Angiopoietin-1 promotes tumor angiogenesis in a rat glioma model

Abstract

Angiopoietins have been implicated in playing an important role in blood vessel formation, remodeling, maturation, and maintenance. However, the role of angiopoietins in tumor angiogenesis remains uncertain. In this study, expression of human angiopoietin-1 (hAng-1) and angiopoietin (hAng-2) was amplified in the rat glioma cell line GS9L by stable transfection using an inducible tet-off system. Transfected cells were implanted intracerebrally into syngenic Fischer 344 rats. We demonstrated by means of magnetic resonance imaging that increased hAng-1 expression promoted a significant in vivo growth of intracerebral gliomas in rats. Overexpression of hAng-1 resulted in more numerous, more highly branched vessels, which were covered by pericytes. On the other hand, tumors derived from hAng-2-overexpressing cells were smaller than empty-plasmid control tumors. The tumor vasculature in these tumors was composed of aberrant small vascular cords, which were associated with few mural cells. Our results indicate that in the presence of hAng-1, tumors induce a more functional vascular network, which led to better tumor perfusion and growth. On the other hand, overexpression of hAng-2 led to less intact tumor vessels, inhibited capillary sprouting, and impaired tumor growth.

Figure 1

Overexpression of hAng-1 and hAng-2 in GS9L cells. A: Equal amounts of total protein obtained from cell fractions were loaded and analyzed by SDS-PAGE followed by Western blotting and detection of angiopoietins with a goat anti-human Ang-1 or a goat anti-human Ang-2 antibody. The membranes were subsequently stripped and incubated with an anti-tubulin antibody for loading control. No expression of angiopoietins was detected in GS9L parental cells. In selected clones Western blot confirmed the overexpression of hAng-1 and hAng-2 protein. B: GS9L and stable-transfected cells were cultivated under normoxic and hypoxic conditions. hAng-2 protein levels in serum-free conditioned media were evaluated using ELISA. A 19-fold increase in levels of secreted hAng-2 was observed in supernatants obtained from transfected cells. Hypoxia increases hAng-2 protein levels in conditioned media. In GS9L-hAng-2 cells expression levels at normoxia was already at detection limit of the test and further increase under hypoxia could not be detected.

Figure 2

Intracranial glioma growth in syngenic rats. Wild-type and transfected cells (10⁵ in 10 μl PBS) were injected intracerebrally in syngenic Fischer rats. Nine days after tumor implantation, rats were anesthetized and brains were scanned in a Bruker 7.0 Tesla without and with contrast injection. Tumor volume was calculated by determining the area of contrast-enhanced region in each image. The area in each section was multiplied by the slice thickness. Final volume represents the sum of all enhanced areas in a given tumor. Each bar represents the mean and SD of tumors from rats injected with the corresponding GS9L wild-type (n = 6), GS9L-vector (n = 6), GS9L-hAng-1 (n = 8), and GS9L-hAng-2 (n = 6). (*, P < 0.05 relative to the wild-type control tumors).

Figure 3

In situ hybridization analysis of Ang-1, Ang-2, Tie-2, and VEGF mRNA in wild-type, hAng-2-, and hAng-1-transfected tumors. At day 9 after tumor implantation, small amounts of Ang-1 mRNA are expressed in control and hAng-2-transfected tumors. Only a subset of tumor cells (about 20%) expressed Ang-1 mRNA in hAng-1-transfected tumors suggesting a silencing of the transgene expression in vivo in these tumors. However, compared with the low constitutive expression of Ang-1 in control tumors, the overexpression of hAng-1 in transfected tumors is considerable. Ang-2 mRNA is expressed in control tumors, particularly associated with vessels localized at the tumor borders (arrows). hAng-2-overexpressing tumors showed a robust up-regulation of Ang-2 mRNA. There was no increase in Ang-2 expression in hAng-1-transfected tumors. Many tumor vessels are marked by Tie-2 expression (arrows). A slight up-regulation of Tie-2 mRNA is observed in hAng-2-overexpressing tumors. All tumors showed high expression levels of VEGF mRNA. Almost all tumor cells expressed VEGF mRNA at this tumor stage. hAng-1 or hAng-2 overexpression did not affect the expression patterns of VEGF as assessed by in situ hybridization. Sense control for Ang-1, Ang-2, Tie-2, and VEGF. Bars, 50 μm.

Figure 4

Confocal analysis of the vascularization of intracranial tumors 9 days after tumor implantation. Vibratome slices were stained with a rabbit anti-vWF (red). Transfected cells (vector, hAng-1, and hAng-2) expressed the EGFP (green). Tumor derived from parental and vector-transfected cells displayed an irregular vasculature, with vessels of uneven diameter and several shunts. EGFP expression in hAng-1-overexpressing tumors correlates with the finding of in situ hybridization, confirming that only a subset of tumor cells expressed the transgene. Nevertheless, hAng-1-overexpressing tumors are highly vascularized with highly branched vessels compared to the control tumors. hAng-2-overexpressing tumors, in contrast, displayed poorly built vessels consisting of small vessels with narrow lumen and few branching. Bars, 20 μm.

