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Biomedical subjects

D Marmé

Publications and source records attributed to D Marmé.

At least 37 records · Page 2Linked to original sources
PubMed1997

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.

3T3 Cells↗
PubMed1997

Mapping of the sites for ligand binding and receptor dimerization at the extracellular domain of the vascular endothelial growth factor receptor FLT-1.

The vascular endothelial growth factor (VEGF) receptor FLT-1 has been shown to be involved in vasculogenesis and angiogenesis. The receptor is characterized by seven Ig-like loops within the extracellular domain. Upon VEGF binding FLT-1 becomes phosphorylated, which has been thought to be preceded by receptor dimerization. To further investigate high affinity binding of VEGF to FLT-1 and ligand-induced receptor dimerization, we expressed in Sf9 cells the entire extracellular domain comprising all seven Ig-like loops: sFLT-1(7) and several truncated mutants consisting of loop one, one and two, one to three, one to four, and one to five. The corresponding proteins, named sFLT-1(1), (2), (3), (4), and (5) were purified. Only mutants sFLT-1(3) to (7) were able to bind 125I-VEGF with high affinity. No binding of VEGF was observed with sFLT-1(1) and sFLT-1(2), indicating that the first three Ig-like loops are involved in high affinity binding of VEGF. The binding of VEGF to sFLT-1(3) could be competed with placenta growth factor (PlGF), a VEGF-related ligand, suggesting that high affinity binding of VEGF and PlGF is mediated by the same or closely related contact sites on sFLT-1. Deglycosylation of the sFLT-1(3), (4), (5), and (7) did not abolish VEGF binding. Furthermore, unglycosylated sFLT-1(3), expressed in Escherichia coli, was able to bind VEGF with similar affinity as sFLT-1(3) or sFLT-1(7), both expressed in Sf9 cells. This indicates that receptor glycosylation is not essential for high affinity binding. Dimerization of the extracellular domains of FLT-1 upon addition of VEGF was detected with all mutants containing the Ig-like loop four. Although sFLT-1(3) was able to bind VEGF, dimerization of this mutant was inefficient, indicating that sites on Ig-like loop four are essential to stabilize receptor dimers.

Animals↗
PubMed1996

Cloning and functional analysis of the hematopoietic cell-specific phospholipase C(gamma)2 promoter.

Phospholipase C(gamma)2 (PLCgamma2) is a phospholipid-converting enzyme which, upon receptor stimulation, is activated within membrane-bound signalling complexes. In contrast to the highly ubiquitous PLCgamma1, PLCgamma2 is expressed predominantly in B-lymphocytes. Associated with antigen-coupling receptors it is activated by tyrosine phosphorylation after the triggering of B-cell surface immunoglobulin. We have cloned and sequenced the human PLCgamma2 promoter. Primer extension analysis reveals the existence of a major transcriptional start site. The TATA-less promoter contains G+C-rich stretches with a cluster of contiguous SP1 consensus sites, an NF1, and an AP2 site between bp -220 to -70. A construct containing the region from -189 to +78 confers full promoter activity, as shown by fusion to a luciferase reporter gene construct. The distal part of the promoter between bp -662 to -293 containing an SRE, EBF and CACCC box contributed negatively to promoter activity in the B-cell line Raji but not in three adherent cell lines. In Raji cells, PLCgamma2 mRNA is expressed at low levels with a half life greater than 4 h. After treatment with serum, TPA, retinoic acid, or with 5-azacytidine increased levels of PLCgamma2 mRNA were induced in B-cells.

3T3 Cells↗
PubMed1996

VEGF121 induces proliferation of vascular endothelial cells and expression of flk-1 without affecting lymphatic vessels of chorioallantoic membrane.

