

Northern Blot Analysis-- Northern blot containing poly(A+) RNA from mouse tissues was bought from CLONTECH. Hybridization was performed with a VEGF 115-specific probe that was obtained by Klew reaction using a 130-bp fragment as template and VEGF 115-specific primer. Hybridization was performed in rapid hybridization buffer (Amersham Corp.). The membrane was striped in boiling 0.5% SDS for 5-10 min and reprobed with VEGF 164 probe. Finally, the membrane was probed with beta -actin for loading control.

RT-PCR-- Total RNA was extracted from various tissues and cells with Isogen reagent (NIPPON Gene Corp., Japan). RNA was reverse transcribed using Superscript reverse transcriptase at 37 °C for 1 h (18). PCR was performed with VEGF 164 sense (5'- GATCAAGTTCATGGACGTCT-3') and VEGF 115-specific antisense (5'-CCAAAAGTTTCCCAGGCA-3') primers using Taq polymerase for 40 cycles (at 94, 58, and 72 °C) of amplification. The control PCR was performed with glyceraldehyde-3-phosphate dehydrogenase sense (5'-TTCATTGACCTCAACTACATG-3') and antisense (5'-GTGGCAGTGATGGCATGGAC-3') primers. Mouse genomic DNA (CLONTECH) was used for control PCRs. The ectopic gene expression in NIH 3T3 was examined by PCR with the sense (5'-TGTTGCCTTTACTTCTAGGCCTGTACGGAA-3') and antisense (5'-CAGCACTGATCCACGATGCCGCGCTTCTGC-3') primers from the vector sequences at an annealing temperature of 58 °C. VEGF 120 RT-PCR was performed with sense (5'-TCCAGGAGTACCCCGACGAG-3') and antisense (5'-TCACCGCCTTGGCTCGTCAC-3') primers using Taq polymerase (at 94, 58, and 72 °C).

Recombinant VEGF 115-- The purified DNA containing the full open reading frame for VEGF was cloned in pQE-32 vector (Qiagen Inc.), which was then expressed in M15 Escherichia coli cells in the presence of isopropyl-beta -thiogalactopyranoside. The bacterial pellet was sonicated and boiled in SDS-PAGE sample buffer (21). After centrifugation at 15000 rpm, the protein sample was resolved on 15% SDS-PAGE followed by Western analysis (22) with anti-His tag antibody (Qiagen Inc.) and anti-VEGF antibody (a kind gift from Dr. S. Kondo) (23).

Southern Blot Analysis of P1 Clone-- Mouse P1 library was screened by PCR primers specific for VEGF 115 (sense, 5'-GTGGGCACCTGAGGCACA-3', and antisense, 5'- CCAAAAGTTTCCCAGGCA-3'; amplicon size, 110 bp); VEGF 164 (sense, 5'-CACTGTGAGCCTTGTTCAGAG-3', and antisense, 5'- CTGCAAGTACGTTCGTTTAA-3'; amplicon size, 131 bp) (Genome Systems Inc.). Three P1 clones were obtained by these two independent screenings. Each of these three clones (3 µg) was digested with EcoRI-SalI-SphI, HindIII-PvuII-SpeI, and EcoRI-PstI-XhoI at 37 °C overnight and hybridized with VEGF 115- and VEGF 164-specific probes obtained by the above described PCR (24). A common probe for VEGF 164 and VEGF 115 was prepared by PCR amplification of exon 3 by sense (5'-GATCAAGTTCATGGACGTCT-3') and antisense (5'-ATGTTGCTCTCTGACGTGGG-3') primers; amplicon size, 186 bp. Southern hybridization was carried out at 65 °C for 24 h in 5× saline/sodium/phosphate/EDTA, 5× Denhardt's solution, and 0.5% SDS. Filters were washed twice with 1× saline/sodium/phosphate/EDTA and 1.0% SDS for 15 min at 65 °C. The membrane was exposed to Kodak XAR-2 film with an intensifying screen at -70 °C for 1-2 days.

Transfection Assay-- The VEGF 115 was first cloned and confirmed for its sequence in pT7-Blue vector (Novagen). EcoRI-PstI fragment from pT7-Blue vector was next cloned into expression vector CMVneo+ (a kind gift from Olivia M. Pereira-Smith, Baylor College of Medicine). Plasmids containing accurate sequence for VEGF were obtained. Expression vector was mixed with Tfx-50 Reagent (Promega), and added to the NIH 3T3 culture at 60% confluency. Transfectants were selected in 500 µg/ml G418-supplemented growth medium. About 6-10 clones were isolated from each transfection by cloning rings, which were subsequently maintained in 100 µg/ml G418-supplemented medium.

