Karin Weindel

Vascular endothelial growth factor in the sera and effusions of patients with malignant and nonmalignant disease

Clinical data clearly indicate a correlation between tumor neovascularization, aggressiveness of tumor growth, and metastatic spread. One of the key factors capable of stimulating tumor angiogenesis is vascular endothelial growth factor (VEGF). Using an immunoassay for VEGF, we assessed the levels of soluble VEGF in the sera and effusions of patients with malignant and nonmalignant disease as well as in the sera of healthy controls.

Both v-Ha-Ras and v-Raf Stimulate Expression of the Vascular Endothelial Growth Factor in NIH 3T3 Cells

Stimulation of NIH 3T3 cells with platelet-derived growth factor (PDGF)-BB and 12-O-tetradecanoylphorbol-13-acetate (TPA) enhances vascular endothelial growth factor (VEGF) gene expression. To address the question of whether Ras and Raf are involved in the induction of VEGF gene expression by PDGF and TPA, we examined the effects of both factors on NIH 3T3 cells stably transfected with v-Ha-ras or v-raf. In serum-starved NIH 3T3 cells, only low levels of mRNA expression can be detected, whereas both ras and raf transformed cell lines express enhanced levels of a 4.3-kilobase VEGF transcript. Stimulation with PDGF or TPA resulted in increased VEGF mRNA in all cell lines, with highest levels found in the transformed cells. Immunofluorescence studies confirmed that the elevated VEGF mRNA expression correlated with enhanced protein levels. Positive immunofluorescence signals could be detected in v-Ha-ras or v-raf transformed cell lines but not in unstimulated NIH 3T3 cells. VEGF from conditioned medium of v-raf transformed NIH 3T3 cells was partially purified by chromatography on heparin-Sepharose. Biological activity of this VEGF protein was demonstrated by competition with binding of recombinant I-VEGF to human umbilical vein endothelial cells and by its ability to stimulate proliferation of these cells.

AIDS-associated kaposi's sarcoma cells in culture express vascular endothelial growth factor

PCR cloning and cDNA sequencing have been used to identify mRNAs of two splice products of the vascular endothelial growth factor (VEGF) gene, VEGF121 and VEGF165, in cells isolated from Kaposis sarcomas (KS) of AIDS patients (AIDS-KS). As demonstrated by Northern blot analysis, AIDS-KS cells as well as tumor cells show a high expression level of the VEGF gene as compared to primary human vascular cells like smooth muscle cells or endothelial cells. In addition to the lower expression of the gene, vascular cells express a 3.9 kb band together with a 3.2 kb band instead of a 3.9 kb and a 4.3 kb band in AIDS-KS cells. Our data suggest that the angiogenic properties of AIDS-KS cells might be mediated by the secretion of this growth factor and that this factor alone or in combination with other endothelial mitogens may be involved in endothelial proliferation associated with Kaposis sarcoma.

Detection and quantification of vascular endothelial growth factor/vascular permeability factor in brain tumor tissue and cyst fluid: the key to angiogenesis?

In primary malignant brain tumors increased vascularity and marked edema strongly suggest a possible role of the vascular endothelial growth factor/vascular permeability factor (VEGF/VPF). This was confirmed by earlier in situ hybridization studies, by analysis of the expression of the mitogen in different subsets of glioblastoma cells, and by the fact that the VEGF/VPF receptor flt-1 (fms-like tyrosine kinase) is up-regulated in tumor cells in vivo. To assess and quantify the expression of the VEGF/VPF gene and of the receptor gene, 26 surgical specimens of brain tumor tissue from 24 patients were analyzed. In most malignant gliomas, the expression level of the VEGF/VPF gene is elevated and can be increased up to 20- to 50-fold in comparison with low-grade tumors. Using polymerase chain reaction-based amplification, it could be shown that the messenger RNAs of three different VEGF/VPF forms are synthesized in tumor tissue samples. Northern blot studies revealed that in some samples a significant expression of the gene coding for placenta growth factor, a growth factor closely related to VEGF/VPF, was observed. In addition, using a radioreceptor assay it was possible to detect high VEGF/VPF-like activity in the cyst fluids of brain tumors, indicating the accumulation of the mitogen and permeability factor in brain tumor cysts. Further investigations revealed that astrocytoma and glioblastoma cells in culture express the VEGF/VPF gene and secrete the VEGF/VPF protein, whereas gene expression of the two known VEGF/VPF receptors, kinase insert domain-containing receptor and flt-1, could not be detected. These data support previous reports, which stated that VEGF/VPF acts as a paracrine growth and permeability factor in brain tumors and may contribute to tumor growth by initiating tumor angiogenesis.

