The oral multitarget tumour growth inhibitor, ZK 304709, inhibits growth of pancreatic neuroendocrine tumours in an orthotopic mouse model.

BACKGROUND AND AIMS: Current systemic therapies for neuroendocrine tumours (NETs) do not provide sufficient control of tumour growth. However, efficient evaluation of novel drugs is hindered by the lack of a suitable preclinical animal model. Here an orthotopic mouse model of pancreatic NET is established and used to study the action of ZK 304709, a first in class, oral multitarget tumour growth inhibitor. ZK 304709 is an inhibitor of cyclin-dependent kinases (Cdks) 1, 2, 4, 7 and 9, vascular endothelial growth factor receptor-type kinases (VEGF-RTKs) 1-3 and platelet-derived growth factor receptor-type kinase beta (PDGF-RTKss). METHODS: BON and QGP-1 human NET cells were used to study proliferation, survival and cell cycle distribution in vitro. For induction of orthotopic NETs, BON cells were injected into the pancreas of NMRI(nu/nu) mice. Primary tumour growth and metastatic spread were recorded after 9 weeks, and apoptosis, microvessel density and lymphatic vessel density were determined. RESULTS: ZK 304709 dose-dependently suppressed proliferation and colony formation of NET cells. Direct effects on NET cells were consistent with Cdk inhibition and involved G(2) cell cycle arrest and apoptosis induction, which was associated with reduced expression of MCL1 (myeloid cell leukaemia sequence 1), survivin and hypoxia-inducible factor 1alpha (HIF1alpha). Apoptosis similarly occurred in vivo in ZK 304709-treated orthotopic BON tumours, resulting in a 80% reduction of primary tumour growth. In contrast, treatment with lanreotide or 5-fluorouracil and streptozotocin failed to inhibit tumour gowth. ZK 304709 also reduced tumour microvessel density, implicating antiangiogenic mechanisms. CONCLUSION: BON orthotopic tumours provide an informative model for preclinical drug evaluation in NETs. In this model, ZK 304709 achieved efficacious tumour growth control via induction of apoptosis and inhibition of tumour-induced angiogenesis.

Molecular and pharmacodynamic characteristics of the novel multi-target tumor growth inhibitor ZK 304709.

Loss of cell cycle control and tumor-induced neovascularization are major drivers of human tumor growth. The multi-target tumor growth inhibitor ZK 304709 is a nanomolar inhibitor of cyclin-dependent kinases 1, 2, 4, 7 and 9, as well as vascular endothelial growth factor receptor tyrosine kinase 1-3 and of platelet-derived growth factor receptor beta tyrosine kinase. The multi-targeted mode of action of ZK 304709 acting on cell cycle and angiogenesis resulted in superior efficacy compared to standard chemotherapeutic compounds both in s.c. human tumor xenografts as well as orthotopic human pancreatic carcinoma models.

Vascular endothelial growth factor up-regulates its receptor fms-like tyrosine kinase 1 (FLT-1) and a soluble variant of FLT-1 in human vascular endothelial cells.

The growth of solid tumors and the formation of metastases are dependent on neoangiogenesis. One of the most important factors in inducing the formation of new blood vessels is the vascular endothelial growth factor (VEGF), which acts specifically on endothelial cells. VEGF is expressed and secreted by almost all solid tumors. The molecular mechanisms leading to enhanced production of this angiogenic mitogen are manyfold and have been elucidated to some degree. Two VEGF receptors, fms-like tyrosine kinase 1 (FLT-1) and KDR, have been identified almost specifically on human endothelial cells. They are expressed preferentially in the proliferating endothelium of vessels lining and/or penetrating solid tumors, whereas they are almost undetectable by convenient methods in vessels of healthy tissue. However, the underlying mechanisms are not understood. We could show that media conditioned by various cancer cell lines grown under hypoxic conditions were able to up-regulate expression of FLT-1 mRNA and protein but not of KDR mRNA. Furthermore, up-regulation of a shorter mRNA species was observed that most probably codes for the soluble variant of FLT-1. These effects were completely inhibited by VEGF-neutralizing extracellular VEGF receptor domains. The effect could be mimicked by adding recombinant VEGF instead of conditioned cancer cell medium to the endothelial cell cultures. Both mutant VEGF, which activates only KDR, and placenta growth factor, which activates only FLT-1, were able to enhance FLT-1 expression. VEGF-stimulated FLT-1 mRNA expression was inhibited by actinomycin D. These data suggest that VEGF itself is the main factor secreted by tumor cells that is able to enhance the expression of its receptor FLT-1 and of a soluble variant of FLT-1 in endothelial cells.

