Heparan Sulfate Proteoglycans as Regulators of Fibroblast Growth Factor-2 Signaling in Brain Endothelial Cells

SPECIFIC ROLE FOR GLYPICAN-1 IN GLIOMA ANGIOGENESIS*

Dianhua Qiao, Kristy Meyer, Christoph MundhenkeDagger, Sally A. Drew, and Andreas Friedl§

From the Department of Pathology and Laboratory Medicine, University of Wisconsin, Madison, Wisconsin 53562-8550

Received for publication, November 4, 2002, and in revised form, February 17, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Fibroblast growth factor-2 (FGF2) is a potent angiogenic factor in gliomas. Heparan sulfate promotes ligand binding to receptor tyrosine kinase and regulates signaling. The goal of this study was to examine the contribution of heparan sulfate proteoglycans (HSPGs) to glioma angiogenesis. Here we show that all brain endothelial cell HSPGs carry heparan sulfate chains similarly capable of forming a ternary complex with FGF2 and fibroblast growth factor receptor-1c and of promoting a mitogenic signal. Immunohistochemical analysis revealed that glypican-1 was overexpressed in glioma vessel endothelial cells, whereas this cell-surface HSPG was consistently undetectable in normal brain vessels. To determine the effect of increased glypican-1 expression on FGF2 signaling, we transfected normal brain endothelial cells, which express low base-line levels of glypican-1, with this proteoglycan. Glypican-1 expression enhanced growth of brain endothelial cells and sensitized them to FGF2-induced mitogenesis despite the fact that glypican-1 remained a minor proteoglycan. In contrast, overexpression of syndecan-1 had no effect on growth or FGF2 sensitivity. We conclude that the glypican-1 core protein has a specific role in FGF2 signaling. Glypican-1 overexpression may contribute to angiogenesis and the radiation resistance characteristic of this malignancy.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

High-grade gliomas are characterized by a highly aggressive clinical course, active angiogenesis, and endothelial cell proliferation. Fibroblast growth factor-2 (FGF2)1 is one of the most potent stimulators of angiogenesis in wound healing and tumor growth. FGF2 induces all the important components of blood vessel growth, including degradation of the extracellular matrix, endothelial cell migration and proliferation, and differentiation into vascular tubes. FGFs signal through transmembrane receptor tyrosine kinases, the FGF receptors (FGFRs) (1). Of the four known FGFRs, FGFR1 is the one most prevalently found on endothelial cells (2).

Stable binding of the FGF ligand to the receptor tyrosine kinase and sustained signaling require the presence of heparan sulfate (HS) glycosaminoglycans (3, 4). The participation of HS polysaccharides in the ternary FGF receptor signaling complex is facilitated through HS-binding motifs on both the FGF ligand and the receptor tyrosine kinase (5). HS chains are characterized by complex sulfation patterns resulting in distinct protein-binding domains, although it is currently unclear how the assembly of such domains is regulated. Experimental evidence indicates that stimulatory as well as inhibitory moieties are embedded within HS chains and that their relative balance determines the net effect on FGF signaling (6). Cells have the ability to rapidly adjust their HS to respond to a changing microenvironment (7). HS proteoglycan (HSPG) alterations have also been reported to occur during malignant progression. Recently, we described HS alterations in breast carcinomas, resulting in increased FGF2 binding and enhanced ternary FGF receptor complex assembly (8).

In vivo, the vast majority of HS exists in covalent linkage to core proteins, the HSPGs. HSPGs can be divided into cell-surface forms (syndecans and glypicans) and secreted extracellular matrix forms (e.g. perlecan). Literature reports on which HSPGs promote FGF2 signaling are conflicting. One study identified perlecan as the sole stimulatory HSPG, whereas syndecans and glypicans were found to inhibit FGF signaling (9). Others reported that syndecans and glypicans also promote FGF2 signaling (10). These divergent results suggest that HSPGs with different core proteins assume this role depending on cell type and functional context. The question of whether different HSPG core proteins produced by one cell type can carry HS chains with different effects (stimulatory versus inhibitory) on FGF2 signaling is currently unresolved.