Detection and Quantification of Vascular Endothelial Growth Factor/Vascular Permeability Factor in Brain Tumor Tissue and Cyst Fluid

In primary malignant brain tumors increased vascularity and marked edema strongly suggest a possible role of the vascular endothelial growth factor/vascular permeability factor (VEGF/VPF). This was confirmed by earlier in situ hybridization studies, by analysis of the expression of the mitogen in different subsets of glioblastoma cells, and by the fact that the VEGF/VPF receptor flt-1 (fms-like tyrosine kinase) is up-regulated in tumor cells in vivo. To assess and quantify the expression of the VEGF/VPF gene and of the receptor gene, 26 surgical specimens of brain tumor tissue from 24 patients were analyzed. In most malignant gliomas, the expression level of the VEGF/VPF gene is elevated and can be increased up to 20- to 50-fold in comparison with low-grade tumors. Using polymerase chain reaction-based amplification, it could be shown that the messenger RNAs of three different VEGF/VPF forms are synthesized in tumor tissue samples. Northern blot studies revealed that in some samples a significant expression of the gene coding for placenta growth factor, a growth factor closely related to VEGF/VPF, was observed. In addition, using a radioreceptor assay it was possible to detect high VEGF/VPF-like activity in the cyst fluids of brain tumors, indicating the accumulation of the mitogen and permeability factor in brain tumor cysts. Further investigations revealed that astrocytoma and glioblastoma cells in culture express the VEGF/VPF gene and secrete the VEGF/VPF protein, whereas gene expression of the two known VEGF/VPF receptors, kinase insert domain-containing receptor and flt-1, could not be detected. These data support previous reports, which stated that VEGF/VPF acts as a paracrine growth and permeability factor in brain tumors and may contribute to tumor growth by initiating tumor angiogenesis.

Expression of Angiopoietin-2 in Endothelial Cells Is Controlled by Positive and Negative Regulatory Promoter Elements

Objective—Angiopoietin-2 (Ang-2) is a non-signal transducing ligand of the endothelial receptor tyrosine kinase Tie-2. Ang-2 is produced by endothelial cells and acts as an autocrine regulator mediating vascular destabilization by inhibiting Angiopoietin-1-mediated Tie-2 activation. To examine the transcriptional regulation of Ang-2, we studied the Ang-2 promoter in endothelial cells and nonendothelial cells. Methods and Results—The human Ang-2 promoter contains a 585-bp region around the transcriptional start site (−109 to +476) that is sufficient to control endothelial cell-specific and cytokine-dependent Ang-2 expression. Strong repressor elements of Ang-2-promoter activity are located in the 5′-region of the promoter and in the first intron. The Ets family transcription factors Ets-1 and Elf-1 act as strong enhancers of endothelial cell Ang-2-promoter activity. Ets-binding sites −4 and −7 act as positive regulators, whereas Ets-binding site −3 acts as negative regulator. Demethylation experiments revealed that the Ang-2 gene (in contrast to the Tie-2 gene) is not controlled by imprinting. Conclusions—The data determine unique positive and negative regulatory mechanisms of endothelial cell Ang-2 expression and provide further evidence for the critical role of Ang-2 as a key autocrine regulator of vascular stability and responsiveness.

Identification of a soluble form of the angiopoietin receptor TIE-2 released from endothelial cells and present in human blood

The transmembrane tyrosine kinase TIE-2, the receptor for the angiopoietins-1 and -2, has been shown to be involved in angiogenic processes. Investigating the regulation of TIE-2 expression on endothelial cells, we found that stimulators such as PMA induce a decrease of TIE-2 protein from the cell surface without affecting TIE-2 mRNA. In conditioned media of PMA stimulated endothelial cells, a soluble form of this receptor comprising parts of the extracellular domain can be detected. Using a sandwich ELISA, we were able to detect and quantify TIE-2 receptors in cell lysates (representing the whole transmembrane receptor) and in cell culture supernatants (representing a soluble form of this receptor, sTIE-2). Several factors influencing this shedding process e.g. basic FGF could be identified. Finally, the soluble form of TIE-2 could also be detected in human biological fluids such as sera and plasma from healthy controls.

Activated Neu/ErbB-2 induces expression of the vascular endothelial growth factor gene by functional activation of the transcription factor Sp 1.

The neu(c-erbB-2or HER2) proto-oncogene which encodes a receptor protein homologous to the epidermal growth factor receptor is overexpressed in 20%–30% of human breast and ovarian cancers. Oncogenic activation of Neu can also occur through multiple molecular mechanisms, including a point mutation in the transmembrane domain, deletion of the extracellular domain and short in-frame deletions of 7–12 amino acids in the extracellular region proximal to the transmembrane domain. Because of the highly vascularized phenotype of breast and ovarian cancers and the contribution of the Neu receptor to the development and progression of these tumors, we investigated the effect of Neu on the expression of the tumor angiogenesis factor VEGF. Expression of various activated Neu receptors but not wild-type Neu in Rat-1 cells, leads to increased VEGF expression on mRNA as well as on protein level. This effect is mediated by transcriptional activation of the VEGF promoter via a cluster of Sp 1 binding sites. Molecular analysis of the activation mechanism of Sp 1 revealed that neither the VEGF promoter binding activity of Sp 1 nor the expression of Sp 1 is affected by Neu transformation of the cells. Instead, functional Neu-induced transactivation of Sp 1 was observed by using a GAL4-based transactivation assay. These results demonstrate that functional changes of the transcription factor Sp 1 mediates a Neu-signaling cascade leading to VEGF promoter activation.

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

Abstract 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 μg/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 μg 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 β5 to β6 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.