The alpha -Helical Domain Near the Amino Terminus Is Essential for Dimerization of Vascular Endothelial Growth Factor*

Gerhard SiemeisterDagger , Dieter Marmé, and Georg Martiny-Baron

From the Institute of Molecular Medicine, Tumor Biology Center, D-79106 Freiburg, Germany

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen and a key mediator of aberrant endothelial cell proliferation and vascular permeability in a variety of human pathological situations such as tumor angiogenesis, diabetic retinopathy, or psoriasis. By amino-terminal deletion analysis and by site-directed mutagenesis we have identified a new domain within the amino-terminal alpha -helix that is essential for dimerization of VEGF. VEGF121 variants containing amino acids 8 to 121 or 14 to 121, respectively, either expressed in Escherichia coli and refolded in vitro, or expressed in Chinese hamster ovary cells, were in a dimeric conformation and showed full binding activity to VEGF receptors and stimulation of endothelial cell proliferation as compared with wild-type VEGF. In contrast, a VEGF121 variant covering amino acids 18 to 121, as well as a variant in which the hydrophobic amino acids Val14, Val15, Phe17, and Met18 within the amphipathic alpha -helix near the amino terminus were replaced by serine, failed to form biological active VEGF dimers. From these data we conclude that a domain between amino acids His12 and Asp19 within the amino-terminal alpha -helix is essential for formation of VEGF dimers, and we propose hydrophobic interactions between VEGF monomers to stabilize or favor dimerization.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Vascular endothelial growth factor (VEGF),1 also known as vascular permeability factor, is a mitogen that specifically regulates endothelial cell function. The biological activities of VEGF (for recent reviews, see Refs. 1-4) include stimulation of endothelial cell growth and migration, rapid enhancement of microvascular permeability in vivo, promotion of vasculogenesis and angiogenesis, and induction of differentiation of embryonic stem cells to hematopoietic precursors (5). Recent experiments of targeted disruption of the VEGF gene have demonstrated its essential role for vascular development in the embryo (6, 7). Even inactivation of a single VEGF allele results in defective development of large vessels, defective capillary sprouting, and embryonic lethality. Aberrant elevated expression of VEGF has been observed in a variety of human pathological situations such as tumor angiogenesis (8), diabetic retinopathy (9), rheumatoid arthritis (10), or psoriasis (11). Neutralizing of VEGF by antibodies or recombinant soluble receptor domains have shown therapeutic potential as agents capable of suppressing tumor growth (12) and retinal neovascularization (13). Two homologous cell-surface receptors of the tyrosine kinase family, Flt-1 (VEGFR-1) and KDR (VEGFR-2), bind VEGF with high affinity (14, 15).

VEGF is a homodimeric glycosylated protein that exists in five different isoforms of 121, 145, 165, 189, and 206 amino acids of which the amino-terminal 114 amino acids are identical. Together with placenta growth factor (PlGF) (16) and the recently described VEGF-B (17) and VEGF-C (18) VEGF builds a family of related growth factors which show structural homology to PDGF. In particular, the cysteines building up the structural fold of the proteins consisting of three intramolecular disulfide brigdes, and two intermolecular disulfide brigdes cross-linking the polypeptide chains, are conserved for these growth factors (19, 20). The very recently solved crystal structure of VEGF (21) confirmed the overall structural similarity of VEGF and PDGF. Alignment of VEGF121 and PDGF-B/v-sis amino acid sequences showed a 25% identity of the region Cys26 to Cys104 of VEGF and Cys16 to Cys99 of mature PDGF-B. This region of PDGF-B/v-sis, the minimal v-sis transforming domain, was described to retain biological activity of the growth factor (22, 23). In case of VEGF, it had previously been shown that a plasmin digested 110-amino acid amino-terminal fragment, which was cleaved between Arg110 and Ala111, retains full biological activity as compared with the VEGF121 isoform (24).