Published reports on the role of HSPGs in angiogenesis are sparse (11). Quiescent as well as angiogenically active blood vessels contain ample amounts of FGF2. This suggests that FGF2-mediated angiogenesis is regulated by additional factors such as HSPGs. Perlecan has been described as a pro-angiogenic molecule (12) as well as an inhibitor of FGF2 signaling in blood vessels (13). Other investigators have attributed an exclusive role to syndecan-4 in FGF2-induced angiogenesis and ascribed a special role to the core protein of this HSPG (14).

The goal of this study was to examine the contribution of different brain endothelial cell HSPGs to FGF2 signaling and glioma angiogenesis. We found that despite the fact that all endothelial cell HSPGs have the potential to promote binding of FGF2 to FGFR1c through their HS chains and to promote signaling, specific HSPGs appear to carry out the function of principal FGF2 co-receptors in tumor angiogenesis. Specifically, glypican-1 is dramatically up-regulated in glioma vessels, leading to enhanced growth and endothelial sensitivity to FGF2 stimulation.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents-- Yeast-derived human recombinant FGF2 was kindly provided by Dr. Brad Olwin (University of Colorado, Boulder, CO). Anti-syndecan-1 antibody (B-B4) was purchased from Serotec (Raleigh, NC). Antibodies to syndecan-2 (10H4) (15), syndecan-3 (1C7) (16), syndecan-1 and -3 (2E9) (16), syndecan-4 (8G3) (17), and glypican-1 (S1) (18) were a generous gift from Dr. Guido David (University of Leuven, Leuven, Belgium). Rabbit polyclonal anti-syndecan-4 antibody was a gift from Dr. Alan Rapraeger (University of Wisconsin) (19). Neomarkers anti-perlecan antibody was purchased from LabVision (Freemont, CA), and anti-Delta HS antibody (3G10) (20) was from Seikagaku America (Falmouth, MA). This antibody reacts with unsaturated uronate/HS "stubs" generated by heparitinase treatment. Polyclonal rabbit anti-von Willebrand factor (vWF) antibody was purchased from Dako Corp. (Carpinteria, CA). This antibody reacts with the Factor VIII·vWF macromolecular complex.

Cell Culture and FGF2 Response Assays-- Dr. Robert Auerbach (University of Wisconsin, Madison, WI) kindly provided mouse brain endothelial (MBE) cells (21). GM7373 immortalized bovine endothelial cells were obtained from the Coriell Institute for Medical Research (Camden, NJ) (22). Both cell types were grown in Dulbecco's modified Eagle's medium supplemented with 10% calf serum. Human umbilical vein endothelial cells (HUVECs) and human dermal microvascular endothelial cells (HMVECs) were from Cambrex Corp. (East Rutherford, NJ). Human saphenous vein endothelial cells (HSVECs) were purchased from VEC Technologies (Rensselaer, NY). All human primary endothelial cells were grown on gelatin-coated tissue culture dishes in EGM-2 endothelial cell growth medium (Cambrex Corp.). Proliferation of adherent cells was measured with a bromodeoxyuridine (BrdUrd) incorporation assay (BrdUrd colorimetric, Roche Diagnostics) according to the manufacturer's instructions. The cells were starved in serum-free medium for 24 h and then treated with FGF2 for 24 h with BrdUrd label added for the final 4 h. The urokinase assay was adapted from Presta et al. (22). Briefly, GM7373 cells were treated with FGF2 for 24 h. Cell lysates were prepared by sonication. Ten µg of protein from cell lysate were incubated with bovine plasminogen (40 µg/ml) and Spectrozyme PL chromogenic substrate (330 µM). Absorbance at 405 nm was measured at 10-min intervals. Human high-molecular mass urokinase served as a standard. All urokinase assay reagents were purchased from American Diagnostica, Inc. (Greenwich, CT). FR1c-11 cells (BaF3 cells transfected with the FGFR1c isoform; kindly provided by Dr. David Ornitz, Washington University, St. Louis, MO) were maintained in RPMI 1640 medium supplemented with 10% calf serum and 10% WEHI-3 cell-conditioned medium as source of interleukin-3. For the growth assays, cells (20,000/well) were added to 96-well tissue culture plates in RPMI 1640 medium lacking interleukin-3 and incubated with various combinations of FGF2 and HSPGs for 72 h. The tetrazolium compound-based CellTiter 96 aqueous cell viability/proliferation assay (Promega, Madison, WI) was performed according to the manufacturer's instructions at the end of the incubation period.