Assay for Mitogenic Activity-- The individual transfectants were selected in Dulbecco's modified Eagle's minimal essential medium supplemented with 10% FCS and 500 µg/ml G418. Upon confluence, the cells were cultured without G418 for a day, and the cultured medium was collected and assayed for its mitogenic activity by incorporation of [3H]thymidine into DNA of endothelial cells. Endothelial cells were plated at 2 × 104 cells/0.5 ml/well into 24-well tissue culture plates (Falcon) coated with fibronectin. After incubation in a growth medium with FGF-1 for 24 h, the medium was replaced with MCDB151 medium supplemented with 15% FCS (starvation medium). At 24 h, endothelial cells were subjected to the fresh starvation medium supplemented (1:10) with the test sample (conditioned medium or its purified fractions). At 16 h after the initial addition of the test sample, [3H]thymidine was added and the cells were labeled for the next 6 h. The cells were then washed with cold phosphate-buffered saline and trypsinized at 37 °C. The solution was collected on a glass fiber filter and washed using a cell harvester (LABO-MASH, Labo Science Co.). The radioactivities were counted by a liquid scintillation counter.

    RESULTS AND DISCUSSION
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Abstract
Introduction
Procedures
Results & Discussion
References

VEGF 115 Cloning and Expression Studies-- A 304-bp fragment amplified from RS-4 cells (an immortal clone from CD1-ICR embryonic fibroblasts) reacted to the VEGF probe on Southern analysis by RT-PCR with VEGF primers, indicating that this may represent a fourth form of VEGF (18). Sequence analysis of this band indeed showed 36.7% homology to the 411-nucleotide sequence of VEGF 164 obtained in the same RT-PCR amplification (18). 151 of 304 nucleotides aligned completely to the VEGF 164 sequence, and 153 nucleotides showed a novel identity. By the use of a sense primer from 5' upstream sequence of VEGF 164 and an antisense primer from the novel sequence from our RT-PCR amplified 304-bp VEGF fragment, a second RT-PCR was performed on the RNA from RS-4 cells. A 458-bp band was obtained that has a single open reading frame of 423 bp, yielding a protein of 141 amino acid residues that contained a signal sequence of 26 amino acid residues, reported earlier for three known forms of VEGF (9); it thus can yield a mature protein of 115 amino acid residues, named VEGF 115 (Fig. 1A). This protein is identical to the three known forms of VEGF (9) for 104 amino acid residues, including 26 amino acid residues of the signal sequence, and has 37 novel amino acids at the carboxyl terminus (Fig. 1B). Structural analysis of VEGF 115 revealed its similarity to VEGF 120, except that the carboxyl-terminal novel sequence that confers it has a beta  sheet structure instead of a turn present in VEGF 120 (Fig. 1C). VEGF 188 and VEGF 164 have been characterized as basic proteins with high affinity for heparin. In contrast, VEGF 120 is a weakly acidic protein with no affinity for heparin (25). Similar to VEGF 120, VEGF 115 did not show binding to heparin (data not shown).


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Fig. 1.   VEGF 115 sequence. A, the nucleotide sequence of VEGF 115 is shown with the deduced amino acid sequence of 141 residues. Signal peptide sequence (26 residues) is marked by underlining. B, amino acid alignments of the four forms of VEGF. C, secondary structure of the four forms of VEGF. Structural differences between VEGF 120 and VEGF 115 are shown by the boxes.

We next analyzed VEGF 115 expression in various mouse tissues. VEGF 115-specific sequence reacted to a single transcript of approximately 4.0 kb in multiple mouse tissues (Fig. 2A). The filter was striped and reprobed with VEGF 164 probe. The latter reacted to four transcripts, including the one recognized by VEGF 115 sequence (data not shown). To exclude the differences in RNA loading on Northern blot, the RT-PCR with VEGF 115-specific primers was performed on mouse tissue RNA. The highest level of expression was detected in both spleen and kidney (Fig. 2B), in contrast to the VEGF 120 and VEGF 164 expression, which were found to be predominant in spleen (Fig. 2) and kidney (26), respectively. Mouse genomic DNA was used for PCRs in parallel. It did not yield any equivalent amplification (data not shown), supporting the idea that the amplified band did not result from contamination of genomic DNA. The presence of VEGF 115 transcript in tissues that also harbor VEGF 120 and VEGF 164 suggests that VEGF 115 is not a tissue-specific isoform of the other three known forms of VEGF. High expression of VEGF in kidney has been attributed to the extensive vascularization of this tissue. In addition, a high level of expression of VEGF 115 in spleen and testis may relate to the proliferative potential of these tissues. VEGF 115 RT-PCR was performed on RNA from normal (CMEF) and an immortal (RS-4) mouse fibroblasts. A significant increase in expression level of VEGF 115 was detected during spontaneous immortalization of CMEFs (Fig. 3), whereas VEGF 120 amplification from the same cells yielded substantially different results. The data suggested a functional relevance of VEGF 115 to spontaneous immortalization or, in other words, a divisional phenotype of cells. We have previously reported that FGF-replacing activity for HUVECs is present in the conditioned medium of RS-4 cells, which represent immortalized stage II as defined by their growth properties, p81/ezrin, and p53 expression and is not detected in CMEFs (normal) and NIH 3T3 cells that represent immortalized stage I (18). A high expression of VEGF 115 in RS-4 cells but not in CMEFs (Fig. 3) and NIH 3T3 (data not shown) thus suggests that VEGF 115 may correspond to the FGF-replacing factor in the conditioned medium of RS-4 cells.