To analyze the contribution of amino acids Ala1 to Tyr25, which are located amino-terminal with respect to the homology region of VEGF to the minimal v-sis transforming domain, to structure and/or biological activity of VEGF, we generated amino-terminal truncated VEGF121 variants. Here were show that an amino-terminal domain between amino acids His12 and Asp19 is essential for in vitro dimerization of VEGF and for functional expression of VEGF in vivo. As the conversion of hydrophobic amino acids within this domain to serine impairs formation of VEGF dimers we propose hydrophobic interactions between VEGF monomers to stabilize or favor formation of dimers.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Construction and Bacterial Expression of VEGF Mutant Proteins-- cDNAs encoding amino-terminal truncated VEGF proteins were generated by PCR technique using the 5'-primers VEGF-8 (5'-GAGCCATGGAGAATCATCACGAAGTGGTGAAG-3'), VEGF-14 (5'-CACGCCATGGTGAAGTTCATGGATGTCT-3'), and VEGF-18 (5'-AAGTCCATGGATGTCTATCAGCGCAGC-3'), respectively, the 3'-primer VEGF-Bam (5'-AGTGGATCCCCCCCTGGTGA-3'), and cloned human VEGF121 cDNA (25) as template. Mutant VEGF-V14S/V15S/F17S/M18S was generated by PCR using 5'-primer 207 (5'-TCGGGCCTCCGAAACCATGA-3') and 3'-primer VEGF-N1 (5'-GGCTCCTGAAGCTGCTAAGCACTACTAAGACGGGAGG-3') for amplification of the 5'-fragment and 5'-primer VEGF-N2 (5'-AATCGTCGAAGTCCTCGGATGTCTATCAGCGCAGCTA-3') and 3'-primer VEGF-Bam for amplification of the 3'-fragment. The fragments were purified by agarose gel electrophoresis and used in an equimolar ratio as template for fusion-PCR using 5'-primer 207 and 3'-primer VEGF-Bam. VEGF variant C61S was generated by amplification of the VEGF121 cDNA cloned in pBluescript vector using the 5'-primer C61S (5'-GGCTGCTCCAATGACGAGGGC-3') and the 3'-primer 252c (5'-CCCGCATCGCATCAGGGGCAC-3'). The resulting PCR fragment containing mutant VEGF sequence and vector sequence was phosphorylated by T4 polynucelotide kinase (Pharmacia, Freiburg, Germany), gel purified, religated, and transformed into Escherichia coli XL1-blue. The mutant VEGF cDNAs were cloned into the His-pET vector (26) via NcoI and BamHI sites, and transformed into E. coli BL21DE3 (27). All constructs were verified by DNA sequencing. Solubilization of VEGF proteins from inclusion bodies, refolding, and purification was performed essentially as described for wild-type VEGF121 (26).

Transfection Analysis-- For the construction of eukaryotic expression plasmids NcoI/BamHI fragments encoding mutant VEGF variants were fused to the VEGF signal sequence by substitution of the NcoI/BamHI restriction fragment of wild-type VEGF121 in a plasmid, which contains the entire VEGF121 coding region cloned into the SmaI site of pBluescript (25). The cDNA fragments were released by EcoRI/XbaI restriction endonuclease digestion and ligated into pCI-neo expression vector (Promega, Heidelberg, Germany) which provides a cytomegalovirus promoter and enhancer. Transient transfection of chinese hamster ovary (CHO) cells was performed in six-well plates containing approximately 2 × 105 cells/well, which were incubated at 37 °C overnight in the presence of 2 µg/well of calcium phosphate-precipitated VEGF-variant/pCI-neo DNA. For determination of transfection efficiency and protein secretion 1 µg/well pSBC-2/SEAP expression vector DNA (28) encoding secreted placental alkaline phosphatase (SEAP) was cotransfected. Cell culture supernatant was replaced with serum-free medium, and cells were incubated for 48 h at 37 °C. Conditioned media (2 ml) were harvested, centrifuged, and stored at -80 °C. Total RNA was prepared from transfected cells using an RNeasy kit (Quiagen, Hilden, Germany). Relative SEAP activity was determined as optical density at 405 nm of heat-inactivated (5 min at 65 °C) aliquots (100 µl) of conditioned media, which were incubated for 30-60 min at room temperature with 100 µl of SEAP-buffer (1 M diethanolamine, 10 mM homoarginine, 1.5 mM MgCl2, 23 mM p-nitrophenyl phosphate). Aliquots (30 µl) of conditioned media were electrophoresed on 15% SDS-polyacrylamide gels under nonreducing conditions, electrotransferred to nitrocellulose Hybond-N membranes (Amersham, Braunschweig, Germany), probed with the polyclonal antiserum K7.16 (29) raised against human VEGF, and detected using the ECL detection system (Amersham, Braunschweig, Germany). Semiquantitative reverse-transcriptase PCR for determination of VEGF RNA in transfected cells was performed by converting 1 µg of total RNA to cDNA using a first-strand cDNA synthesis kit (Pharmacia, Freiburg, Germany) with random hexanucleotide primers followed by PCR amplification of a 247-base pair VEGF cDNA fragment and a 397-base pair GAPDH cDNA fragment simultaneously using the primers VEGF-E3 (5'-GGTGGACATCTTCCAGGAGTACCC-3'), VEGF-E5R (5'-TTCTTGTCTTGCTCTATCTTTCTTTG-3'), GAPDH1 (5'-AGCGAGACCCCACTAACATCAAA-3'), and GAPDH2 (5'-GTGGATGCAGGGATGATGTTCTG-3').

