Cell type-specific expression of angiopoietin-1 and angiopoietin-2 suggests a role in glioblastoma angiogenesis

Abstract

Glioblastomas are highly vascular tumors which overexpress the angiogenesis factor vascular endothelial growth factor (VEGF). VEGF and its receptors, VEGF-R1 and VEGF-R2, have been shown to be necessary for embryonic angiogenesis as well as for tumor angiogenesis. Recently, the angiopoietin/Tie2 receptor system has been shown to exert functions in the cardiovascular system that are distinct from VEGF but are also critical for normal vascular development. To assess the potential role of Tie2 and its ligands angiopoietin-1 and angiopoietin-2 in tumor vascularization, we analyzed their expression pattern in human gliomas. Tie-2 was up-regulated in tumor endothelium compared to normal human brain tissue. We further observed cell type-specific up-regulation of the message for both angiopoietin-1 and angiopoietin-2 in gliomas. Whereas Ang-1 mRNA was expressed in tumor cells, Ang-2 mRNA was detected in endothelial cells of a subset of glioblastoma blood vessels. Small capillaries with few periendothelial support cells showed strong expression of Angiopoietin-2, whereas larger glioblastoma vessels with many periendothelial support cells showed little or no expression. Although the function of Tie2 and its ligands in tumor angiogenesis remains a subject of speculation, our findings are in agreement with a recently proposed hypothesis that in the presence of VEGF, local production of Ang-2 might promote angiogenesis.

Figure 1.

Immunohistochemistry for Tie2 in normal brain (a,b), astrocytoma (c,d), and glioblastoma (e,f). a, c, and e show immunoreactivity for Tie2. b, d and f represent control experiments with blocked antibody. Magnification, ×400; scale = 10 μm.

Figure 2.

Northern blot analysis of Ang-1 and Ang-2 mRNA expression in normal brain, low-grade gliomas, and high-grade gliomas. Total RNA samples of 10 μg prepared from normal brain (lane1), three glioblastomas WHO grade IV (lanes 2–4), one anaplastic astrocytoma WHO grade III (lane 5), two astrocytomas WHO grade II (lanes 6–7), and one pilocytic astrocytoma WHO grade I (lane 8) were probed for the indicated transcripts. Ethidium bromide-stained gel indicates amount of RNA in each lane.

Figure 3.

In situ hybridization analysis of Ang-1 and Ang-2 mRNA expression in human brain and in glioblastoma. a-d: Ang-1 in situ hybridization in cerebellum (a) and in glioblastoma (c). Arrow in a denotes blood vessel expressing Ang-1. b and d: Ang-1 sense-control hybridization. Magnification, ×100. e-f: Ang-2 in situ hybridization in cerebellum (e) and in glioblastoma (g, i, j). g and i: Ang-2 expression in small vessels. Arrowhead in (i) denotes vessel sprout, (j) no Ang-2 expression in large vessel. f and h represent Ang-2 sense control hybridization. Magnification, ×100 (e-i); ×200 (j). MCL, molecular cell layer; PCL, Purkinje’s cell layer; GCL, granular cell layer; N, necrosis

Figure 4.

Smooth muscle actin expression in normal brain (a) and in glioblastoma (b). a: Few SMA-positive cells in postcapillary venule (arrow). b: Extensive recruitment of SMA-positive cells to the tumor vasculature in glioblastoma. Magnification, ×200 (a), (scale = 20 μm); ×400 (b). c,d: Double labeling in situ hybridization (ISH) and immunohistochemistry (IHC) for Ang-2 mRNA and vWF expression in glioblastoma. Ang-2 mRNA is expressed in a subset of vWF-positive endothelial cells (c). Note overlapping ISH and IHC staining for Ang-2 (dark brown) and vWF (orange). L: Lumen of large vessel with vWF-positive endothelial cells not expressing Ang-2. Magnification, ×100. (d) Blow-up of vessel marked in (c). Ang-2 mRNA expression (dark brown staining, arrows) is localized in vWF-positive (orange staining) vascular endothelial cell. Magnification, ×630.