Platelet-derived growth factor-B enhances glioma angiogenesis by stimulating vascular endothelial growth factor expression in tumor endothelia and by promoting pericyte recruitment

Abstract

Platelet-derived growth factor (PDGF)-B and its receptor (PDGF-R) beta are overexpressed in human gliomas and responsible for recruiting peri-endothelial cells to vessels. To establish the role of PDGF-B in glioma angiogenesis, we overexpressed PDGF-B in U87MG glioma cells. Although PDGF-B stimulated tyrosine phosphorylation of PDGF-Rbeta in U87MG cells, treatment with recombinant PDGF-B or overexpression of PDGF-B in U87MG cells had no effect on their proliferation. However, an increase of secreted PDGF-B in conditioned media of U87MG/PDGF-B cells promoted migration of endothelial cells expressing PDGF-R beta, whereas conditioned media from U87MG cells did not increase the cell migration. In mice, overexpression of PDGF-B in U87MG cells enhanced intracranial glioma formation by stimulating vascular endothelial growth factor (VEGF) expression in neovessels and by attracting vessel-associated pericytes. When PDGF-B and VEGF were overexpressed simultaneously by U87MG tumors, there was a marked increase of capillary-associated pericytes as seen in U87MG/VEGF(165)/PDGF-B gliomas. As a result of pericyte recruitment, vessels induced by VEGF in tumor vicinity migrated into the central regions of these tumors. These data suggest that PDGF-B is a paracrine factor in U87MG gliomas, and that PDGF-B enhances glioma angiogenesis, at least in part, by stimulating VEGF expression in tumor endothelia and by recruiting pericytes to neovessels.

Figure 1.

PDGF-B activates endogenous PDGF-Rβ in U87MG glioma cells. PAE, PAE-PDGF-Rβ, and U87MG cells were serum-starved overnight. The cells were then treated with 50 ng/ml of a human recombinant PDGF-B protein at 37°C for 15 minutes. The cell lysates were subjected to immunoblotting for detection of tyrosine phosphorylation on PDGF-Rβ. The membrane was then reprobed with an anti-human PDGF-Rβ antibody. The PDGF-Rβ protein runs as a 180-kd band in a 7.5% SDS-PAGE gel (bottom). Duplicate experiments yielded identical results.

Figure 2.

Expression of exogenous PDGF-B in U87MG glioma cells. A: Northern blot analysis. Top: Samples of the parental U87MG and two types of PDGF-B-overexpressing cells are shown. In each class, four individual PDGF-B-expressing clones are presented. Endogenous (•, 3.8 kb) and exogenous (▸, 0.8 kb) PDGF-B transcripts and their descriptions are in the text. Bottom: Methylene blue staining of the membrane after RNA was transferred into the membrane. ▪: 18 S and 28 S ribosomal RNA species. Duplicate experiments yielded similar results. B: PDGF-B ELISA analyses. Cells (3 × 10⁵) of the parental U87MG- or PDGF-B-expressing cells or VEGF₁₆₅- or VEGF₁₆₅/PDGF-B-expressing cells were seeded onto 12-well plates in triplicate. On the next day, the media was replaced with DMEM/0.5% bovine serum albumin/1% dialyzed fetal bovine serum for another 24 hours. CM was collected after the cells were cultured for an additional 48 hours. Each bar represents the mean ± SEM of three triplicates. Identical experiments were also performed using CM of other PDGF-B-overexpressing clones, B-26, B-34, V/B-11, and V/B-33. The assays were performed two additional times using cells of various passage numbers with similar results.

Figure 3.

PDGF-B enhances U87MG glioma angiogenesis by increasing VEGF expression in tumor ECs and by recruiting vessel-associated pericytes. A: Immunohistochemical analyses of gliomas formed by the parental U87MG- and PDGF-B-expressing cells. The analyses were performed with a rat monoclonal anti-CD31 antibody (a and b), a polyclonal anti-desmin antibody (c and d), a polyclonal anti-VEGF antibody (red color) together with the anti-CD31 antibody (green color, e and f) and a polyclonal anti-PDGF-B antibody (g and h). Arrows show positive staining for blood vessels (a and b) or pericytes (c and d). In e and f, arrows indicate blood vessels in the tumors. Arrowheads indicate VEGF staining. In f, most of the vessels were stained by both the anti-VEGF and the anti-CD31 (vessels) antibodies (orange color). a, c, b, and d: Immunohistochemical staining in identical areasof serial sections from the same individual mouse brains. Six or more individual tumor samples of each type were analyzed. Experiments were repeated two additional times with similar results. B: Increased vessel densities and recruitment of vessel-associated pericytes in U87MG PDGF-B-expressing gliomas. Quantitative analyses of immunohistochemistry data that are shown in a, b, c, and d were done as described in Materials and Methods. The representative immunohistochemical stains from the parental U87MG- and PDGF-B-expressing gliomas are shown in a and b (vessel stains) or c and d (desmin stains). In each analysis, 10 to 15 random areas within the same tissue section were examined. The mean values of five to seven serial sections from six to seven separate mouse brains in each group were used for the quantitative analyses. Data are presented as means ± SD. Numbers above each column are the numbers of mice analyzed in each group. Numbers in the parentheses under the x axis are the fold difference of the densities found in U87MG- and PDGF-B-expressing gliomas compared with that in parental U87MG tumors. Original magnifications in A: ×200 (a, b); ×400 (c, d, e, f, g, and h).

Figure 4.

Overexpression of PDGF-B increased both tumor and EC proliferation. A: Immunohistochemical analyses of gliomas formed by parental U87MG- and PDGF-B-expressing cells. The analyses were performed with a monoclonal anti-BrdUrd antibody together with a polyclonal anti-von Willebrand factor antibody (a and b). Arrows show positive staining of proliferative nuclei in blood vessels. Arrowheads indicate BrdUrd staining only in the nuclei of tumor cells. Three to five serial sections from four to six individual tumor samples of each type were analyzed independently. Experiments were repeated two additional times with similar results. B: Increased proliferative index in both total cell (left columns) and ECs (right columns) in the PDGF-B-expressing gliomas. Quantitative analyses of immunohistochemistry data were done as described in Materials and Methods. Original magnification: ×400 (A).

Figure 5.

PDGF-B augments U87MG glioma angiogenesis by promoting vessel migration and recruiting vessel-associated pericytes. A: Immunohistochemical analyses of gliomas formed by either VEGF₁₆₅- or VEGF₁₆₅/PDGF-B-expressing cells. The analyses were performed with the anti-CD31 antibody (a and b), the polyclonal anti-desmin antibody (c and d), the polyclonal anti-VEGF antibody (e and f), and the polyclonal anti-PDGF-B antibody (g and h). Arrows show positive staining for blood vessels (a and b) or pericytes (c and d) or VEGF (e and f) or PDGF-B (g and h). Areas shown in a, c, and b, d or e, g, and f, h are immunohistochemical staining in identical areas from the same individual mouse brains. Five serial sections from five or six individual tumor samples of each type were analyzed independently. Experiments were repeated two independent times with similar results. B: Increased recruitment of vessel-associated pericytes in U87MG/VEGF/PDGF-B-expressing gliomas. Quantitative analyses of immunohistochemistry data were performed as described in Materials and Methods. Original magnifications in A: ×40 (a, b, c, and d); ×400 (e, f, g, and h).