Vascular Endothelial Growth Factor/vascular Permeability Factor Research Articles

Antisense Oligonucleotides Inhibit Vascular Endothelial Growth Factor/Vascular Permeability Factor Expression in Normal Human Epidermal Keratinocytes

In psoriatic lesions, epidermal keratinocytes overexpress vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) and transforming growth factor alpha (TGF-alpha). TGF-alpha has been shown to induce VEGF/VPF in normal human epidermal keratinocytes in vitro. By using a 19-mer antisense phosphorothioate oligodeoxynucleotide (PS-ODN) complementary to bases 6-24 relative to the translational start site of the VEGF/VPF mRNA, the control sense and mismatched PS-ODNs, we examined modulation of VEGF/VPF induction by TGF-alpha in vitro. Normal human epidermal keratinocytes were treated with PS-ODNs and Lipofectin for 8 h prior to the addition of TGF-alpha. Inhibition was assayed at the level of secreted protein by capture ELISA and mRNA expression was assayed by Northern blot analysis. The anti-sense PS-ODN was capable of inhibiting VEGF/VPF RNA and protein to near-basal levels. This inhibition was concentration dependent. No effect was observed with the sense or mismatch control PS-ODNs. These studies suggest that antisense oligonucleotide technology may be a potential therapy for the inhibition of angiogenesis associated with certain skin disorders such as psoriasis.

VASCULAR ENDOTHELIAL GROWTH FACTOR/VASCULAR PERMEABILITY FACTOR (VEGF/VPF) IS AN AUTOCRINE GROWTH FACTOR FOR AIDS-KS

Background: Kaposi's sarcoma (KS) is the most common tumor associated with HIV-1 infection. KS develops in approximately 30% of AIDS cases. The principal features of KS tumor are abnormal vascularization and the proliferation of endothelial cells and spindle(tumor) cells. Cells derived from Kaposi's sarcoma induce vascular lesions and display enhanced vascular permeability when inoculated subcutaneously in the nude mouse. This finding suggests that angiogenesis and capillary permeability play an important role in the development and progression of Kaposi's sarcoma. Methods: Several phosphothionate modified VEGF antisense oligonucleotides from different regions of VEGF were synthesized. Several control and AIDS-KS cell lines were treated with various concentration of VEGF antisense oligonucleotides and both DNA synthesis and cell proliferation was measured. VEGF mRNA was also examined by Northern and RT-PCR. VEGF protein was measured by ELISA. Expression of VEGF receptors (Flt-1 and KDR) were examined by RT-PCR. The in vivo studies were carried out in Balb/c Nu+/NU+ athymic nude mice. The VEGF antisense or scrambled VEGF oligonucleotides were injected intra-peritoneally daily for five consecutive days and the size of tumors were examined on day 14. Results: We found that all AIDS-KS cell lines expressed high levels of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF). AIDS-KS cells and primary tumor tissues also expressed high levels of Flt-1 and KDR, the receptors for VEGF, while the skin of the same patients did not show receptor expression. We further demonstrated that of the five different VEGF antisense oligonucleotides two of them (AS-1 and AS-3) specifically blocked VEGF mRNA and protein production and inhibited KS cell growth in a dose dependent manner. The IC50 of AS-1 and AS-3 oligonucleotides in AIDS-KS cells were 2.6 µM and 2.45 µM respectively. VEGF antisense oligonucleotides AS-1 and AS-3 were also examined on various control cell lines and the data showed that the antisense growth inhibitory effects were limited only to the KS cell lines. We have also studied both VEGF antisense (AS-1 and AS-3) and scrambled oligonucleotidesin vivo and found that both antisense oligonucleotides inhibited tumor growth in nude mice while the scrambled oligonucleotide had no significant effect. Conclusion: These results show that VEGF is an autocrine growth factor for AIDS-KS cells. Inhibitors of VEGF or its cognate receptors may thus be candidates for therapeutic intervention.