Cell Transfections-- A 2.5-kb mouse glypican-1 cDNA fragment (a generous gift from Dr. Guido David) was cloned into the mammalian expression vector pcDNA3.1/Myc-HisA (Invitrogen). A 1.3-kb mouse syndecan-1 cDNA sequence was cloned into the pIRESneo3 mammalian expression vector (Clontech) under the control of the cytomegalovirus promoter/enhancer. The internal ribosome entry site in this vector permits simultaneous expression of syndecan-1 and the selection marker neomycin phosphotransferase (Neo) from one mRNA. This vector was chosen because measurable syndecan-1 overexpression could not be achieved with the pcDNA3.1 vector. MBE cells were stably transfected with either construct or the respective empty vector using TransIT-LT1 transfection reagent (Mirus, Madison, WI). After selection with G418 (500 µg/ml), cell extracts were subjected to HSPG Western blot analysis with antibody 3G10 to determine the levels of HSPG core proteins. Proliferation assays were carried out on mixed transfected populations to avoid clonal selection bias.

HSPG Extractions and Complex Precipitations-- Total endothelial cell HSPGs were purified as described (8, 23). For selective isolation of cell-associated and extracellular matrix HSPGs, we modified a procedure described by Elkin et al. (24). Cells were grown for 3 days in 150-mm dishes. After rinsing, the cell monolayer was dissolved in cold cell lysis buffer (20 mM NH4OH and 0.5% (w/v) Triton X-100 in phosphate-buffered saline, pH 10) for 1.5 min. After removal of the lysate, the remaining extracellular matrix layer was washed four times with Hepes-buffered saline and extracted with 3 ml of buffer containing 10 mM Tris, M urea, 0.1% (w/v) Triton X-100, 1 mM Na2SO4, 1 mM phenylmethylsulfonyl fluoride, and 1 mM N-ethylmaleimide, pH 8.0, for 5 min on ice and scraped into a polypropylene bottle. All extractions were enriched for proteoglycans by DEAE ion-exchange chromatography as described (8). HSPGs were quantified by dot blotting after heparitinase digestion and stained with antibody 3G10 using commercially available HSPGs (Sigma) as standards.

To examine the ability of HSPGs to bind to FGF2, the growth factor was biotinylated while bound to heparin-agarose to protect its HS-binding site and then immobilized on streptavidin-agarose beads. MBE cell HSPGs were incubated with immobilized FGF2 and eluted with 2 M NaCl. Fractionation of HSPGs according to their ability to promote binding of FGF2 to FGFR1 was performed as described (8). All HSPG preparations were digested with heparitinase and chondroitinase to remove all glycosaminoglycan chains prior to analysis on a 3.5-15% (w/v) Tris borate-polyacrylamide gradient gel. The membranes were stained with anti-Delta HS antibody 3G10 or anti-core protein antibodies (see above). A horseradish peroxidase-conjugated secondary antibody and SuperSignal West Femto maximum sensitivity substrate (Pierce) were used for chemiluminescent detection.