Binding Assays-- Recombinant extracellular domain of human VEGF receptor Flt-1 (30) and KDR,2 respectively, were coated onto Maxisorb plates (1 µg/well) and were incubated with biotinylated VEGF165 (10 ng/ml) in the presence of increased concentrations of VEGF121 proteins as described previously (30).

Thymidine Incorporation Assays-- Quiescent HUVE cells were stimulated with increased concentrations of VEGF121 proteins. After 18 h of VEGF-incubation [3H]thymidine (0.5 µCi) was added and the incubation was continued for additional 6 h. The cells were washed and the incorporation of radioactivity was determined by scintillation counting.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Characterization of Amino-terminal VEGF121 Variants Expressed in E. coli-- Amino-terminal truncated VEGF121 variants were expressed in E. coli using the T7 RNA-polymerase driven pET system (27). The proteins were solubilized from inclusion body material, refolded, and finally purified by Ni2+-affinity chromatography essentially as described previously for wild-type VEGF121 (26). The E. coli derived "wild-type" VEGF121 in fact covered amino acids Met3 to Arg121 of mature human VEGF121 and showed virtually identical biological activity as compared with human VEGF121 covering amino acids 1 to 121 produced by recombinant techniques in Sf9 insect cells (26). The VEGF variants VEGF121Delta 1-7 contained amino acids 8 to 121 of mature human VEGF121 with Gly8 changed to Met, and Gln9 changed to Glu due to insertion of an NcoI restriction endonuclease site, VEGF121Delta 1-13 contained amino acids 14-121 with Val14 changed to Met, and VEGF121Delta 1-17 contained amino acids 18-121 (Fig. 1A). For control, VEGF121-C61S was prepared, a monomeric VEGF mutant with Cys61 replaced by serine which did not bind to endothelial cells and showed no significant biological activity (19). An additional amino-terminal His6 tag facilitated affinity purification of the recombinant proteins. On nonreducing SDS gels VEGF121, VEGF121Delta 1-7, and VEGF121Delta 1-13 migrated with apparent molecular masses of approximately 34, 32, and 30 kDa, respectively, which correspond to the molecular mass of VEGF121 dimers (Fig. 1B, lanes 1, 3, and 4). In contrast, VEGF121Delta 1-17 (Fig. 1B, lane 5), as well as the control, VEGF121-C61S (Fig. 1B, lane 2), migrated as monomers of approximately 16 kDa. Upon electrophoresis on reducing SDS gels all of the VEGF variants migrated as monomeric forms of approximately 16-17 kDa (not shown).


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Fig. 1.   SDS-polyacrylamide gel electrophoresis analysis of amino-terminal VEGF variants. A, amino-terminal amino acid sequences of VEGF variants expressed in E. coli. The numbering corresponds to mature human VEGF; vector-derived amino-terminal His-tag sequence is shown in italics. B, VEGF121 (1), amino-terminal truncated variants VEGF121Delta 1-7 (3), VEGF121Delta 1-13 (4), and VEGF121Delta 1-17 (5), and the monomeric VEGF mutant VEGF121-C61S (2) were expressed in E. coli, refolded from inclusion body material, purified by Ni2+-affinity chromatography, and electrophoresed on a 15% nonreducing SDS-polyacrylamide gel. C, comparison of electrophoretic mobility of a mutant VEGF121 in which the hydrophobic amino acids Val14, Val15, Phe17, and Met18 had been replaced by serine (VEGF121-Phi /S; lane 4) to VEGF121 (1), VEGF121Delta 1-13 (2), and VEGF121Delta 1- 17 (3).