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Fig. 2.   Expression analysis of VEGF 115. A, Northern analysis with VEGF 115-specific probe showing a single transcript of approximately 4.0 kb in mouse tissues. beta -Actin probing for loading control is shown in the lower panel. B, VEGF 115 and VEGF 120 RT-PCR in mouse tissues. Primers used for VEGF 115 RT-PCR are indicated by arrows and are detailed under "Experimental Procedures," along with the primers used for VEGF 120. The ethidium bromide signal of PCR-amplified DNA fragments and their quantification is shown.


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Fig. 3.   Expression levels of VEGF 115 and VEGF 120 in normal (CMEF) and immortal (RS-4) mouse fibroblasts. RT-PCR amplification of VEGF 115 and VEGF 120 from cells at different population doublings is shown as the ethidium bromide signal. Its quantitation was performed with respect to glyceraldehyde-3-phosphate dehydrogenase amplification from the same samples.

VEGF 115 Protein and Its Biological Activity-- A 423-bp open reading frame, as detected from the sequence analysis of the VEGF cDNA clone, was confirmed by its cloning in a bacterial expression vector, pQE32 (Qiagen) and expression as a His-tagged 17-kDa protein by isopropyl-beta -thiogalactopyranoside induction in bacteria (Fig. 4B). The recombinant protein was also detected with anti-VEGF antibody (Fig. 4A) in addition to an anti-His tag antibody confirming its identity as a new form of VEGF protein.


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Fig. 4.   Recombinant VEGF 115. Western analysis of recombinant VEGF with anti-VEGF antibody (A) and anti-His antibody (B). Lanes 1, 2, and 3 were loaded with VEGF 164 (100 ng; Pepro Technology, Inc.), isopropyl-beta -thiogalactopyranoside-unstimulated bacterial extracts, and isopropyl-beta -thiogalactopyranoside-stimulated bacterial extracts with pQE/VEGF 115 construct, respectively. VEGF 164 and VEGF 115 are shown by the arrow and arrowhead, respectively.

VEGF 115 was cloned in mammalian expression vector pCMVneo+ and transfected to NIH 3T3 cells. Stable transfectants were selected in G418 (500 µg/ml) supplemented growth medium and were examined for VEGF 115 expression by RT-PCR with vector primers (Fig. 5A). A 642-bp band was clearly detected in VEGF transfectants, whereas controls (vector-transfected cells) did not exhibit any equivalent RT-PCR amplification. The amplified band was confirmed to be VEGF 115 by its sequence analysis. The conditioned medium from three stable transfectants was further analyzed for its growth-promoting effect on HUVECs. The latter require FGF or VEGF for their entry into the cell cycle. We had earlier detected an FGF/VEGF-replacing activity in the conditioned medium of RS-4 but not NIH 3T3 cells. We found that HUVECs did not grow when cultured in the presence of conditioned medium of control transfectants. However, when supplemented with the conditioned medium (1:10) from VEGF-transfected NIH 3T3 clones, HUVECs exhibited [3H]TdR incorporation (Fig. 5B) and also showed an increase in cell number. The conditioned medium from a VEGF 164 stable transfectant was also analyzed for comparative mitogenic activity. The effect of conditioned medium from VEGF 115 and VEGF 164 was significant as compared with the control. Although the effect of VEGF 115 appeared to be higher than that of VEGF 164 when an equal amount of the conditioned medium was used for mitogenic assay for HUVECs, the expression levels of VEGF 115 and VEGF 164 and the amount of respective proteins in conditioned medium of the transfectants were not estimated due to the lack of VEGF 115-specific antibodies at present. Therefore, comparative mitogenicity of VEGF 115 and VEGF 164 could not be estimated from the present data. Supplementation of growth medium of HUVECs with 1:10 of the conditioned medium from VEGF 115 transfectants exhibited an effect equivalent to the supplementation of the medium with FGF-1 (3 ng/ml). Therefore, the data that can be identified as VEGF 115 correspond to the FGF-1-replacing factor identified from the conditioned medium of RS-4 cells in our earlier study.