FGF·Receptor Complex Reconstitution in Situ-- Residual human tissue was obtained fresh from the surgical pathology laboratory, embedded in OCT compound (Sakura, Torrance, CA), snap-frozen, and stored at -70 °C. Binding assays were carried out essentially as described previously (25, 26). Briefly, the sections were first incubated with FGF2 (10 nM) and then with the soluble recombinant FGFR1c extracellular domain linked to alkaline phosphatase (referred to as FR1-AP; 30 nM) (3). Immobilized FR1-AP was detected with monoclonal anti-AP antibody (Sigma) and visualized with Alexa 546-conjugated donkey anti-mouse antiserum (Molecular Probes, Inc., Eugene, OR). Rabbit polyclonal anti-vWF antiserum (1:2000) was used to localize endothelial cells in tissue sections, and 4,6-diamidino-2-phenylindole was applied as a nuclear counterstain.

Immunohistochemistry-- Syndecan-1 and -4 and glypican-1 staining was performed on paraffin-embedded tissues as described (8). Staining for syndecan-2 and -3 was carried out on cryostat sections because antibodies 10H4 and 1C7 did not produce a detectable signal on paraffin sections. In addition, cryosections were stained for glypican-1 with antibody S1 to confirm the paraffin section results. Antibodies 10H4 (10 µg/ml), 1C7 (10 µg/ml), and S1 (5 µg/ml) were detected with Alexa 546-conjugated secondary antibody. Rabbit polyclonal anti-vWF antibody was used to localize endothelial cells in tissue sections (see above). All fluorescence microscopy was done using an Olympus BX51 microscope equipped with a SPOT RT slider chilled CCD digital camera (Diagnostic Instruments, Sterling Heights, MI).

Statistics-- Growth responses at different concentrations were compared using the Wilcoxon rank sum test. Correlations between staining results were examined by regression analysis using the StatView statistics program.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Heparan Sulfate Is Required for FGF2 Signaling in Endothelial Cells-- A number of studies on a variety of cell types have demonstrated that HS is necessary for stable binding of FGF2 to FGFRs and for signaling. To establish an HS requirement for FGF2 signaling in endothelial cells, we evaluated the effect of abolishing HS on the key angiogenic and FGF2-stimulated events mitogenesis and urokinase production. MBE cells show a robust proliferative response to FGF2. In GM7373 bovine endothelial cells, FGF2 prominently induces urokinase-type plasminogen activator, a crucial enzyme responsible for extracellular matrix degradation during angiogenesis (27). Experimental evidence points toward divergent signaling pathways involved in the regulation of these two cellular responses (22). Functional HS was abolished by treatment with sodium chlorate, a competitive inhibitor of glycosaminoglycan sulfation (28). Chlorate treatment resulted in a significant inhibition of MBE cell proliferation (Fig. 1A) and complete suppression of urokinase production by GM7373 bovine endothelial cells (Fig. 1B). Importantly, these responses were recovered in either cell type when HS sulfation was restored with sodium sulfate or by providing soluble heparin as an HS substitute to the cells in culture.


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Fig. 1.   HS requirement for FGF2 signaling in endothelial cells. Endothelial cell lines were grown for 48 h in sulfate-free medium supplemented with sodium chlorate (30 mM) to abolish sulfation of HS. After 24 h of serum starvation, the cells were treated with FGF2 (100 pM) for 24 h in the presence or absence of SO4 (10 mM) or heparin (1 µM) as indicated. A, proliferation of MBE cells measured by BrdUrd incorporation. Results are expressed as -fold stimulation over base-line growth. Bars indicate the means of six measurements. Error bars indicate S.E. B, urokinase-type plasminogen activator (uPA) production by GM7373 bovine endothelial cells. Urokinase was measured with a two-step chromogenic enzyme assay in cell lysates standardized according to protein concentration. Bars indicate means of triplicates. Error bars indicate S.E.