Hypoxia Regulates the Expression of Vascular Permeability Factor/Vascular Endothelial Growth Factor (VPF/VEGF) and its Receptors in Human Skin

Tissue hypoxia is a characteristic feature of malignant tumors and healing wounds, conditions that are associated with angiogenesis and with increased expression of vascular permeability factor (VPF; also called vascular endothelial growth factor, VEGF), a selective endothelial cell mitogen inducing microvascular hyperpermeability in vivo. We investigated the regulation of VPF/VEGF and its receptors by tissue hypoxia in normal human skin explants and in cultured skin cells in vitro. VPF/VEGF mRNA expression was dramatically upregulated in epidermal keratinocytes, dermal fibroblasts, and dermal microvessels after 24 h of skin organ culture. Hypoxia also enhanced the expression of VPF/VEGF in cultured epidermal keratinocytes and dermal microvascular endothelial cells (predominantly VPF/VEGF121 and VPF/VEGF165) and in dermal fibroblasts (additional upregulation of VPF/VEGF189). The expression of the VPF/VEGF receptor Flt-1 was selectively induced on dermal microvessels in skin explant cultures and in dermal endothelial cell monolayer cultures under hypoxic conditions. In contrast, the KDR receptor was downregulated by hypoxia. These results suggest that hypoxia likely regulates cutaneous angiogenesis and microvascular permeability by two distinct mechanisms: (i) Induction of VPF/VEGF in epithelial and mesenchymal cells, including endothelial cells. (ii) Differential modulation of VPF/VEGF receptor expression by microvascular endothelial cells. These mechanisms may be of importance in the pathogenesis of healing wounds and some malignant tumors that are commonly characterized by hypoxia and overexpression of VPF/VEGF.

Transforming growth factor-alpha-induced transcriptional activation of the vascular permeability factor (VPF/VEGF) gene requires AP-2-dependent DNA binding and transactivation.

The endothelial cell-specific mitogen vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) represents a central regulator of cutaneous angiogenesis. Increased VPF/VEGF expression has recently been reported in psoriatic skin and healing wounds, both conditions in which transforming growth factor-alpha (TGF alpha) and its ligand, the epidermal growth factor receptor, are markedly up-regulated. Since TGF alpha strongly induces VPF/VEGF synthesis in keratinocytes, TGF alpha-mediated VPF/VEGF expression is likely to play a significant role in the initiation and maintenance of increased vascular hyperpermeability and hyperproliferation in skin biology. The objectives of the present studies were to determine the molecular mechanisms responsible for TGF alpha-induced transcriptional activation of the VPF/VEGF gene. We have identified a GC-rich TGF alpha-responsive region between -88 bp and -65 bp of the VPF/VEGF promoter that is necessary for constitutive and TGF alpha-inducible transcriptional activation. In electrophoretic mobility shift assays, this region binds Sp1-dependent protein complexes constitutively and an additional TGF alpha-inducible protein complex that is distinct from Sp1 protein. Both AP-2 and Egr-1 transcription factors were detected as components of the TGF alpha-inducible protein complex in supershift EMSA studies. In co-transfection studies, an AP-2 but not an Egr-1 expression vector activated VPF/VEGF transcription, thus indicating that AP-2 protein is functionally important in TGF alpha-induced VPF/VEGF gene expression. By clarifying regulatory mechanisms that are critical for angiogenic processes in the skin, these studies may form the basis for new therapeutic strategies to modulate VPF/VEGF expression in cutaneous inflammation and wound healing.

Time-dependent vascular regression and permeability changes in established human tumor xenografts induced by an anti-vascular endothelial growth factor/vascular permeability factor antibody.

The hyperpermeability of tumor vessels to macromolecules, compared with normal vessels, is presumably due to vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) released by neoplastic and/or host cells. In addition, VEGF/VPF is a potent angiogenic factor. Removal of this growth factor may reduce the permeability and inhibit tumor angiogenesis. To test these hypotheses, we transplanted a human glioblastoma (U87), a human colon adenocarcinoma (LS174T), and a human melanoma (P-MEL) into two locations in immunodeficient mice: the cranial window and the dorsal skinfold chamber. The mice bearing vascularized tumors were treated with a bolus (0.2 ml) of either a neutralizing antibody (A4.6.1) (492 micrograms/ml) against VEGF/VPF or PBS (control). We found that tumor vascular permeability to albumin in antibody-treated groups was lower than in the matched controls and that the effect of the antibody was time-dependent and influenced by the mode of injection. Tumor vascular permeability did not respond to i.p. injection of the antibody until 4 days posttreatment. However, the permeability was reduced within 6 h after i.v. injection of the same amount of antibody. In addition to the reduction in vascular permeability, the tumor vessels became smaller in diameter and less tortuous after antibody injections and eventually disappeared from the surface after four consecutive treatments in U87 tumors. These results demonstrate that tumor vascular permeability can be reduced by neutralization of endogenous VEGF/ VPF and suggest that angiogenesis and the maintenance of integrity of tumor vessels require the presence of VEGF/VPF in the tissue microenvironment. The latter finding reveals a new mechanism of tumor vessel regression-i.e., blocking the interactions between VEFG/VPF and endothelial cells or inhibiting VEGF/VPF synthesis in solid tumors causes dramatic reduction in vessel diameter, which may block the passage of blood elements and thus lead to vascular regression.