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Fig. 5.   Biological activity of VEGF 115. A, detection of VEGF 115 from NIH 3T3 transfectants by RT-PCR with vector primers. RT-PCR with vector primers yielded a 642-bp band in NIH 3T3/VEGF 115 transfectants (lane 2) but not in control NIH 3T3/vector (lane 1) (a); glyceraldehyde-3-phosphate dehydrogenase-PCR from the two samples yielded equal amplification (b). B, detection of FGF replacing activity for HUVECs in the conditioned medium of transfectants. Lanes represent [3H]thymidine labeling index of HUVECs when cultured in growth medium-GM (MCDB 151) (lane 1), GM supplemented with 15% FCS (lane 2), conditioned medium from NIH 3T3/vector (lane 3), NIH 3T3/VEGF 164 transfectants (lane 4), NIH 3T3/VEGF 115 transfectants (lane 5), and 15% FCS plus 10 ng/ml FGF (lane 6). Each bar represents standard deviations from at least three independent experiments.

VEGF 115, a Novel Alternatively Spliced Form of VEGF Gene-- Mouse P1 library was screened by PCR with primers specific for VEGF 115 and VEGF 164 (Genome Systems, Inc.). These two independent screenings resulted in an isolation of three clones in each case that were analyzed on Southern blot with common probes, as well as probes specific for VEGF 164 and VEGF 115 (Fig. 6). Hybridization of the EcoRI-SalI-SphI, HindIII-PvuII-SpeI, and EcoRI-PstI-XhoI-digested P1 clone with the region of cDNA that is common between VEGF 164 and VEGF 115 (exon 3) yielded only a single band with each of the enzymes, indicating that it is a single copy gene (Fig. 6B), as has been reported earlier (8). Furthermore, the three P1 isolates gave identical result on Southern analysis, indicating that the three clones are identical and represent a mouse VEGF gene. An identical blot was probed with VEGF 115-specific probe (novel sequence). It hybridized to a single band in each of the three digests described above, and the bands were identical for three P1 clones. The pattern was also similar to the one obtained with the exon 3 probe. This result strongly suggests that VEGF 164- and 115-specific sequences align closely on the mouse gene, especially when it is considered that the Southern blot assay was performed on P1 DNA digested with a combination of three restriction enzymes in each lane (Fig. 6). A third, identical blot was hybridized to a VEGF 164-specific (exon 7) probe. It also identified a single band from each of the three above digests. As expected from the VEGF gene structure (8), the size of the band was different from the one identified by exon 3 and VEGF 115 probes. The possibility of occurrence of two genes in a tandem repeat can be ruled out by the fact that the signal on Southern blot with VEGF 164 and VEGF 115 common probes (exon 3) was not double as compared with the one obtained by VEGF 164-specific probes. Thus, these data are strongly suggestive of VEGF 115 as a novel alternatively spliced form of mouse VEGF.


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Fig. 6.   Southern analysis of VEGF P1 clone with VEGF 164 and VEGF 115 common and specific probes. P1 vector DNA, pAD10sacBII (control) and three clones (1, 2, and 3) isolated by PCR screenings were digested with EcoRI-SalI-SphI (lane a), HindIII-PvuII-SpeI (lane b), or EcoRI-PstI-XhoI (lane c) and were probed with VEGF 115 (A), exon 3 (B), and exon 7 (C) probes. A single band was detected in each of the lanes. An identical pattern of hybridization was obtained with exon 3 and VEGF 115 probes, whereas exon 7 gave a different pattern.

Taken together, our results have shown that FGF replacing activity for HUVECs in the conditional medium of RS-4 cells is caused by a novel alternatively spliced form of VEGF, VEGF 115. In addition, an angiogenic function that has been demonstrated for different forms of VEGF, a proliferation-associated function of VEGF 115, is suggested from the up-regulation of VEGF 115 with spontaneous immortalization of fibroblasts and high expression in spleen and testis. A precise mechanism of alternative splicing awaits intron characterization of murine VEGF gene.

    ACKNOWLEDGEMENTS

We thank Dr. J. Fukami for helpful discussions, T. Matsuda for technical assistance, and Dr. S. Kondo (Toagosei Chemical Industry, Co., Ltd.) for anti-VEGF antibody.

    FOOTNOTES

* This work was supported in part by a Center of Excellence, Japan, grant.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. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) MMV50279.

§ To whom correspondence should be addressed. Tel.: 81 298 54 6502; Fax: 81 298 54 6095.

1 The abbreviations used are: VEGF, vascular endothelial growth factor; bp, base pair(s); FGF, fibroblast growth factor; HUVEC, human umbilical vein endothelial cell; CMEF, CD1-ICR mouse embryonic fibroblast; RT, reverse transcription; PCR, polymerase chain reaction; FCS, fetal calf serum.

    REFERENCES
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Abstract
Introduction
Procedures
Results & Discussion
References

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