All Endothelial Cell HSPGs Are Capable of Acting as FGF2 Co-receptors-- HS exists almost exclusively in covalent linkage to a heterogeneous group of core proteins. As a first step in determining which HSPGs participate in the FGF2 co-stimulator role, we measured HSPG expression in different endothelial cells. Microvascular MBE cells, primary HMVECs, and human umbilical and saphenous vein endothelial cells were analyzed by Western blotting. Removal of all glycosaminoglycan chains by treatment with heparitinase and chondroitinase allowed the detection of discrete bands on SDS-polyacrylamide gradient gels. Visualization of all HSPGs was possible using an antibody (clone 3G10) directed against the HS stubs (anti-Delta HS) remaining on the core proteins after heparitinase digestion (Fig. 2A, lanes 1-4) (20). The signal generated with this antibody would be expected to be a function of the amount of core protein and the number of HS attachment sites per molecule. However, the possibility of biased heparitinase activity or 3G10 reactivity against different HSPGs cannot entirely be excluded. With this semiquantitative method, we estimated that syndecan-2 and -4 were the most prevalent cell-surface HSPGs produced by all of the endothelial cell types examined, whereas syndecan-1 and -3 and glypican-1 represented minor HSPG fractions in some cell types or were undetectable in others. A relatively strong band representing the secreted extracellular matrix HSPG perlecan was also observed in all cells examined. The identity of most bands was established in MBE cell extracts using core protein-specific antibodies (Fig. 2A, lanes 5 and 7-9). Glypican-1 was identified in HMVEC extracts (Fig. 2A, lane 6) for lack of a mouse-reactive antibody. We noted a slightly delayed migration of human syndecan-4 (Fig. 2A, lanes 2-4) compared with murine syndecan-4 (lane 1), likely due to the species difference. Overall, MBE cells produced the largest amount of HSPGs. Syndecan-2 was the predominant HSPG in microvascular cells (MBE cells and HMVECs), whereas syndecan-4 predominated in large vein endothelial cells (human umbilical and saphenous vein endothelial cells).


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Fig. 2.   Characterization of endothelial cell HSPGs as FGF2 co-receptors. A, spectrum of HSPGs produced by different endothelial cell types. HSPGs extracted from different endothelial cell types were treated with heparitinase and chondroitinase (see "Materials and Methods") to degrade glycosaminoglycan chains and then analyzed by SDS-PAGE using a 3.5-15% (w/v) gradient gel. Lanes 1-4, 5.0 × 105 cell equivalents stained with anti-HS stub (anti-Delta HS) antibody 3G10, which allows the detection of all HSPGs; lanes 5-9, 5.0 × 105 cell equivalents stained with anti-core protein antibodies as indicated. Antibody 2E9, used for staining of lane 8, reacted with the cytoplasmic domains of both syndecan-1 and -3 (see "Materials and Methods" for details). Lanes 5 and 7-9 contained extracts from MBE cells, and lane 6 contained extracts from HMVECs. HUVEC, human umbilical vein endothelial cells; HSVEC, human saphenous vein endothelial cells; Perl, perlecan; Glyp-1, glypican-1; Synd-2, syndecan-2; Synd-1, -3, syn- decan-1 and -3; Synd-4, syndecan-4. B, fractionation of brain endothelial cell HSPGs according to their ability to bind FGF2. HSPGs isolated from MBE cells (1.2 × 106 cell equivalents) were incubated with FGF2-coated agarose beads. Bound material was eluted with 2 M NaCl. After concentration on DEAE beads, the complex eluate (C) and supernatant (S) were analyzed as described for A. The membrane was probed with anti-Delta HS antibody 3G10. A total HSPG (T) loading control (lane 1), a beads-only (without FGF2) control (lanes 4 and 5), and competition with heparin (1 mg/ml; lanes 6 and 7) are shown as indicated. C, fractionation of brain endothelial cell HSPGs according to their ability to promote binding of FGF2 to FGFR1. In the presence of FGF2 (16 nM), HSPGs isolated from MBE cells (1.2 × 106 cell equivalents) were incubated with FR1-AP fusion protein immobilized on agarose beads. After washing with NaCl (concentrations as indicated) and digestion with heparitinase and chondroitinase, HSPGs complexed to the beads were analyzed by SDS-PAGE, blotting the membrane with anti-Delta HS antibody (lanes 4-6). A total HSPG (T) loading control (5.0 × 105 cell equivalents) is shown (lane 1). Negative controls included digestion of the HSPG preparation with heparitinase prior to complex formation (lane 2) and omission of FGF2 from the binding reaction (lane 3).