Reversion of deregulated expression of vascular endothelial growth factor in human renal carcinoma cells by von Hippel-Lindau tumor suppressor protein.

Mutations or loss of both alleles of the von Hippel-Lindau (VHL) tumor suppressor gene has been documented in sporadic renal cell carcinomas and neoplasms that arise in individuals having the VHL syndrome. The well-vascularized phenotype of tumors that form in VHL disease let us consider vascular endothelial growth factor (VEGF) as a mediator of tumor growth in VHL disease. Human renal carcinoma cells that either lacked endogenous wild-type VHL or were transfected with an inactive mutant VHL showed deregulated expression of VEGF on the mRNA and protein level that was reverted by introduction of wild-type VHL. Stimulation of proliferation of endothelial cells by conditioned medium of cells expressing mutant VHL was almost abolished by neutralizing the VEGF. In contrast, expression of basic fibroblast growth factor and of c-myc proto-oncogene was not affected by VHL. Our data suggest VEGF as the key tumor angiogenesis factor in VHL disease.

Two independent mechanisms essential for tumor angiogenesis: inhibition of human melanoma xenograft growth by interfering with either the vascular endothelial growth factor receptor pathway or the Tie-2 pathway.

Protein ligands and receptor tyrosine kinases that specifically regulate endothelial cell function are mainly involved in physiological as well as in disease-related angiogenesis. These ligand/receptor systems include the vascular endothelial growth factor (VEGF) and the angiopoietin (Ang) families, and their receptors, the VEGF receptor family and the tyrosine kinase with immunoglobulin-like and epidermal growth factor homology domains (Tie) family. In the present study, the contribution of these endothelium-specific ligand/receptor systems to tumor angiogenesis was evaluated. A375v human melanoma cells, which express at least the angiogenic growth factors VEGF, VEGF-C, and Ang-1, were stably transfected to overexpress the extracellular ligand-binding domains of the endothelium-specific receptor tyrosine kinases fms-like tyrosine kinase-1 (Flt-1), Flt-4, Tie-1, and Tie-2, respectively. In vitro proliferation and colony formation assays confirmed that expression of the extracellular receptor domains inhibited neither tumor cell mitogenesis nor the ability to produce anchorage-independent growth. Nude mouse xenografts revealed that interference with either the VEGF receptor pathway or the Tie-2 pathway resulted in a significant inhibition of tumor growth and tumor angiogenesis. In contrast, interference with the Flt-4 pathway or the Tie-1 pathway was without significant effect. Our results show that both the VEGF receptor pathway and the Tie-2 pathway are essential for A375v melanoma xenograft growth. The inhibition of the VEGF receptor pathway cannot be compensated by the Tie-2 pathway, nor vice versa. These findings suggest that the VEGF receptor pathway and the Tie-2 pathway have to be considered as two independent mediators essential for the process of in vivo angiogenesis.

PTK787/ZK 222584, a novel and potent inhibitor of vascular endothelial growth factor receptor tyrosine kinases, impairs vascular endothelial growth factor-induced responses and tumor growth after oral administration.

PTK787/ZK 222584 (1-[4-chloroanilino]-4-[4-pyridylmethyl] phthalazine succinate) is a potent inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinases, active in the submicromolar range. It also inhibits other class III kinases, such as the platelet-derived growth factor (PDGF) receptor beta tyrosine kinase, c-Kit, and c-Fms, but at higher concentrations. It is not active against kinases from other receptor families, such as epidermal growth factor receptor, fibroblast growth factor receptor-1, c-Met, and Tie-2, or intracellular kinases such as c-Src, c-Abl, and protein kinase C-alpha. PTK787/ZK 222584 inhibits VEGF-induced autophosphorylation of kinase insert domain-containing receptor (KDR), endothelial cell proliferation, migration, and survival in the nanomolar range in cell-based assays. In concentrations up to 1 microM, PTK787/ZK 222584 does not have any cytotoxic or antiproliferative effect on cells that do not express VEGF receptors. After oral dosing (50 mg/kg) to mice, plasma concentrations of PTK787/ZK 222584 remain above 1 microM for more than 8 h. PTK787/ZK 222584 induces dose-dependent inhibition of VEGF and PDGF-induced angiogenesis in a growth factor implant model, as well as a tumor cell-driven angiogenesis model after once-daily oral dosing (25-100 mg/kg). In the same dose range, it also inhibits the growth of several human carcinomas, grown s.c. in nude mice, as well as a murine renal carcinoma and its metastases in a syngeneic, orthotopic model. Histological examination of tumors revealed inhibition of microvessel formation in the interior of the tumor. PTK787/ZK 222584 is very well tolerated and does not impair wound healing. It also does not have any significant effects on circulating blood cells or bone marrow leukocytes as a single agent or impair hematopoetic recovery after concomitant cytotoxic anti-cancer agent challenge. This novel compound has therapeutic potential for the treatment of solid tumors and other diseases where angiogenesis plays an important role.