Inspection of the amino-terminal amino acid sequence using a structure predicting software (31) revealed an interspersed sequence of charged (His12, Glu13, Lys16, and Asp19) and hydrophobic (Val14, Val15, Phe17, and Met18) residues which might anticipate an amphipathic alpha -helical conformation (Fig. 2). Deletion of this domain apparently prevents dimerization of VEGF, at least in vitro. The interaction of the hydrophobic interface of the proposed amphipathic alpha -helix of one VEGF monomer either with the corresponding domain or with another hydrophobic stretch of the other VEGF monomer might stabilize or favor dimerization of VEGF. To further support this model we constructed a VEGF121 variant (VEGF121-Phi /S) covering amino acids Met3 to Arg121 in which we replaced the hydrophobic amino acids Val14, Val15, Phe17, and Met18 by serine. On nonreducing SDS gels this variant migrated most predominantly as a monomer (Fig. 1C, lane 4) as the deletion mutant VEGF121Delta 1-17 (lane 3) did, and in contrast to dimeric VEGF121 and VEGF121Delta 1-13 (lanes 1 and 2). From these data we concluded that the amino-terminal domain between His12 and Asp19 is essential at least for in vitro dimerization of VEGF.


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Fig. 2.   Amphipathic alpha -helical conformation predicted for the VEGF amino-terminal domain between His12 and Asp19.

Amino-terminal Domain His12 to Asp19 Is Essential for Functional Expression of VEGF in Vivo-- To analyze whether the amino-terminal domain between His12 and Asp19 is also essential for in vivo dimerization of VEGF the variant VEGF121 cDNAs were fused to the VEGF signal sequence, ligated into pCI-neo expression vector providing cytomegalovirus promoter/enhancer sequences, and transiently transfected into CHO cells. Due to fusion to the signal sequence the resulting expression plasmid pCI-neo/VEGF121 encoded the entire human VEGF121 coding region, whereas in construct pCI-neo/VEGF121Delta 3-13 amino acids Met3 to Glu13 had been deleted and Val14 was replaced by Met, and in construct pCI-neo/VEGF121Delta 3-18 amino acids Met3 to Phe17 had been deleted. Construct pCI-neo/VEGF121-Phi /S encoded the entire VEGF121 coding sequence in which the hydrophobic amino acids Val14, Val15, Phe17, and Met18 had been replaced by serine. Immunoblot analysis of conditioned media of the transfected cells using the polyclonal antiserum K7.16 raised against human VEGF protein showed that VEGF121 and VEGF121Delta 3-13 migrated under nonreducing conditions as dimers (Fig. 3A, lanes 1 and 2). In contrast, in conditioned media of pCI-neo/VEGF121Delta 3-18 and pCI-neo/VEGF121-Phi /S, as well as in pCI-neo vector control transfected CHO cells, no VEGF protein was detectable by Western blot analysis (Fig. 3A, lanes 3-5). Western blot analysis of VEGF variants expressed in E. coli showed that the antiserum used was able to recognize all of the various VEGF variants including the monomeric ones (data not shown). Measurement of SEAP activity in conditioned media of the transfected cells revealed efficient transfection of the cells and efficient secretion of proteins even in transfections in which VEGF protein was not detectable in the conditioned medium (Fig. 3B). Using lysates of transfected cells for Western blot analysis neither wild-type VEGF nor mutant VEGF variants were detectable demonstrating that wild-type VEGF and VEGF121Delta 3-13 were secreted efficiently by the cells, and that the VEGF variants VEGF121Delta 3-18 and VEGF121-Phi /S were not accumulated within the cells (not shown). Expression of transfected constructs for the VEGF variants at the level of mRNA was shown by semiquantitative reverse-transcriptase PCR (Fig. 3C). Transfection of human "293" embryonic kidney cells gave similar results (not shown). Taken together these results show that the amino-terminal His12 to Asp19 domain is essential for functional expression of VEGF at least in transfected CHO and 293 cells. Truncation or mutation of this domain either impairs synthesis of VEGF or the cells recognize these variants as aberrant proteins which were apparently degraded.


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Fig. 3.   Transfection analysis of amino-terminal VEGF variants in CHO cells. A, SDS-polyacrylamide gel electrophoresis immunoblot analysis. Conditioned media from CHO cells which were transiently transfected with expression plasmids encoding human VEGF variants VEGF121 (1), VEGF121Delta 3-13 (2), VEGF121Delta 3-17 (3), and VEGF121-Phi /S (4), respectively, and with pCI-neo-vector as a control (5), and which were cotransfected with pSBC-2/SEAP, were electrophoresed on a 15% nonreducing SDS-polyacrylamide gel, transferred to a nitrocellulose membrane, and probed with a polyclonal antiserum raised against human VEGF. B, relative SEAP activity in conditioned media of transfected CHO cells. C, semiquantitative reverse-transcriptase PCR analysis of VEGF mRNA and GAPDH mRNA levels in total RNA isolated from transfected CHO cells.