FGF2-binding ability would be expected to be the minimal requirement for HSPGs to function as FGF2 co-stimulators. To detect any potential differences in FGF2 binding between endothelial HSPGs, we incubated purified MBE cell HSPGs with immobilized FGF2 and analyzed the HSPGs found in this complex. The spectrum of HSPGs that bound to FGF2 (Fig. 2B, lane 3) was similar to that of the loading control (lane 1), indicating that all HSPG core proteins are decorated with FGF2-binding HS chains. No binding was detected when beads lacked FGF2 (Fig. 2B, lane 5) or when heparin was added to the binding reaction as a competitive inhibitor (lane 7).

FGF2 binding is likely necessary for HSPGs to stimulate signaling, but is clearly not sufficient. The ternary receptor complex, which is generally regarded as the active signaling complex, is expected to require additional HS motifs interacting with HS-binding sites on FGFR (5). Because FGFR1 is reportedly the only FGFR expressed by MBE cells (2), and FGFR1c the most prevalent FGFR1 isoform,2 HSPGs isolated from this cell type were incubated with immobilized FGFR1c in the presence of FGF2. HSPGs participating in this ternary complex were analyzed on gradient gels (Fig. 2C, lane 4). Again, the relative abundance of HSPGs found in the complex was similar to that found in the loading control (Fig. 2C, lane 1), suggesting that all core proteins carry HS chains with a similar potential to promote FGF2 binding to FGFR1c. Complex formation was abolished by omitting FGF2 from the reaction (Fig. 2C, lane 3) or by digesting the HSPGs with heparitinase prior to binding (lane 2), demonstrating the requirement for the FGF2 ligand and for intact HS chains. The complexes were similarly resistant to a NaCl concentration of 600 mM, but dissociated progressively when exposed to NaCl concentrations between 1 and 1.4 M (Fig. 2C, lanes 5 and 6). To exclude the possibility that FGF2 bound to recombinant FGFR1c independent of heparan sulfate in these experiments, we immobilized biotinylated FGF2 on streptavidin-coated plates and measured binding of FGFR1c. As expected, FGFR1c binding to immobilized FGF2 was highly dependent on the presence of heparin (data not shown). In summary, this series of in vitro experiments demonstrates that HS chains on all MBE cell HSPGs are similarly capable of binding FGF2 and of promoting binding of FGF2 to FGFR1c in a ternary complex.

Different Classes of Brain Endothelial Cell HSPGs Have a Similar Ability to Promote FGF2 Signaling through FGFR1c-- The formation of a high-affinity ternary FGF·receptor complex does not necessarily assure signal generation. It is possible that a dimerized receptor signaling complex requires additional structural features within HS not examined in the assays used so far. To examine potential differences in the ability of endothelial cell HSPGs to promote receptor activation, HSPGs were fractionated into functional classes using selective extraction protocols (adapted from Ref. 24). The relative purity of cell-associated HSPGs (composed primarily of syndecans and glypican-1) and extracellular matrix HSPGs (composed primarily of perlecan) was tested by Western blot analysis (Fig. 3A). Purified HSPGs were carefully quantified by dot blotting using antibody 3G10 (data not shown). Their activity was tested using FR1c-11 cells, which are BaF3 murine lymphoid cells transfected with FGFR1c (3). FR1c-11 (and parent BaF3) cells lack endogenous HSPGs and therefore depend on an exogenous source of HSPGs for FGF2-induced mitogenesis. HSPG dose-response curves in the presence or absence of FGF2 did not differ significantly between cell-associated, extracellular matrix, and total HSPGs, indicating that HS chains on these different core proteins are similarly capable of promoting an FGF2 signal (Fig. 3B).


