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NG2 proteoglycan promotes angiogenesis-dependent tumor growth in CNS by sequestering angiostatin¹

MARTHA CHEKENYA^*, MARI HJELSTUEN, PER ØYVIND ENGER^*, FRITS THORSEN^*, ANNE L. JACOB^*, BEATRICE PROBST^*, OLAV HARALDSETH, GEOFFREY PILKINGTON^II, ARTHUR BUTT^¶, JOEL M LEVINE^** and ROLF BJERKVIG^*2

^* Department of Anatomy and Cell Biology, University of Bergen, N-5009 Bergen, Norway;
SINTEF Unimed MR Center, N-7465 Trondheim, Norway;
Department of Anesthesia and Medical Imaging, The Norwegian University of Science and Technology, Trondheim, Norway;
^II Experimental NeuroOncology, Department of Neuropathology, Institute of Psychiatry King’s College, London SE5 8AF, UK;
^¶ Center for Neuroscience, Guys’, Kings and St. Thomas School of Biomedical Sciences, King’s College, London SE1 1UL, UK; and
^** Department of Neurobiology and Behavior, State University of New York, Stony Brook, New York, USA

²Correspondence: Department of Anatomy and Cell Biology, University of Bergen, Årstadveien 19, N-5009 Bergen, Norway. E-mail: Rolf.Bjerkvig{at}pki.uib.no

SPECIFIC AIMS

During embryogenesis immature capillaries express NG2 in the brain and spinal cord, but the expression becomes less prominent in the postnatal and mature brain. Our experiments were undertaken to 1) investigate the hypothesis that the NG2 chondroitin sulfate proteoglycan (CSPG) mediates tumor growth by increasing tumor angiogenesis and 2) delineate its mechanism of action in the malignant progression of human brain tumors.

PRINCIPAL FINDINGS

1. NG2 increases glioblastoma cell proliferation in vitro and tumorigenicity in vivo
To investigate the effect of NG2 on the proliferation of glioblastoma multiforme (GBM) cells, we compared the growth of three NG2-transfected U251N clones against wild-type (WT) and sham negative controls in vitro. All three NG2 clones showed significantly enhanced growth rates vs. WT (P<0.001) and control cells (P<0.05). To test whether the growth advantage conferred by NG2 in vitro would affect tumor growth in vivo, we implanted the tumor spheroids intracerebrally into immunodeficient nude rats. NG2 increased the tumorigenicity of the GBM cells: 87.5% of the animals in this group developed intracranial tumors vs. 40% in the WT and sham control groups.

2. NG2 increases tumor volume and intracranial distribution, predisposing to a poorer survival outcome
We used contrast-enhanced MRI with high spatial resolution to localize and determine tumor volumes and distribution within the brain. Tumor tissue could easily be distinguished from normal brain tissue due to the disruption of the blood-brain barrier (Fig. 1 a–c). Representative control tumors, WT (Fig. 1a ), and sham (Fig. 1b ) exhibited homogeneously contrast-enhancing small tumors. The NG2 tumors were eightfold larger and more heterogeneous (Fig. 1c ), with contrast-enhancing peripheral ‘cuffs’ surrounding dark hypodense necrotic regions (P=0.032). Histological analyses of viable tumor tissue confirmed higher cellular density in the NG2-expressing tumors than controls (P<0.001). These tumors contained more mitotic figures than WT (P=0.001) and sham control tumors (P=0.0003), (Fig. 1a-c , lower panel). Ki67 (MIB-1) immunolabeling allowed us to assess tumor and endothelial cell proliferation. The NG2 tumors were significantly more proliferative than either the sham (P<0.001) or WT control tumors (P<0.02). The NG2 recipient animals (n=14) had a significantly shorter survival (median survival of 53 ± 3 days; 95% CI: 47–59 days) than control animals (n=19), with a median survival of 125 ± 24 days (95% CI: 77–173 days); Log Rank_13,16; df = 1; P < 0.0001).