Sp1 recognition sites in the proximal promoter of the human vascular endothelial growth factor gene are essential for platelet-derived growth factor-induced gene expression.

Stimulation of NIH3T3 cells with platelet-derived growth factor (PDGF)-BB enhances expression of vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen and a key mediator of tumor angiogenesis. Here, we identified cis-acting VEGF promoter elements and trans-acting factors which are involved in PDGF-stimulated VEGF expression. By 5'-deletion and transient transfection analysis, a G + C-rich region at -85 to -50 of the human VEGF promoter was shown to be necessary and sufficient for both PDGF inducible and basal expression. The region contains three potential recognition sites for Sp1 transcription factors, which overlap with two Egr-1 sites. Mutations that abolish the ability of Sp1 to interact with the VEGF promoter element also abrogate expression induced by PDGF. Mutations of the potential Egr-1 binding sites did not affect PDGF responsiveness. Gel shift and antibody supershift analyses showed that Sp1 and Sp3 interact constitutively with the VEGF promoter element. Our data strongly suggest that enhanced VEGF gene expression in PDGF-induced NIH3T3 cells is mediated by Sp1 and/or Sp3 transcription factors bound to the -85 to -50 promoter region of the VEGF gene.

Targeting of endothelial KDR receptors with 3G2 immunoliposomes in vitro.

Immunoliposomes (IL) containing anti-angiogenic drugs directed selectively to the easily accessible kinase insert domain containing receptor (KDR) vascular endothelial growth factor (VEGF), which is predominantly expressed on tumour vessels are a promising tool to inhibit tumour angiogenesis. To explore this strategy, we have prepared fluorescent-labelled IL presenting antibodies against the KDR receptor (3G2) on their surface. 3G2-IL were composed of egg phosphatidylcholine and cholesterol (6:4), containing 2 mol% of the new thiol reactive linker lipid O-(3-cholesteryloxycarbonyl)propionyl-O'-m-maleimido-benzoyl tetraethylene glycol. Specific binding of 3G2-IL to immobilised recombinant KDR was used to show the maintenance of sufficient immunoreactivity of 3G2 antibodies upon the coupling procedure. 3G2-IL bound to Chinese hamster ovarian (CHO) cells stably transfected to overexpress KDR to a five times higher amount as compared to mock-transfected CHO cells. Subsequently, specific binding of 3G2-IL to KDR could also be demonstrated on KDR expressing cells, human umbilical vein endothelial cells and human microvascular endothelial cells, whereas only low binding of 3G2-IL to NIH-3T3 mouse fibroblast cells, which do not express KDR, was found. The binding of 3G2-IL to KDR receptors could not be blocked by VEGF, suggesting that the binding site for VEGF is not identical with the epitope recognised by 3G2. We could demonstrate that 3G2-IL is able to bind in vitro even in the presence of high levels of VEGF.

Sequence and expression of a glyceraldehyde-3-phosphate dehydrogenase-encoding gene from quail embryo fibroblasts.

Using differential hybridization techniques, a cDNA library derived from a line of v-myc-transformed quail embryo fibroblasts was screened for clones whose expression was elevated in transformed, as compared with normal, cells. One of the isolated clones contained the entire coding region of the quail glyceraldehyde-3-phosphate dehydrogenase-encoding gene (GAPDH). A comparison of the deduced 333-amino-acid (aa) sequence of quail GAPDH with that of the only other avian (chicken) GAPDH sequence known, and with those of mammalian counterparts indicates the strong aa sequence conservation of this glycolytic enzyme. GAPDH is expressed in all transformed and non-transformed quail and chicken embryo fibroblasts and macrophages tested, with a moderate elevation of expression in most of the transformed cell lines. In the avian genomes, GAPDH is present in a single copy, in contrast to the high number of GAPDH-related sequences in mammalian species.