Influence of Microvessel Density and Vascular Permeability Factor/Vascular Endothelial Growth Factor Expression on Prognosis in Vulvar Cancer

Microvessel density (MVD) and expression of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), acting as a highly specific inducer of angiogenesis, were evaluated in tissue specimens of 25 patients with squamous cell cancer of the vulva. MVD was quantified by immunostaining for factor VIII-related antigen at one field of 0.25 mm2. VPF/VEGF expression was evaluated immunohistochemically using a monoclonal anti-VEGF antibody. FIGO stages I, II, and III were diagnosed in 12, 7, and 6 patients, respectively. MVD >20/field was found in 10 of 25 tumors and moderate or strong expression of VPF/VEGF in 10 of 25 tumors. High MVD was significantly more frequent in tumors with moderate or strong VPF/VEGF expression compared to tumors with no or weak VPF/VEGF expression (P= 0.01). Overall survival rates of patients with tumors of high MVD (P= 0.01) and strong VPF/VEGF expression (P< 0.01) were significantly poorer compared to those patients with low MVD or poor VPF/VEGF expression. Strong VPF/VEGF expression and high MVD are considered important parameters of tumor angiogenesis and therefore are related to poor survival probability in vulvar cancer patients.

Correlation of vascular permeability factor/vascular endothelial growth factor with extraretinal neovascularization in the rat.

To determine whether neovascularization was spatially correlated with the distribution of messenger RNA for vascular permeability factor/vascular endothelial growth factor (VPF/VEGF). Neonatal rats were raised 8 days in 80% oxygen with daily intervals of relative hypoxia in room air, then transferred to room air for 5, 7, or 10 additional days. In situ hybridization for VPF/VEGF expression and avascular area were examined in retinas from oxygen-exposed animals and room-air controls. Severity of neovascularization was scored. The inner nuclear layer of oxygen-exposed retinas exhibited a continuous intense band of VPF/VEGF messenger RNA expression across the peripheral avascular zone that dropped sharply in vascular retina. Neovascularization occurred adjacent to regions of greatest expression. Controls had much lower expression and smaller avascular regions. The VPF/VEGF messenger RNA expression was most intense in Müller cells, scattered astrocytes, and amacrine cells, strong in retinal pigment epithelium, and moderate in the remaining inner nuclear layer and ganglion cell layer. The expression of VPF/VEGF message was spatially and quantitatively correlated with the neovascularization.

Negative regulation of hypoxia-inducible genes by the von Hippel-Lindau protein.

Inactivation of the von Hippel-Lindau protein (pVHL) has been implicated in the pathogenesis of renal carcinomas and central nervous system hemangioblastomas. These are highly vascular tumors which overproduce angiogenic peptides such as vascular endothelial growth factor/vascular permeability factor (VEGF/VPF). Renal carcinoma cells lacking wild-type pVHL were found to produce mRNAs encoding VEGF/VPF, the glucose transporter GLUT1, and the platelet-derived growth factor B chain under both normoxic and hypoxic conditions. Reintroduction of wild-type, but not mutant, pVHL into these cells specifically inhibited the production of these mRNAs under normoxic conditions, thus restoring their previously described hypoxia-inducible profile. Thus, pVHL appears to play a critical role in the transduction of signals generated by changes in ambient oxygen tension.

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.

Nuclease-resistant nucleic acid ligands to vascular permeability factor/vascular endothelial growth factor