Binding of VEGF Variants to VEGF Receptors and Stimulation of Proliferation of HUVE Cells-- A PDGF-B variant in which the two cysteines involved in interchain disulfide bonds had been converted to serine, migrated as a monomer on non-reducing SDS gels, but exists as a noncovalent dimer at pH 4-7 in solution and shows similar mitogenic activity as compared with wild-type PDGF-BB (31, 32). To analyze the biological activity of the amino-terminal VEGF121 variants receptor binding assays and proliferation assays were performed. Binding of the VEGF variants expressed in E. coli to recombinant extracellular domain of human VEGF receptors Flt-1 (Fig. 4A) and KDR (Fig. 4B), respectively, was studied by competition assays with biotinylated VEGF165. The dimeric VEGF variants VEGF121Delta 1-7 and VEGF121Delta 1-13 competed with biotinylated VEGF for binding of both of the VEGF receptors in an almost undistinguishable manner as compared with wild-type VEGF121. Binding of the monomeric variant VEGF121Delta 1-17, as well as the monomeric control VEGF121-C61S, to the receptors was strongly impaired. Growth of HUVE cells was stimulated by the VEGF variants VEGF121Delta 1-7 and VEGF121Delta 1-13 in a dose dependent manner which was almost undistinguishable from wild-type VEGF121 stimulated growth of the cells. The monomeric variants VEGF121Delta 1-17 and VEGF121-C61S failed to induce proliferation of HUVE cells (Fig. 4C). Taken together these results show that truncation or mutation of the VEGF amino-terminal alpha -helical domain prevents the formation of stable VEGF dimers although the cysteines involved in formation of the core structure of VEGF had not been affected. Dimerization of VEGF had previously been shown to be a prerequisite for biological activity (19).


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Fig. 4.   Competition binding of VEGF variants to VEGF receptors and stimulation of endothelial cells. Recombinant extracellular domain of VEGF receptors Flt-1 (A) and KDR (B), respectively, coated onto Maxisorb plates were incubated with biotinylated VEGF165 (10 ng/ml) in the presence of increased concentrations of various VEGF121 variants expressed in E. coli. C, quiescent HUVE cells were stimulated with increased concentrations of various VEGF121 variants, and growth factor stimulated DNA synthesis was measured as [3H]thymidine incorporation. VEGF variants used were VEGF121 (filled circles), VEGF121Delta 1-7 (open triangles), VEGF121Delta 1-13 (open squares), VEGF121Delta 1-17 (open diamonds), and VEGF121-C61S (filled triangles).

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

The analysis of amino-terminal truncated VEGF121 variants revealed a domain between amino acids His12 and Asp19 that is essential for in vitro dimerization of VEGF and for functional expression of VEGF in vivo. This domain showed an interspersed sequence of charged and hydrophobic amino acids which may anticipate an amphipathic alpha -helical conformation. We postulate that the interaction of hydrophobic interfaces stabilize or favor dimerization of VEGF. Conversion of the hydrophobic amino acids to serine by site-directed mutagenesis, which results in VEGF monomers, supports this model. The very recently by Muller et al. (21) solved crystal structure of VEGF indeed revealed that the amino acids near the amino terminus anticipate an alpha -helical conformation. The crystal structure shows that hydrophobic amino acids from this alpha -helix together with residues from helix alpha 2, loop regions beta 1-beta 3, and beta 5-beta 6, and sheet beta 6 of the other monomer form a small hydrophobic core that presumably stabilizes the central structure of the VEGF dimer. In addition, they found that a Phe17 to alanine mutant VEGF displayed on a phage surface lost KDR receptor binding implicating a contribution of Phe17 to receptor binding. As the conformation of the Phe17 right-arrow Ala mutant displayed on the phage surface was not investigated, our results implicate that loss of KDR binding is more likely due to impaired dimerization of the mutant VEGF.