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Fig. 3.   Activity of different brain endothelial cell HSPGs as promoters of FGF2 signaling. Total, cell-associated (CA), and extracellular matrix (ECM) HSPGs were selectively extracted from MBE cell cultures (see "Materials and Methods" for details). A, the relative purity of the preparations was tested by Western blot analysis using anti-Delta HS antibody 3G10 for detection. Note that cell-associated HSPGs were greatly enriched for syndecan-1, -2, -3, and -4 and contained only small amounts of perlecan. In contrast, extracellular matrix HSPGs consisted almost exclusively of perlecan. B, total and selectively extracted HSPG preparations were carefully quantified, and the concentrations were adjusted. Increasing concentrations of HSPGs were added to FR1c-11 cells (HSPG-deficient murine lymphoid cells transfect with FGFR1c) in the presence and absence of FGF2 (10 nM). Cell number was determined after 72 h using a tetrazolium compound-based assay.

Human Glioma Vessel HSPGs Have an Increased Ability to Form the Ternary FGF2·HSPG·FGFR1c Complex Compared with Normal Brain Vessel HSPGs: Glypican-1 Is Overexpressed in Human Glioma Vessels-- To examine whether vascular HSPG binding activities are altered in gliomas, we reconstituted the FGF2·HSPG·FGFR1 complex in situ on frozen or paraffin sections of human tissue samples. Biopsy samples from grade IV astrocytomas (glioblastoma multiforme) and non-neoplastic brain controls (temporal lobectomy samples and normal white matter adjacent to tumors) were compared. In these experiments, binding of soluble FGFR1c to HSPGs within the tissue was measured in the presence of FGF2. FGFR1c binding was notably increased in glioma vessels (Fig. 4, A and E) compared with normal brain vessels (Fig. 4, D and H) in 7 of 11 cases examined (64%). No recognizable morphologic features distinguished FGFR1c "binding" from "non-binding" cases. The binding signal was abolished by enzymatic degradation of HS with heparitinase (Fig. 4, B and F), demonstrating HS dependence, or by omitting the FGF2 incubation step (Fig. 4, C and G). This result indicates either a qualitative or quantitative alteration of glioma vessel HSPGs in the majority of tumors. Paraffin sections stained with anti-HS stub antibody 3G10 demonstrated an increase in total HSPG content in glioma vessels compared with normal brain vessels (Fig. 5, A and B), suggesting that increased vascular HSPG content rather than altered HS structure is responsible for the elevated binding activity. Regression analysis of individual cases revealed a significant association between FR1-AP binding and antibody 3G10 staining intensity (p = 0.047).


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Fig. 4.   Reconstitution of the FGF2·HSPG·FGFR1 complex in situ. Frozen sections of glioma tissues were first incubated with FGF2 (10 nM) and after a washing step with soluble FR1-AP fusion protein (30 nM). In this assay, the tissue section served as a source of HSPGs. Bound FR1-AP was detected by immunofluorescence using an anti-AP antibody followed by Alexa 546-conjugated secondary antibody (red channel). A and E, FR1-AP binding in high-grade glioma vessels. B and F, negative control for FR1-AP binding in high-grade glioma vessels. The tissue section was treated with heparitinase (Hep'ase) prior to the FGF2 binding step to identify the tissue binding sites as HSPGs. C and G, negative control for FR1-AP binding in high-grade glioma vessels. The FGF2 incubation step was omitted to demonstrate FGF2 dependence of FR1-AP binding. D and H, FR1-AP binding in normal brain vessels. The granular red stain in the brain parenchyma represents autofluorescence. E-H are merged images in which endothelial cells (ECs) were identified by immunofluorescence staining with anti-vWF antibody followed by Alexa 488-conjugated secondary antibody (green channel). Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI; blue channel). Magnification is ×400 in all panels.