Background: Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is a potent inducer of new blood vessel growth (angiogenesis) that contributes to the pathology of many angiogenesis-associated disease states such as psoriasis, rheumatoid arthritis and cancer. Few molecular entities capable of binding to VPF/VEGF with high affinity and specificity have been described to date. Results: Nuclease-resistant 2′-amino-2′-deoxypyrimidine nucleotide RNA (2′-aminopyrimidine RNA) ligands that bind to VPF/VEGF with high affinity have been identified by iterative rounds of affinity-selection/amplification from two independent random libraries. The sequence information that confers high affinity binding toVPF/VEGF is contained in a contiguous stretch of 24 nucleotides, 5′- CCCUGAUGGUAGACGCCGGGGUG-3′ (2′-aminopyrimidine nucleotides are designated with italic letters). Of the 14 ribopurines in this minimal ligand, 10 can be substituted with the corresponding 2′-O-methylpurine nucleotides without a reduction in binding affinity to VPF/VEGF. In fact, the 2′ O-methyl substitution at permissive positions leads to a ∼17-fold improvement in the binding affinity to VPF/VEGF The higher affinity results from the reduction in the dissociation rate constant of the 2′-O-methyl-substituted RNA ligand from the protein compared to the unsubstituted ligand. The 2′-O-methyl-substituted minimal ligand, which folds into a bulged hairpin motif, is also more thermally stable than the unsubstituted ligand. Nuclease resistance of the ligand is further improved by the 2′-O-methyl substitutions and the addition of short phosphorothioate caps to the 3′- and 5′-ends. Conclusions: We have used the SELEX ( s ystematic e volution of ligands by e xponential enrichment) process in conjunction with post-SELEX modifications to define a highly nuclease-resistant oligonucleotide that binds to VPF/VEGF with high affinity and specificity.

Keratinocyte-Derived Vascular Permeability Factor (Vascular Endothelial Growth Factor) Is a Potent Mitogen for Dermal Microvascular Endothelial Cells

Expression of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is markedly increased in the epidermis of lesional psoriatic skin and in healing skin wounds. In this study, we characterized the effects of several cytokines and growth factors on the expression and secretion of VPF/VEGF mRNA and protein by cultured human epidermal keratinocytes, as well as the effect of VPF/VEGF on the growth of cultured human dermal microvascular endothelial cells. Transforming growth factor-alpha, epidermal growth factor, and phorbol myristate acetate markedly stimulated VPF/VEGF mRNA expression by cultured keratinocytes; as in psoriatic skin, the three most common VPF/VEGF isoforms (encoding proteins of 121, 165, and 189 amino acids) were upregulated to an equal extent. Transforming growth factor (TGF)-alpha, epidermal growth factor, and phorbol myristate acetate also enhanced the secretion of VPF/VEGF by keratinocytes; in contrast, a number of other cytokines including interleukin (IL)-1, IL-6, IL-8, tumor necrosis factor-alpha, interferon-gamma, and transforming growth factor-beta did not induce VPF/VEGF secretion. The VPF/VEGF secreted by keratinocytes was biologically active in that, like recombinant human VPF/VEGF, it potently stimulated dermal endothelial cell proliferation. Scatchard analysis revealed two high-affinity VPF/VEGF binding sites on dermal endothelial cells with dissociation constants of 51 pM and 2.9 pM. These results suggest that the avascular epidermis has the capacity to regulate dermal angiogenesis and microvascular permeability by a paracrine mechanism involving the secretion of VPF/VEGF. Similar mechanisms may be anticipated in a variety of inflammatory and neoplastic skin diseases characterized by microvascular hyperpermeability, edema, and angiogenesis.

Hypoxic induction of human vascular endothelial growth factor expression through c-Src activation.

Angiogenesis, the formation of new microvasculature by capillary sprouting, is crucial for tumour development. Hypoxic regions of solid tumours produce the powerful and directly acting angiogenic protein VEGF/VPF (vascular endothelial growth factor/vascular permeability factor). We now investigate the signal transduction pathway involved in hypoxic induction of VEGF expression. Hypoxia is known to induce a tyrosine kinase cascade that results in the activation of nitrogen-fixation genes in Rhizobium meliloti, and activation of tyrosine kinases is critical in signalling triggered by growth factors and ultraviolet light. We show here that genistein, an inhibitor of protein tyrosine kinase, blocks VEGF induction. Hypoxia increases the kinase activity of pp60c-src and its phosphorylation on tyrosine 416 but does not activate Fyn or Yes. Expression of either a dominant-negative mutant form of c-Src or of Raf-1 markedly reduces VEGF induction. VEGF induction by hypoxia in c-src(-) cells is impaired, although there is a compensatory activation of Fyn. Our results provide an insight into hypoxia-triggered intracellular signalling, define VEGF as a new downstream target for c-SRC, and suggest a role for c-SRc in promoting angiogenesis.