View larger version (69K): [in this window] [in a new window]   Figure 1. NG2 increases the tumor burden. High spatial resolution T1-weighted spin echo sequence was used to determine tumor volumes. a) WT and b) sham negative control tumors were small and homogeneously enhancing. c) NG2 tumors were larger and more heterogeneous, with contrast-enhancing peripheral ‘cuffs’ surrounding dark hypodense regions inside the tumor corresponding to necrosis on H&E histological sections. WT and sham tumors (a, b, lower panels) were moderately cellular compared with the densely cellular NG2 tumors (P<0.001). NG2 tumors contained more mitotic figures than WT (P=0.001) and sham control tumors (P=0.0003) (c, arrowheads, lower panel); x400; scale bar: 60 µm).

3. NG2 augments tumor growth by promoting tumor angiogenesis in vivo
The growth-promoting effects observed with NG2 suggested increased tumor angiogenesis as a possible underlying mechanism. To test this hypothesis, we used functional MRI to determine the vascular volume fraction and vascular permeability of the NG2-positive and control tumors. Cerebral blood flow indicated that the NG2-positive tumors had a significantly increased vascular volume compared with sham and WT control tumors (P=0.031), (Fig. 2 a). There were no significant differences in vascular permeability (P=0.35). Quantification of microvessel density (MVD) using von Willebrand Factor (vWF) and CD31 confirmed the MRI findings that NG2 tumors were significantly more vascularized than the WT and sham control tumors: P = 0.001 and P = 0.004, respectively (Fig. 2b ). NG2 was stably expressed in vivo on tumor cells and their neovasculature (Fig. 2c ). NG2 immunoreactivity was restricted to the pericyte and basement membrane components of the tumor vasculature (Fig. 2c , insert). NG2 tumors were colossally vascularized (Fig. 2d ); 3-dimensional (3D) reconstruction of the confocal images confirmed a highly interconnected and chaotic vascular architecture (Fig. 2e ). The sham and WT tumors expressed low levels of NG2 on vasculature (Fig. 2f, g ). Immunolabeling for vascular antigens vWF revealed that control tumors were less vascularized than the NG2 tumors (Fig. 2h ).

View larger version (66K): [in this window] [in a new window]   Figure 2. NG2 promotes tumor blood flow and tumor angiogenesis in vivo. Gadolinium concentration time curves were used to assess the vascular volume fraction (D0) and vascular permeability (K2) of the tumor sublines. Typical concentration-time curves for NG2 (circle), sham (square), and WT (triangle) are shown. a) NG2 tumors had a significantly higher vascular volume fraction than the sham and WT control tumors. There was no significant difference in vascular permeability between the tumor sublines; P > 0.05. b) MVD counts of vWF-positive endothelial cells confirmed higher vascular densities in the NG2 tumors than the controls. Data represent mean MVD/µm3 ± SE, *Considered statistically significant using the Student’s t test, P < 0.05. c) Stable NG2 expression on the tumor cells and their neovasculature in vivo (brown, x400, scale bar: 60 µm). c, insert) NG2 immunoreactivity on tumor cells (black arrowhead) the pericyte and basement membrane components of the tumor vasculature (white arrowheads), x1000, scale bar: 40 µm. Harris hematoxylin nuclear counterstain (blue). d) vWF-positive endothelial cells on neovasculature in the corresponding NG2 tumor (green). e) 3D reconstruction of the tumor section in panel d x630, scale bar: 125 µm. f) Background NG2 expression in the sham tumors (green). g) NG2 expression in the WT tumor, x160, scale bar: 50 µm. h) Representative vWF-positive endothelial cells on vessels in the control tumors.