Characterization of a new potent, in vivo neutralizing monoclonal antibody to human vascular endothelial growth factor.

Vascular endothelial growth factor (VEGF) is an important mediator of tumor-induced angiogenesis and represents a potential target for anticancer therapy. Therefore, we prepared a panel of monoclonal antibodies (mAb) against both the VEGF121 and VEGF165 isoforms. Three of them completely neutralized the mitogenic stimulation by VEGF of human umbilical vein endothelial cells at mAb concentrations below 0.1 microg/ml. The most potent one, with a dissociation constant (Kd) of 8 pM, inhibited, in a dose-dependent manner, VEGF-induced angiogenesis in a growth factor implant model in mice. A complete inhibition of the angiogenic response was obtained by daily intraperitoneal injections of 10 microg mAb/mouse. Angiogenesis induced by basic fibroblast growth factor was not inhibited by the mAb. Epitope mapping of the mAb, performed by competitive enzyme-linked immunosorbent assay and Western blot analysis, showed that it did not bind to the reduced and denatured monomer of VEGF. Substitutions of three residues (Q87R, G88K, Q89K), located on the major surface loop beta5 to beta6 of VEGF, resulted in the complete loss of binding (more than 400-fold reduction). The results suggest that the mAb binds primarily to a conformation-dependent epitope on the VEGF dimeric form, encompassing one of the loop regions involved in KDR receptor binding. The mAb with its strong neutralizing properties represents a useful agent for effective blocking of VEGF-mediated tumor neovascularization.

Novel antibodies directed against the extracellular domain of the human VEGF-receptor type II.

Vascular endothelial cell growth factor (VEGF) plays a pivotal role in the regulation of angiogenesis by binding to its cognate receptor molecule type II (VEGFr-II, KDR). VEGFr-II is an endothelial cell-specific transmembrane tyrosine kinase important for vascular endothelial cell development and differentiation during embryogenesis, angiogenic processes under physiological conditions, and various diseases. An increasing number of reports indicate that VEGF/VEGFr-II also play a fundamental role for tumor angiogenesis. We present the generation and in vitro characterization of the monoclonal antibodies 2-7-9 and 2-10-1. Both antibodies are highly specific for VEGFr-II as demonstrated by Western blotting and immunoprecipitation. MAbs 2-10-1 and 2-7-9 bind to a disulphide bridge-stabilized epitope within domains 6 and 7 of the human VEGFr-II with an affinity of 8 and 80 nM, respectively. Furthermore, the antibodies are suitable for immunohistochemistry and ELISA techniques. Because both antibodies recognize their epitope on living cells, they also have the potential for drug targeting and diagnostic purposes.

Characterization of novel MPS1 inhibitors with preclinical anticancer activity.

Monopolar spindle 1 (MPS1), a mitotic kinase that is overexpressed in several human cancers, contributes to the alignment of chromosomes to the metaphase plate as well as to the execution of the spindle assembly checkpoint (SAC). Here, we report the identification and functional characterization of three novel inhibitors of MPS1 of two independent structural classes, N-(4-{2-[(2-cyanophenyl)amino][1,2,4]triazolo[1,5-a]pyridin-6-yl}phenyl)-2-phenylacetamide (Mps-BAY1) (a triazolopyridine), N-cyclopropyl-4-{8-[(2-methylpropyl)amino]-6-(quinolin-5-yl)imidazo[1,2-a]pyrazin-3-yl}benzamide (Mps-BAY2a) and N-cyclopropyl-4-{8-(isobutylamino)imidazo[1,2-a]pyrazin-3-yl}benzamide (Mps-BAY2b) (two imidazopyrazines). By selectively inactivating MPS1, these small inhibitors can arrest the proliferation of cancer cells, causing their polyploidization and/or their demise. Cancer cells treated with Mps-BAY1 or Mps-BAY2a manifested multiple signs of mitotic perturbation including inefficient chromosomal congression during metaphase, unscheduled SAC inactivation and severe anaphase defects. Videomicroscopic cell fate profiling of histone 2B-green fluorescent protein-expressing cells revealed the capacity of MPS1 inhibitors to subvert the correct timing of mitosis as they induce a premature anaphase entry in the context of misaligned metaphase plates. Hence, in the presence of MPS1 inhibitors, cells either divided in a bipolar (but often asymmetric) manner or entered one or more rounds of abortive mitoses, generating gross aneuploidy and polyploidy, respectively. In both cases, cells ultimately succumbed to the mitotic catastrophe-induced activation of the mitochondrial pathway of apoptosis. Of note, low doses of MPS1 inhibitors and paclitaxel (a microtubular poison) synergized at increasing the frequency of chromosome misalignments and missegregations in the context of SAC inactivation. This resulted in massive polyploidization followed by the activation of mitotic catastrophe. A synergistic interaction between paclitaxel and MPS1 inhibitors could also be demonstrated in vivo, as the combination of these agents efficiently reduced the growth of tumor xenografts and exerted superior antineoplastic effects compared with either compound employed alone. Altogether, these results suggest that MPS1 inhibitors may exert robust anticancer activity, either as standalone therapeutic interventions or combined with microtubule-targeting chemicals.