Vascular endothelial growth factor in human glioma cell lines: induced secretion by EGF, PDGF-BB, and bFGF

Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) is an endothelial cell-specific mitogen that is structurally related to platelet-derived growth factor (PDGF). Vascular endothelial growth factor/vascular permeability factor induces angiogenesis in vivo and may play a critical role in tumor angiogenesis. Using immunohistochemical analysis, the authors demonstrated the presence of VEGF/VPF protein in surgical specimens of glioblastoma multiforme and cultured glioma cells. By means of an enzyme-linked immunosorbent assay (ELISA) of cell supernatants, the authors showed that VEGF/VPF is variably secreted by all nine cultured human malignant glioma cell lines (CH-235MG, D-37MG, D-54MG, D-65MG, U-87MG, U-105MG, U-138MG, U-251MG, U-373MG) and by a single meningioma cell line (CH-157MN). An immunocytochemical survey of these cell lines revealed a cytoplasmic and cell-surface distribution of VEGF/VPF. In the U-105MG glioma cell line, VEGF/VPF secretion was induced with physiological concentrations of epidermal growth factor, PDGF-BB, or basic fibroblast growth factor, but not with PDGF-AA. Moreover, it was observed that activation of convergent growth factor signaling pathways led to increased glioma VEGF secretion. Similar results were obtained using these growth factor combinations in the D-54MG glioma cell line. The data obtained suggest a potential role for VEGF/VPF in tumor hypervascularity and peritumoral edema. These observations may lead to development of new therapeutic strategies.

Ultrastructural localization of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) to the abluminal plasma membrane and vesiculovacuolar organelles of tumor microvascular endothelium.

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is a cytokine secreted by many animal and human tumors, activated macrophages, keratinocytes, rheumatoid synovial cells, embryonic tissues, and by cultured epithelial and mesenchymal cell lines. It acts selectively on vascular endothelial cells to increase their permeability to circulating macromolecules and to stimulate their replication. Although not detectably expressed by vascular cells in the human and animal tumors we have studied, VPF/VEGF accumulates in the microvessels supplying tumors and certain inflammatory reactions in which VPF/VEGF is also overexpressed. Light microscopic immunohistochemistry lacked the resolution necessary to localize VPF/VEGF precisely in such vessels. Therefore, we used a pre-embedding immunocytochemical method to localize VPF/VEGF at the ultrastructural level in the new blood vessels that are elicited in the peritoneal walls of mice bearing a transplantable mouse ascites tumor of ovarian origin. Intense immunostaining for VPF/VEGF was observed on the abluminal plasma membrane of tumor-associated microvascular endothelial cells and in vesiculovacuolar organelles (VVOs) present in these same endothelial cells. (VVOs are recently described cytoplasmic organelles present in tumor vascular endothelium that provide an important pathway for extravasation of circulating macromolecules.) In contrast to labeling of the abluminal plasma membrane and VVO vesicles and vacuoles, endothelial cytoplasmic organelles, such as multivesicular bodies and Weibel-Palade bodies, and the underlying basal lamina, did not stain with antibodies to VPF/VEGF. The distribution of VPF/VEGF here described corresponds to that anticipated for high-affinity VFP/VEGF receptors, although binding of VPF/VEGF to other endothelial cell surface structures, such as plasma membrane proteoglycans, is also a possibility.

The shortest isoform of human vascular endothelial growth factor/vascular permeability factor (VEGF/VPF 121) produced by Saccharomyces cerevisiae promotes both angiogenesis and vascular permeability

Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) is a multifunctional cytokine that is expressed as four isoforms having 206, 189, 165, and 121 amino acids in humans. We constructed a system that produces the shortest isoform of human VEGF/VPF in Saccharomyces cerevisiae (yVEGF/VPF 121). Active yVEGF/VPF 121 was secreted from the yeast cells as a glycosylated dimeric protein. Various biological activities of the purified yVEGF/VPF 121 were examined. It bound to cell surface receptor(s) and stimulated the growth of human umbilical vein endothelial cells in culture at a dose similar to that of native VEGF/VPF. Purified yVEGF/VPF 121 also induced angiogenesis in mice, and promoted the extravasation of plasma proteins from the blood vessels. These observations demonstrated that the shortest isoform of VEGF/VPF with an amino acid sequence of 121 residues contains enough information necessary to trigger both angiogenesis and the induction of vascular permeability upon binding to its cognate receptor(s).

Vascular endothelial growth factor/vascular permeability factor expression in a mouse model of retinal neovascularization.