We have studied the effect of VEGF(121) homodimer and VEGF(121/165) heterodimer on the chorioallantoic membrane (CAM) of 13-day-old chick embryos. The factors were applied in doses of 2-4 micrograms and the effects were evaluated macroscopically after 2 and 3 days. Histological studies were performed on semi- and ultrathin sections. Proliferation was studied according to the BrdU-anti-BrdU method on whole mounts and sections. The labeling density was quantified in whole mounts. The fractal dimension, D, of the vascular tree was assessed as a value for vascular bifurcation density. Both forms of VEGF induce brush-like vessel formation in the precapillary region. New capillaries are found in the stroma of the CAM, which normally does not contain capillaries. Our results show that VEGF(121) is a specific endothelial cell mitogen. A fourfold increase of BrdU-labeled endothelial cells is found after VEGF(121) application. The fractal dimension of the vascular tree increases from 1.26 in the controls to 1.44 (VEGF(121)) and 1.41 (VEGF(121/165)). The endothelial cells of the newly formed capillaries possess many mitochondria and micropinocytotic vesicles, but no fenestrations. These capillaries are obviously formed by intussusceptive microvascular growth. Signs of sprouting are almost absent. An effect on the lymphatic vessels of the CAM is not detectable. Compared to VEGF(165) and VEGF(121/165), VEGF(121) diffuses over a slightly greater distance. Using in situ hybridization, VEGF receptor-2 (flk-1/Quek1) and the homologous flt-4 (Quek2) receptor were studied in the CAM of normal quail embryos and after VEGF(121) application on the CAM of 11-day-old quail embryos. During normal development, flk-1 expression becomes restricted to vascular endothelial cells of large vessels in the stroma of the CAM. VEGF(121) application induces expression of flk-1 in capillaries that normally do not express the receptor. In the normal development of the CAM, flt-4 becomes restricted to endothelial cells of vessels that appear to be lymphatic vessels. Application of VEGF(121) does not alter flt-4 expression.

Allantois↗
PubMed1996

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.

Allantois↗
PubMed1996

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.

Carcinoma, Renal Cell↗
PubMed1996

Migration of human monocytes in response to vascular endothelial growth factor (VEGF) is mediated via the VEGF receptor flt-1.

Treatment of human monocytes with vascular endothelial growth factor (VEGF) isolated from tumor cell supernatants was reported to induce monocyte activation and migration. In this study we show that recombinant human VEGF165, and VEGF121 had a maximal effect on human monocyte migration at 65 to 250 pmol/L. Chemotactic activity of VEGF165 was inhibited by a specific antiserum against VEGF, by heat treatment of VEGF165, and by protein kinase inhibitors. In addition, we could show that VEGF-stimulated monocyte migration is mediated by a pertussis toxin-sensitive GTP-binding protein. Placenta growth factor (PlGF152), a heparin-binding growth factor related to VEGF, was also chemotactic for monocytes at concentrations between 2.5 and 25 pmol/L. In accordance with these findings, human monocytes showed specific and saturable binding for 125I-VEGF165 (half-maximal binding at 1 to 1.5 nmol/L). Using Northern blot analysis, we further could show that human monocytes express only the gene for the VEGF receptor type, flt-1, but not for the second known VEGF receptor, KDR. Resting monocytes expressed low levels of flt-1 gene only. Brief exposure (2 to 4 hours) of human monocytes to lipopolysaccharide, a prototypic monocyte activator, led to a significant upregulation of the flt-1 mRNA level. The results presented here suggest that monocyte chemotaxis in response to VEGF and most likely to PlGF152 is mediated by flt-1 and thus show a possible function for the VEGF-receptor flt-1.

Binding, Competitive↗
PubMed1996

Tumor angiogenesis: the pivotal role of vascular endothelial growth factor.

There is considerable evidence that vascular endothelial growth factor is involved in the vascularization and growth of primary tumors and in the formation of metastases. The expression of vascular endothelial growth factor depends on activated oncogenes and inactivated tumor-suppressor genes as well as several other factors, e.g., growth factors, hypoxia, and tumor promoters. Substantial expression of vascular endothelial growth factor receptors is mainly restricted to tumor vessels. The causal involvement of this angiogenic factor in the progression of the disease has been successfully evaluated using monoclonal antibodies against vascular endothelial growth factor, dominant negative receptor mutants, and antisense oligonucleotides against the messenger RNA of vascular endothelial growth factor. Thus, the vascular endothelial growth factor-signaling system seems to be an appropriate target for inhibition of tumor angiogenesis and metastasis formation.

Animals↗
PubMed1995

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 125I-VEGF165 to human umbilical vein endothelial cells and by its ability to stimulate proliferation of these cells.

3T3 Cells↗
PubMed1995

[Are there alternative forms of therapy in breast carcinoma? Status and perspectives for the treatment of metastasized breast carcinoma].

The emergence of new cytotoxic agents and techniques for treatment of systemic disease as single modalities or in combination with irradiation and surgery will impact on the use of such agents in the management of systemic breast cancer. Metastatic breast carcinoma, unlike other solid tumors, is highly responsive to chemotherapy, response rates of 50 to 70% have been reported consistently, although there has not been a significant improvement on long-term survival of these patients in the last ten years. New therapeutic approaches include cytotoxic and hormonal agents, growth and differentiation factors, monoclonal antibodies, hematopoietic stem cell support, conquest of tumor cell resistance by MDR-modulation, genetic manipulation, identification of new targets on the tumor surface, synthesis of target-oriented designer-drugs and inhibition of tumor angiogenesis. In breast cancer the tumor growth correlates with vascularization and angiogenesis. Tumor angiogenesis is stimulated by the vascular endothelial growth factor (VEGF). Microvessel density is a significant predictor of survival among node-negative women, who are at risk for having occult metastases at presentation. These patients could then be given systemic adjuvant therapy. Animal experiments show promising inhibition of tumor growth in nude mice after application of antibodies against VEGF. Other methods of manipulation of molecular mechanisms of angiogenesis are under investigation.