The His12 to Asp19 domain is highly conserved between human, sheep (34), porcine (35), bovine (36), and mouse (37) VEGF (Fig. 5). Similar interspersed sequences of charged and hydrophobic amino acids are located amino-terminal with respect to the v-sis homology regions of the VEGF-related growth factors PlGF (16), VEGF-B (17), and VEGF-C (18, 38). Heterodimerization of VEGF and PlGF has shown to occur in vitro (39, 40) and in vivo (41), as well as heterodimerization of VEGF and VEGF-B was reported for cells expressing both growth factors (17). Heterodimerization of the members of the family of VEGF-related growth factors is thought to contribute to a fine tuning of angiogenic stimuli (41). Although the involvement of the amino-terminal domain in the heterodimerization between various VEGF-related growth factor monomers has not been investigated so far, the similarity of the amino-terminal domains of the VEGF-related proteins suggest that heterodimerization would be supported by these domains. Heterodimerization of VEGF and PDGF-B has not been observed so far although the eight cysteines building the core structure are perfectly conserved. One of the most significant differences in the crystal structure of VEGF (21) and PDGF-B (42) is the structure of the amino terminus which is extended in PDGF rather than alpha-helical as in VEGF, whereas the monomer topology and side-by-side dimer association is highly similar for both proteins. In the PDGF-BB dimer the extended amino terminus of one chain makes contact to the other PDGF chain, but in contrast to VEGF, the amino terminus is not essential for dimerization of PDGF as it was shown by mutagenic analyses which resulted in the identification of the minimal v-sis transforming domain (22, 23). Analysis of the crystal structure of transforming growth factor (TGF)-beta 2 (43), which is besides PDGF, VEGF, and nerve growth factor, another member of the cystine knot family of growth factors, revealed a close contact of alpha -helix H3 of one TGF-beta 2 chain to beta -strand structures of the other chain in the TGF-beta 2 dimer, which presumably stabilizes the dimeric structure. alpha -Helix H3 is located within TGF-beta 2 at a position that correlates to loop/turn II in PDGF-B and VEGF.


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Fig. 5.   Alignment of VEGF, PlGF, VEGF-B, and VEGF-C. The amino-terminal 25 amino acids of mature human VEGF are shown aligned with the corresponding sequence of sheep VEGF, porcine VEGF, bovine VEGF, mouse VEGF, human PlGF, human VEGF-B, and human VEGF-C as derived using the FASTP software (53). The His12 to Asp19 domain is boxed, hydrophobic amino acids within this domain are shaded. Identical amino acids with respect to the human VEGF sequence are indicated by colons (:). Conservative amino acid substitutions are indicated by full stops (.).

Interference with the VEGF/VEGF-receptor system is generally viewed as an attractive target for therapeutical intervention in a variety of human pathological situations which involve elevated VEGF expression and aberrant endothelial cell proliferation such as tumor angiogenesis, diabetic retinopathy, rheumatoid arthritis, or psoriasis. Enhanced VEGF expression is induced by various factors and growth conditions including growth factors (see Finkenzeller et al. (44), and references therein), activated oncogenes (28, 45, 46), inactivated tumor suppressor genes (47, 48), and hypoxia (49). Diverse mechanisms acting at the level of promoter activation (44), mRNA stabilization (50, 51), and translational regulation (52) have been shown to be involved in up-regulation of VEGF expression. The multiplicity of factors and mechanisms involved in VEGF expression hamper the development of therapeutical approaches directed to reduce enhanced expression of the growth factor in pathological settings. As VEGF dimerization is an event that is necessary for biological activity of the growth factor, but is independent from the diverse mechanisms of regulation of gene expression and translation, prevention of dimerization by interference with the amino-terminal domain may be a promising strategy for therapeutical down-regulation of VEGF expression.

    ACKNOWLEDGEMENTS

We thank Steffi Koidl, Gabi Bader, and Katja Mohrs for excellent technical assistance.

    FOOTNOTES

* This work was supported by Kirstins Weg.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.

Dagger To whom correspondence should be addressed: Institute of Molecular Medicine, Tumor Biology Center, Breisacher Str. 117, D-79106 Freiburg, Germany. Tel.: 49-761-206-1711; Fax: 49-761-206-1705.

1 The abbreviations used are: VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor; CHO, chinese hamster ovary; Flt-1, fms-like tyrosine kinase-1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HUVE, human umbilical vein endothelial; KDR, kinase domain receptor; PCR, polymerase chain reaction; PDGF, platelet-derived growth factor; PlGF, placenta growth factor; SEAP, secreted placental alkaline phosphatase; TGF, transforming growth factor.

2 G. Martiny-Baron, B. Barleon, F. Totzke, D. Marmé, and G. Siemeister, unpublished result.

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Abstract
Introduction
Procedures
Results
Discussion
References

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