4. NG2 specifically promotes tumor angiogenesis in vivo by binding and sequestering endogenous angiostatin
To investigate mechanisms by which expression of NG2 influenced tumor angiogenesis, we compared the expression of endogenous levels of angiostatin in tumor tissue and cerebrospinal fluid (CSF) from WT and NG2 tumor-bearing rats. The control tumors contained high levels of angiostatin localized predominantly to blood vessels (P=0.005). We observed a striking diminution of angiostatin in tumor tissue and CSF from the NG2 recipient animals. To test whether NG2 might be mediating these effects by binding and sequestering angiostatin, we exposed NG2-transfected and WT cells to 5 µg/ml human angiostatin in vitro. NG2-expressing cells bound angiostatin and control cells did not. NG2 coimmunoprecipitated with angiostatin, extending the evidence of a physical association of NG2 to angiostatin in this biological microenvironment.

CONCLUSIONS AND SIGNIFICANCE

This study demonstrates that the NG2 CSPG stimulates tumor growth and neovascularization and that the molecule binds and sequesters angiostatin.

The mechanism by which NG2 augments tumor growth in vivo is suggested by its apparent multiple high-affinity binding sites for plasminogen and its proteolytic fragment, angiostatin. The latter profoundly inhibits neovascularization, which is critical for tumor growth. Our work shows that the NG2-expressing tumors have an abundant vascular network not present in corresponding NG2 negative tumors (Fig. 3 a, b). We show that NG2 proteoglycan binds and sequesters angiostatin, which may neutralize angiostatin’s inhibitory effects on endothelial cell proliferation and migration (Fig. 3a ). Indeed, coimmunoprecipitation of NG2 with angiostatin provides evidence of a physical interaction between the two molecules (Fig. 3a ). NG2 tumor-bearing animals lacked circulating endogenous angiostatin in the brain. Thus, increased tumor angiogenesis in the NG2-expressing tumors may be explained by NG2 interfering with the function of angiostatin in regulating tumor blood vessel formation.

View larger version (64K): [in this window] [in a new window]   Figure 3. Schematic representation of the hypothesized involvement of NG2 in tumor angiogenesis. a) NG2-expressing tumor cells bind and sequester angiostatin (red bullets). Inserts: Cell surface NG2 expression in one of several clones. Lanes: 1, NG2-expressing cells coimmunoprecipitate with angiostatin. b) NG2 binding to angiostatin and interferes with the latter’s inhibitory actions on endothelial cell proliferation, resulting in increased tumor growth, luxuriant vascular sprouting. Insert: 3D reconstruction of vWF-positive endothelial cells on vascular structures. c) The NG2 negative tumor cells. d) NG2 negative tumors contain abundant levels of endogenous angiostatin (red bullets on vessels), which inhibits endothelial cell proliferation, resulting in reduced vascular density and tumor angiogenesis. Insert: reduced vWF-positive vascular.

Angiogenesis inhibitors also regulate vascular maturation via mechanisms regulating proteolysis and recruitment of mural cells required for elastogenesis of mature vessels. Immature vessels contain NG2-positive pericytes, suggesting a role for this cell population in modulating endothelial cell proliferation by blocking the inhibitory effects of angiostatin during vessel development.

MRI blood volume measurements revealed a higher vascular volume fraction in the NG2 tumors. NG2 tumors were extensively perfused since the vascular volume estimations consisted only of functional (i.e., blood perfused) vessels that allow the passage of a contrast agent. Our finding reinforces the need for multimodal treatment strategies targeting the tumor vessels. The literature demonstrating angiostatin as a functional and therapeutic protein has been inconsistent. High dosages with multiple injections for prolonged treatment periods have been required to show an effect of angiostatin on tumor growth in animal studies. This compound is now being tested in clinical trials. The interactions of NG2 with angiostatin may have additional implications for the efficacy of angiostatin as a therapeutic agent for human brain tumors as well as other tumor types that constitutively overexpress NG2.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0632fje; to cite this article, use FASEB J. (February 12, 2002) 10.1096/fj.01-0632fje

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