Breast Neoplasms↗
PubMed1995

Hypoxia-induced transcription of the vascular endothelial growth factor gene is independent of functional AP-1 transcription factor.

In this study, we investigated the functional role of the transcription factor AP-1 in hypoxia-induced expression of the vascular endothelial growth factor (VEGF) by using dexamethasone as an inhibitor of AP-1 activity. Phorbol ester and platelet-derived growth factor (PDGF) cause an increase in VEGF mRNA expression, which is strongly suppressed in the presence of dexamethasone, whereas hypoxia-induced VEGF expression is not inhibited by dexamethasone. Studies using a VEGF promoter luciferase construct show that the phorbol ester and PDGF-induced VEGF expression is mediated at least in part by transcriptional activation of the VEGF promoter, whereas no transcriptional activation is seen under hypoxic conditions. In contrast, hypoxia leads to an increase in VEGF mRNA stability, as confirmed by experiments using actinomycin D as an inhibitor of transcription. These results indicate that hypoxia-induced VEGF expression is independent of AP-1 mediated transcription.

3T3 Cells↗
PubMed1995

Proliferation pattern of capillary endothelial cells in chorioallantoic membrane development indicates local growth control, which is counteracted by vascular endothelial growth factor application.

The density and distribution of whole mount BrdU-anti-BrdU labeled endothelial cells (days 6-15) in the chick chorioallantoic membrane (CAM) was analyzed with computer-assisted microscopy. A significant loss of proliferative activity was noted after day 10: the density of labeled nuclei (in 10(-2) mm-2) decreased from a median 7.78 (days 6, 8, 10) to 2.42 (days 12, 14, 15). CAMs initially showed random patterns of labeled endothelial cells, but changed to clearly focal patterns after day 12. A regular arrangement of labeled nuclei was never seen. After application of vascular endothelial growth factor (VEGF) to the day 13 CAM, a significant increase in proliferative activity (11.50) and a random distribution of labeled endothelial cells was observed on day 15. Development of CAM precapillary vessels was assessed in terms of length density (in mm-1, mean +/- standard deviation), which was augmented three-fold from day 6 (1.22 +/- 0.05) to day 14 (3.54 +/- 0.23) and then remained nearly constant. VEGF application from day 13 to 15 raised arterial length per unit area to 4.53 +/- 0.77. It is concluded that normally a local regulation of endothelial proliferation and differentiation develops in the CAM, which doubles capillary endothelial cell density but simultaneously adapts to the decreasing need for endothelial cells, and thus maintains the quasi two-dimensional vessel pattern. However, proliferative foci persist in the capillary layer after day 10, and precapillary vessel density continues to increase until day 14. VEGF enhances DNA synthesis in all capillary endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Allantoin↗
PubMed1995

Regulation of the expression of the VEGF/VPS and its receptors: role in tumor angiogenesis.

Vascular endothelial growth factor (VEGF)/vascular permeability factor (VPS) plays a crucial role for the vascularization of tumors including breast cancers. Tumors produce ample amounts of VEGF, which stimulates the proliferation and migration of endothelial cells (ECs), thereby inducing tumor vascularization by a paracrine mechanism. VEGF receptors (VEGF-Rs) are highly expressed by the ECs in tumor blood vessels. VEGF expression can be induced in various cell types by a number of stimuli including hypoxia, differentiation, growth factors and tumor promoters of the phorbol ester class, such as TPA. The VEGF inductive pathways comprise kinases, oncogenes, tumor suppressor genes, and steroid hormone transcription factors, many of which seem to converge on the activator protein (AP-1) transcription factor. Much less is known about the regulation of VEGF-R expression, which is restricted to ECs. This expression is greatly enhanced in diseased tissue such as solid tumors. So far, it appears that growth factors, cytokines, and tumor promoters are involved in the control of VEGF-R expression. Here we review current knowledge about the regulation of the expression of VEGF and its receptors.

Animals↗
PubMed1995

Oncogenes as inducers of tumor angiogenesis.