Neovascular diseases of the retina are a major cause of blindness worldwide. Hypoxia is thought to be a common precursor to neovascularization in many retinal diseases, but the factors involved in the hypoxic neovascular response have not been fully identified. To investigate the role of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) in retinal neovascularization, the expression of VEGF/VPF mRNA and protein were studied in a mouse model of proliferative retinopathy. RNA (Northern) blot analysis revealed that retinal VEGF/VPF mRNA expression increased 3-fold between 6 and 12 hr of relative retinal hypoxia and remained elevated during the development of neovascularization. In situ hybridization localized VEGF/VPF mRNA to cells bodies in the inner nuclear layer of the retina. Immunohistochemical confocal microscopy demonstrated that VEGF/VPF protein levels increase with a time course similar to that of the mRNA. The cells in the inner nuclear layer of the retina that produce VEGF/VPF were identified morphologically as Müller cells. These data suggest that VEGF/VPF expression in the retina plays a central role in the development of retinal ischemia-induced ocular neovascularization.

Expression of vascular permeability factor/vascular endothelial growth factor in pig cerebral microvascular endothelial cells and its upregulation by adenosine.

Porcine brain-derived microvascular endothelial cells (BMEC) express the mRNA of the polypeptide mitogen vascular permeability factor/vascular endothelial growth factor (VPF/VEGF). The VEGF mRNA expression in BMEC could be upregulated 2.5 fold after 6 h of treatment with 5 microM adenosine and adenosine agonists. Adenosine A1 and A2 receptor antagonists completely abolished the upregulation of the VEGF mRNA caused by adenosine. Agents like forskolin and cAMP phosphodiesterase inhibitors which are known to increase the cAMP level decreased the VEGF mRNA expression slightly whereas agents like phorbolester which activate the proteinkinase C (PKC) pathway enhanced the VEGF mRNA expression 3.2 fold. The specific inhibitor of the PKC bisindolymaleimide (BIM) abolished the upregulation of the VEGF mRNA by adenosine completely. The BMEC conditioned medium stimulated the proliferation of BMEC itself and Western blot analysis of the BMEC conditioned medium using a polyclonal antibody to human VEGF showed one band at 18 kDa which was slightly upregulated after treatment with adenosine. Results suggest that the effect of adenosine on the VEGF mRNA expression is mediated via the A1 receptor and that an activation of the PKC may be involved in the observed effects of adenosine on the VEGF mRNA expression. VEGF produced by BMEC and which is inducible by adenosine may function via the autocrine pathway and may be involved in repair reactions of brain blood vessels and/or the maintenance of these cells.

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.

Vascular endothelial growth factor/vascular permeability factor is detectable in the sera of tumor-bearing mice and cancer patients

We developed an enzyme-linked immunosorbent assay (ELISA) for vascular endothelial growth factor/vascular permeability factor (VEGF/VPF). The assay revealed that VEGF/VPF levels in the sera of mice were significantly increased from undetectable level by s.c. transplantation with a solid tumor. We also measured VEGF/VPF levels in serum specimens obtained from cancer patients with several types of cancers. VEGF/VPF levels in the sera from cancer patients were significantly higher than those in the sera from the individuals with no sign of cancer.

Characterization of vascular permeability factor/vascular endothelial growth factor receptors on mononuclear phagocytes

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is a polypeptide mediator, elaborated by certain tumors and other cell types, that exerts multiple effects on endothelium via interaction with a class of high-affinity binding sites. In this report, the interaction of VPF/VEGF with human mononuclear phagocytes (MPs) is characterized. Radioligand binding studies at 4 degrees C showed the presence of a single class of binding sites, kd approximately 300 to 500 pmol/L (approximately 20 times lower affinity than the high-affinity binding site on endothelial cells [ECs]), the occupancy of which correlated with VPF/VEGF-induced MP migration and expression of tissue factor. These binding results were paralleled by functional experiments which indicated that the same VPF/VEGF preparations were about an order of magnitude less effective in stimulating MP chemotaxis than in inducing EC proliferation. When MPs with surface-bound 125I-VPF/VEGF were warmed to 37 degrees C, endocytosis and degradation occurred. Occupancy of VPF/VEGF binding site resulted in subsequent activation of intracellular signal transduction mechanisms, as shown by an increase in MP intracellular calcium concentration. Cross-linking studies with 125I-VPF/VEGF showed a new high-molecular weight band (corresponding to putative 125I- VPF/VEGF-receptor complex), the appearance of which was blocked by excess unlabeled VPF/VEGF. Consistent with these results, immunoprecipitation of 32PO4-labeled MPs exposed to VPF/VEGF showed a single band of similar mobility, not seen in untreated controls. These results demonstrate that the interaction of VPF/VEGF with MPs, though of lower affinity than that observed with ECs, also results from interaction of the polypeptide with a specific cell-surface protein and leads to activation of intracellular transduction mechanisms.