Organization and nucleotide sequence of ribosomal RNA genes on a circular 73 kbp DNA from the colourless flagellate Astasia longa.

Three tandemly arranged repeats (A, B, C) of 16S and 23S rDNA, and one supplementary (S) 16S rDNA adjacent to the 16S rDNA of repeat A, are present within an 18 kbp segment of a circular 73 kbp DNA from the colourless flagellate Astasia longa. The repeat units are separated by a short region containing a 5S rRNA gene and a gene for tRNA-Val (UAC). Sequence comparisons reveal 78%, 81%, and 67% identical nucleotides of the 23S rDNA (A), the 16S rDNA (B), and the 5S rDNA (A), respectively, with the corresponding genes of the Euglena gracilis chloroplast genome. As in Euglena chloroplasts, the 3'-terminal portion of the 23S rDNA is homologous to the 4.5S rRNA gene of higher plant chloroplast genomes. These results are supportive of a common evolutionary origin for the Astasia 73 kbp DNA and the Euglena 145 kbp chloroplast DNA.

Structure and expression of a gene encoding the large subunit of ribulose-1,5-bisphosphate carboxylase (rbcL) in the colourless euglenoid flagellate Astasia longa.

A gene encoding the large subunit of ribulose-1,5-bisphosphate carboxylase (Rubisco) was identified on a circular 73 kb DNA from the colourless euglenoid flagellate Astasia longa. The rbcL gene of Astasia extends over 3968 bp. It is a split gene interrupted by seven introns as compared to nine intervening sequences in the rbcL gene of the phylogenetically related Euglena gracilis. Coding sequences as well as the positions of the introns within this gene are highly conserved in comparison with the Euglena rbcL except that two introns are missing in Astasia. The alignment of the amino acid sequences deduced from the nucleotide sequences of rbcL of Astasia and Euglena shows 82% identical amino acids whereas 15% of the amino acids represent conservative changes. A 1.5 kb transcript of the rbcL gene was revealed by northern blot analysis of Astasia RNA. By immunoblot analysis the gene product of rbcL was detected as a 53 kDa polypeptide. Genes for components of the chloroplast transcriptional and translational systems encoded by chloroplast DNA of plants and green algae are conserved on the 73 kb DNA of Astasia [24, 25, 26]. From our finding that Astasia obviously is capable of synthesizing the Rubisco large subunit one must conclude that these genes are expressed and form functional plastid transcriptional and translational systems.

Genes for the plastid elongation factor Tu and ribosomal protein S7 and six tRNA genes on the 73 kb DNA from Astasia longa that resembles the chloroplast DNA of Euglena.

The nucleotide sequence of a 6156 bp segment of the circular 73 kb DNA from Astasia longa resembling the chloroplast DNA of Euglena was determined. The genes for the plastid elongation factor Tu (tufA) and the ribosomal protein S7 (rps7), six tRNA genes (trnQ, trnS, trnG, trnM, trnT, trnR), and three open reading frames were identified. These genes show a high degree of sequence similarity (73%-99%) to the corresponding genes on the Euglena chloroplast genome. The tufA gene contains two small AT-rich introns within its coding region. Northern analysis revealed the in vivo transcription of the tufA gene and of a reading frame of 456 codons into monocistronic mRNAs of 1.3 and 1.4 kb, respectively. The arrangement and organization of the genes on the 73 kb DNA of the colourless heterotrophic flagellate Astasia and the chloroplast DNA of autotrophic Euglena are compared.

Genes for ribosomal proteins are retained on the 73 kb DNA from Astasia longa that resembles Euglena chloroplast DNA.