Dominantly acting transforming oncogenes are generally considered to contribute to tumor development and progression by their direct effects on tumor cell proliferation and differentiation. However, the growth of solid tumors beyond 1-2 mm in diameter requires the induction and maintenance of a tumor blood vessel supply, which is attributed in large part to the production of angiogenesis promoting growth factors by tumor cells. The mechanisms which govern the expression of angiogenesis growth factors in tumor cells are largely unknown, but dominantly acting oncogenes may have a much greater impact than hitherto realized. An example of this is the induction of expression of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) by mutant H- or K-ras oncogenes, as well as v-src and v-raf, in transformed fibroblasts or epithelial cells. Besides VEGF/VPF, mutant ras genes are known to upregulate the expression of a variety of other growth factors thought to have direct or indirect stimulating effects on angiogenesis, e.g. TGF-beta and TGF-alpha. This effect may be mediated through the ras-raf-MAP kinase signal transduction pathway, resulting in activation of transcription factors such as AP1, which can then bind to relevant sites in the promoter regions of genes encoding angiogenesis growth factors. In principle, similar events could take place after activation or overexpression of many other oncogenes, especially those which can mediate their function through ras-dependent signal transduction pathways. The regulatory effect of oncogenes on mediators of angiogenesis has some potentially important therapeutic consequences. For example, it strengthens the rationale of pharmacologically targeting oncogene products, such as mutant RAS proteins, as an anti-tumor therapeutic strategy. Such drugs may attack the source of one or more angiogenic growth factors and by doing so, function, at least in part, as anti-angiogenic agents in vivo.

Genes, Tumor Suppressor↗
PubMed1995

VEGF-mediated tumour angiogenesis: a new target for cancer therapy.

Considerable evidence is gathering for the involvement of vascular endothelial growth factor (VEGF) in the vascularization and growth of primary tumours as well as in the formation of metastases. The expression of VEGF depends on activated oncogenes and inactivated tumour suppressor genes as well as several other factors (e.g. growth factors, tumour promoters and hypoxia). Substantial expression of the receptors for VEGF is restricted mainly to the tumour blood vessels. The causal involvement of this angiogenic factor in the progression of disease has been successfully evaluated by means of monoclonal antibodies against VEGF, dominant-negative receptor mutants and the use of antisense oligonucleotides against the VEGF mRNA. Thus, the VEGF signalling system seems to be an appropriate target to inhibit tumour angiogenesis and metastases formation.

Animals↗
PubMed1995

Lack of an effect of novel inhibitors with high specificity for protein kinase C on the action of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate on mouse skin in vivo.

The inhibitory effects of three novel staurosporine-derived compounds were tested with five different types of protein kinases, including protein kinase C (PKC). IC50 values of two of these compounds were found to be 300 to > 5000 times lower for PKC alpha beta gamma (a mixture of the PKC isoenzymes alpha, beta and gamma) than for any of the other protein kinases. The inhibitory action of the most selective inhibitor was tested also with the Ca(2+)-unresponsive PKC isoenzyme delta and was found to suppress PKC alpha beta gamma and PKC delta differentially. The highly specific PKC inhibitors are active both in cell culture and in vivo. They inhibit the PKC-catalyzed phosphorylation of the specific PKC substrate MARCKS in Swiss-3T3 fibroblasts and the okadaic acid-induced edema of the mouse ear. However, the more complex biological processes triggered by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate in mouse skin, such as inflammation, stimulation of cellular hyperproliferation and tumor promotion, remain largely unaffected upon topical application of these compounds.

3T3 Cells↗
PubMed1995

Vascular endothelial growth factor (VEGF) mRNA expression in human tumor models of different histologies.

BACKGROUND: Vascular endothelial growth factor (VEGF) is a polypeptide with specific effects on endothelial cell growth and blood vessel permeability. Recent studies demonstrated a key role for VEGF in tumor neovascularization, which is a prerequisite for tumor proliferation and metastasis. MATERIALS AND METHODS: We studied the expression of VEGF mRNA in a panel of 65 different human tumor xenografts of various histologies using Northern and slot blot analyses. Analyis of vessel density was performed morphologically and after immunohistochemical staining of endothelial cells. RESULTS: High expression levels were observed in 22/65 tumors. In melanoma, colorectal, gastric, breast and lung cancers only single tumors showed strong expression signals, whereas 7/10 renal cell carcinoma (RCC) xenografts demonstrated high levels of VEGF mRNA. Vessel density analysis revealed a clear correlation of VEGF mRNA expression with vascularization in RCC (p = 0.0048). Patient survival time was compared for tumors showing high versus low expression values. The overall 5-year survival rate was significantly lower for patients with high expression of VEGF mRNA (p = 0.0306). CONCLUSIONS: These data support the hypothesis that tumor cells of various histologies secrete VEGF, which acts as a paracrine factor to induce endothelial cell proliferation and vessel formation and mediates tumor progression.

Animals↗