Figure 5

Morphometric analysis of tumor vasculature. Four animals per group were implanted with parental and transfected tumor cells and sacrificed at day 9 post-implantation. Tumor sections (at least three sections per animal) were stained for CD31. A: CD31 immunohistochemistry shows a homogenous distribution of tumor vessels throughout the tumor tissue. B: For assessment of tumor vessel count, random fields (three fields/section) were examined at a ×10 magnification. Blood vessels were counted using Soft Imaging Analysis System and classified according to vessel area (0 to 500 μm², 500 to 2000 μm², and >2000 μm²). Computer-assisted morphometric analysis of CD31-stained vessels disclosed an increase in number of small vessels (area <20 μm²) in hAng-1-overexpressing tumors. C: For assessment of vessel density CD31-stained tumor vessels were evaluated in random fields (five fields per section at a ×20 magnification) using Soft Imaging Analysis System. Analysis of vessel density revealed an increased vessel density in tumors derived from hAng-1 clone compared to the control tumors, whereas a decrease in vessel density was observed in hAng-2-overexpressing tumors. Data are expressed as mean values and SD (*, P < 0.05 relative to the wild-type control tumors). Bar, 100 μm.

Figure 6

Effect of angiopoietin overexpression on pericyte coverage of tumor vessels. A: Double-immunofluorescence stains for vWF (red) and α-SMA (green). Representative fields of wild-type and transfected tumors show increase coverage of tumor vessels in tumors derived from hAng-1-overexpressing cells, while the majority of vessels derived from hAng-2-transfected cells displayed a paucity of SMA-positive periendothelial cells. B: Quantitative analysis of the degree of periendothelial cell-associated vessel. The pericyte coverage index is determined by the percentage of tumor vessels that are surrounded by at least one positive SMA-positive periendothelial cell in random fields of the tumor. Data expressed the mean values and SD of at least six fields of tumors obtained from four animals/group. (*, P < 0.05 relative to the wild-type control tumors). Bars, 50 μm and 20 μm.

Figure 7

Electron micrographs of tumor capillaries in control and tumors overexpressing hAng-1 and hAng-2. Tumor samples were collected at the tumor border and inside the tumor. A and B: Capillaries of vector-transfected tumors are surrounded by pericytes. The perivascular space is enlarged (_*). Some luminal microfolds enlarged the endothelial surface. Endothelial cell nucleus (N); intraluminal red blood cell (RBC); tumor cell (T). The basement membrane is uniformly thick, compact, and closely opposed to the endothelial cells. C and D: hAng-1-overexpressing tumors showed capillaries covered by pericytes also in tumor center. The basal membrane in capillaries inside the tumor is surrounded by a structurally abnormal blurred basement membrane but it still provides complete vessel coverage. Myelin in tumor border (M): pericyte nucleus (PN). E and F: In hAng-2-overexpressing tumors, capillaries at the tumor border were surrounded by a discontinuous basement membrane (arrowheads). Vascular lumen (VL) is filled with fibrin and cell detritus. Pericytes (P) foot has lost the contact with the basement membrane. Intercellular junctions were present (arrows). Endothelial cells were swollen. Complete regression of the endothelial cell layer in a capillary in the center of hAng-2-overexpressing tumor (arrowheads). Bars, 1 μm and 2 μm.

Figure 8

hAng-2 overexpression did not increase tumor cell apoptosis. For detection of apoptotic cells, TUNEL assay was performed. TUNEL staining revealed no major differences between the transfected tumors and the control tumors. Several clusters of tumor cells within the tumor were positively labeled. Bars, 100 μm.

Figure 9

Capillary permeability in GS9L-wt-, GS9L-hAng-1-, and GS9L-hAng-2-derived tumors. A: Rats were injected intravenously with a 2% Evans blue solution and, after 60 minutes, were then perfused with PBS. Brains were then removed and photographed. MRI scans of corresponding slices obtained immediately before the Evans blue injection. B: Quantitative analysis of Evans blue extravasation from brain extracts. Data are expressed as ratio of amount of extravased Evans blue (ng/gram of brain) per tumor volume (mm³). Evans blue was spectrophotometrically quantified at 620 nm. As control for perfusion efficiency, the absorbance of the contralateral hemisphere was subtracted from that of the hemisphere ipsilateral to the tumor. For each group n = 5. P < 0.05 relative to wild-type control tumors.