The nucleotide sequence of a 6.7 kb segment of the circular 73 kb DNA from Astasia longa has been determined. We identified genes for a tRNA-Ile (CAU), a tRNA-Phe (GAA), a tRNA-Cys (GCA) and the ribosomal proteins CS8, CL36, CS14 and CS2, that are normally encoded by plastid genomes. In addition, a gene for the chloroplast ribosomal protein CL5 was found that is not encoded by the plastome in either higher plants or a liverwort, but has recently been identified in Euglena chloroplast DNA. Transcripts of these protein genes, and of an unidentified open reading frame (ORF50), were detected. These results support our previous suggestion that the 73 kb DNA from Astasia is a truncated form of plastid DNA. The 73 kb DNA resembles the chloroplast DNA of Euglena gracilis but contains, almost exclusively, genes for a plastid-type translational (and presumably transcriptional) apparatus.

Molecular biology of insulin resistance.

Insulin resistance is an essential feature of a great variety of clinical disorders, like diabetes mellitus, obesity, essential hypertension, and is primarily due to a defect in hormone action at the cellular level. In the past decade application of novel research techniques including recombinant DNA technology have paved the way to understand the mechanisms of insulin action and its alterations at the molecular level. The first step in insulin action is the activation of the insulin receptor. The insulin receptor is a tetrameric protein consisting of two extracellular alpha- and two transmembrane beta-subunits. Binding of insulin to the alpha-subunit causes autophosphorylation of the intracellular beta-subunit region on tyrosine residues thereby activating the receptor. How the hormonal signal is subsequently transduced within the cell is still quiet unclear. The activated insulin receptor appears to couple to cytosolic receptor substrates which can affect different signaling cascades eliciting the pleiotropic hormone response on cell metabolism and growth. Most proteins involved in the signal transduction pathway of insulin are not known yet, but each of them might play a role in the various forms of insulin resistance. Taking the insulin receptor as an exemplary protein involved in insulin action we review molecular mechanisms regulating insulin receptor activity, gene expression, and the role of natural occurring insulin receptor gene mutations in patients with insulin resistant diabetes mellitus. It is outlined how the combination of both clinical medicine and molecular biology not only helps to understand insulin action and the pathogenesis of insulin resistance, but also leads to new avenues in the differential diagnosis, therapy, and possibly prevention of this heterogenous but most frequent metabolic and endocrine disorder.

The alpha-helical domain near the amino terminus is essential for dimerization of vascular endothelial growth factor.

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.

An antagonistic vascular endothelial growth factor (VEGF) variant inhibits VEGF-stimulated receptor autophosphorylation and proliferation of human endothelial cells.

Vascular endothelial growth factor (VEGF) is a potent mitogen with a unique specificity for endothelial cells 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, rheumatoid arthritis, or psoriasis. VEGF is a symmetric homodimeric molecule with two receptor binding interfaces lying on each pole of the molecule. Herein we report on the construction and recombinant expression of an asymmetric heterodimeric VEGF variant with an intact receptor binding interface at one pole and a mutant receptor binding interface at the second pole of the dimer. This VEGF variant binds to VEGF receptors but fails to induce receptor activation. In competition experiments, the heterodimeric VEGF variant antagonizes VEGF-stimulated receptor autophosphorylation and proliferation of endothelial cells. A 15-fold excess of the heterodimer was sufficient to inhibit VEGF-stimulated endothelial cell proliferation by 50%, and a 100-fold excess resulted in an almost complete inhibition. By using a rational approach that is based on the structure of VEGF, we have shown the feasibility to construct a VEGF variant that acts as an VEGF antagonist.

Expression of biologically active isoforms of the tumor angiogenesis factor VEGF in Escherichia coli.

Vascular endothelial growth factor (VEGF) was identified as an endothelial cell specific mitogen that induces angiogenesis and vascular permeability in vivo. VEGF is a homodimeric protein which contains three intramolecular and two intermolecular disulfide bridges. Here, we report on an efficient procedure for recombinant production of VEGF isoforms VEGF121 and VEGF165 in Escherichia coli. The proteins were solubilized from inclusion bodies, refolded, and purified by chromatographic methods. The final protein products were almost completely in the dimeric conformation, bound to VEGF receptor FLT1 with a Kd of 30 pM, stimulated proliferation of human umbilical vein endothelial cells half-maximally at a concentration of 30 pM, and induced in vivo neovascularization and vascular permeability on the chicken chorioallantoic membrane.