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

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✓ 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.

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Address reprint requests to: Jui-Chang Tsai, M.D., 74, 4F, Chin-Hua Street, Taipei 10606, Taiwan.

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    Graph showing that Alamar blue absorbances and viable cell numbers were linearly related. Graded numbers of U-105MG glioma cells were incubated for 24 hours, after which Alamar blue was added for 30 minutes. Absorbance of supernatants was measured at a wavelength of 562 nm with the background absorbance at 590 nm subtracted. Each data point represented the mean ± standard deviation of quadruplicate cultures.

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    Graph depicting the standard curves of recombinant human vascular endothelial growth factor/vascular permeability factor (rhVEGF/VPF165) serially diluted in Dulbecco's modified Eagle's medium mixed 1:1 with Ham's nutrient mixture F-12 (DMEM/F12) containing 0.8% or 8% fetal bovine serum (FBS), Blotto/Tween-20, human serum, water or human cerebrospinal fluid (CSF) as quantified by enzyme-linked immunosorbent assays (ELISA). Each data point represents the mean ± standard deviation of triplicate samples. Water as a diluent diminished immunoassay sensitivity and various protein-containing diluents yielded similar curves that were linear between 0 to 50 ng/ml with a correlation coefficient of better than 0.99. Coefficient of variation for all protein-containing diluents was below 10%. Sensitivity of the ELISA was estimated to be 2.0 ng/ml.

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    Bar graph showing that various human brain tumor cell lines secreted different amounts of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) as assessed by enzyme-linked immunosorbent assays of 72-hour culture supernatants from 24-well plates (200,000 cells in Dulbecco's modified Eagle's medium mixed 1:1 with Ham's nutrient mixture F-12 + 8% fetal bovine serum, 0.5 ml per well). Each data point represents the mean ± standard deviation of quadruplicate cultures.

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    Graph depicting results of 3-day incubations of graded numbers of cells in 24-well plates in 0.5 ml Dulbecco's modified Eagle's medium mixed 1:1 with Ham's nutrient mixture F-12 + 4% fetal bovine serum (FBS) and vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) in supernatants determined by enzyme-linked immunosorbent assays. The concentration of VEGF/VPF was linearly proportional to the number of cells plated for all cell lines except CH-235MG, which showed an exceptional increase of VEGF/VPF secretion at high cell density.

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    Scatterplots. A: Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) secretion was stimulated by epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and platelet-derived growth factor BB (PDGF-BB) in the U-105MG cell line cultured in 24-well plates (105 cells in 0.5 ml Dulbecco's modified Eagle's medium (DMEM) mixed 1:1 with Ham's nutrient mixture F-12 + 2 mM l-glutamine + 0.8% fetal bovine serum (FBS)) for 3 days. Amounts of VEGF/VPF in 72-hour supernatants revealed that, at the maximum concentrations of EGF, bFGF, or PDGF-BB tested, secretion was enhanced by 85% to 100% above control although PDGF-AA only increased VEGF secretion by 12%. B: An additive rather than a synergistic effect was observed between EGF and bFGF in U-105MG cells cultured in 24-well plates (105 cells/well in 0.5 ml DMEM/F12 + 2 mM l-glutamine + 0.8% FBS) for 3 days. At low concentrations of bFGF, EGF (20 ng/ml) had an additive effect in inducing VEGF/VPF secretion, which was gradually attenuated as bFGF concentrations approached a maximum (50 ng/ml). C: A persistent additive effect on VEGF/VPF secretion was produced by EGF and PDGF-BB in U-105MG cells cultured in 24-well plates (105 cells/well in 0.5 ml DMEM/F12 + 2 mM l-glutamine + 0.8% FBS) for 3 days. Amounts of VEGF/VPF in 72-hour culture supernatants as determined by enzyme-linked immunosorbent assays revealed that EGF (10 ng/ml) had a persistent additive effect on PDGF-BB in inducing VEGF/VPF secretion.

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    Upper: Western blot analysis showing that both rabbit anti-vascular endothelial growth factor (VEGF) antisera (Lane 1) and A4.6.1 anti-VEGF monoclonal antibody (Lane 2), recognized recombinant human VEGF/vascular permeability factor (rhVEGF/VPF165) as a band with a molecular weight of approximately 46 kD. Center: Nonreducing Western blot showing culture supernatants from CH-157MN meningioma cell line (Lanes 1, 3, 5, 7, and 9) and U-105MG glioma cell line (Lanes 2, 4, 6, and 8) using anti-VEGF polyclonal rabbit antisera (Lanes 3 and 4) and A4.6.1 anti-VEGF monoclonal antibody (Lanes 5 and 6) revealed similar bands at 46 kD. This band could be eliminated by preincubation of polyclonal rabbit anti-VEGF antiserum with rhVEGF (2 µg/ml; Lanes 7 and 8) and was not recognized by normal pre-immune rabbit serum (Lanes 1 and 2). No anti-VEGF antiserum was used in Lane 9. This 46-kD band likely represents VEGF165. Lower: Reduced Western blot analysis of tissue lysates generated from meningioma surgical specimens (Lanes 1 to 4) and cell lysates from U-105MG (Lane 5) and CH-157MN (Lane 6) were separated under reducing conditions and revealed an immunoreactive band at 27 kD, which likely represented the VEGF monomer. The 18-kD and 14-kD bands shown were caused by nonspecific reactivity of goat anti—rabbit antiserum.

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    Photomicrographs showing vascular endothelial growth factor (VEGF) immunoreactivity in tumor tissues and cell lines using rabbit anti-VEGF antisera. A: The VEGF immunoperoxidase staining (brown) of a human glioblastoma multiforme was prominent in the cytoplasm of many glioma cells (hematoxylin counterstain). B: Distribution of VEGF in meningiomas appeared more homogenous than in glioblastomas with some increased staining perivascularly and within endothelium. C and D: Glioma cell line U-87MG (C) and meningioma cell line CH-157MN (D) were representative of the immunochemical staining for all cell lines in this study. Immunoreactivity of VEGF in cell lines was prominent in a perinuclear and membrane pattern and was absent in preimmune controls (not shown). Original magnification × 200.

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