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Fig. 5.   Immunohistochemical detection of HSPG core proteins in paraffin sections. A, normal brain vessel stained with anti-Delta HS antibody 3G10; B, high-grade glioma vessel stained with anti-Delta HS antibody 3G10; C, normal brain vessel stained for syndecan-4 (sdc4); D, high-grade glioma vessel stained for syndecan-4; E, normal brain vessel stained for glypican-1 (gpc1); F, high-grade glioma vessel stained for glypican-1; G, high-grade glioma vessel stained with the endothelial marker CD31; H, high-grade glioma vessel stained for syndecan-1 (sdc1). The inset shows positive control tissue (breast duct (Du) with adjacent plasma cells (PC)). Sections shown in A and B were treated with heparitinase prior to antibody incubation. Magnification is ×400. EC, endothelial cells; TC, tumor cells.

Next, we investigated whether the increased binding activity of glioma vessel HSPGs is accompanied by an increase in any particular HSPG core protein. Immunohistochemical analysis of paraffin-embedded human tissues revealed that syndecan-1 was undetectable in normal and glioma vessels (Fig. 5H). Low levels of syndecan-4 were seen in both glioma (Fig. 5D) and normal brain (Fig. 5C) vessels. In contrast, glypican-1 was strikingly overexpressed in glioma vessel endothelial cells (moderate-to-strong staining in five of eight cases, 63%) (Fig. 5F), whereas normal brain vessel endothelial cells consistently lacked this cell-surface HSPG (Fig. 5E).

The study of human tissue samples was expanded to include cryosections. The purpose of these additional labeling experiments was to measure expression of HSPGs undetectable in paraffin sections, to confirm the striking glypican-1 expression pattern seen in the paraffin sections, and to examine co-localization of HSPGs with endothelial cells in more detail. Cryosections were stained with antibodies to syndecan-2 and -3 and glypican-1 (red fluorescence channel). Dual labeling was performed on the same sections with antibody to vWF to identify endothelial cells (green fluorescence channel). When examining the cryosections, blood vessels were initially identified viewing the green channel, and then HSPG expression was evaluated by switching to the red channel. Syndecan-2 was mildly increased in glioma vessel endothelial cells (Fig. 6, A and E) compared with normal brain vessels, where this HSPG was expressed only in low amounts (Fig. 6, I and M). In addition, syndecan-2 was abundantly present in a fibrillar and linear pattern external to tumor vessel endothelial cells, i.e. not directly co-localizing with the vWF signal (Fig. 6E and inset). The cellular origin of syndecan-2 in this location is not clear. Syndecan-3 was moderately expressed in both glioma (Fig. 6, B and F) and normal brain (Fig. 6, J and N) vessels. As in paraffin sections, glypican-1 was abundant in tumor vessel endothelial cells (Fig. 6, C and G), whereas normal brain vessel endothelial cells essentially lacked this HSPG (Fig. 6, K and O). In summary, the immunohistochemistry data are consistent with the hypothesis that increased glypican-1 is responsible for elevated total HSPG levels in glioma vessels, but we cannot exclude the possibility that syndecan-2 and/or HSPGs not included in the analysis also contribute.


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Fig. 6.   Detection of HSPG core proteins in human glioma and normal brain tissues by immunofluorescence. Frozen sections from human high-grade gliomas (glioblastoma multiforme (GBM)) and from normal brain controls (nml) were stained with antibodies to syndecan-2 and -3 and glypican-1 using Alexa 546-conjugated secondary antibody for detection (red channel). An irrelevant mouse anti-hemagglutinin antibody served as a negative control (NC). Blood vessels were localized on the same sections using rabbit anti-vWF antiserum and Alexa 488-conjugated secondary antibody for detection (green channel). Panel pairs display the red channel as a gray-scale image to allow better evaluation of staining intensity (A-D and I-L) and the same images in dual-label mode (anti-core and anti-vWF antibodies) for evaluation of co-localization (E-H and M-P). Insets in E-H and M-P show vascular endothelial cells at higher magnification. A and E, glioma vessels stained for syndecan-2. Please note antibody reactivity surrounding the blood vessel (arrowhead in the inset in E), but only weak-to-moderate staining in the vascular endothelial cells. B and F, glioma vessels stained for syndecan-3. Moderate staining was seen in endothelial cells. C and G, glioma vessels stained for glypican-1. Strong and diffuse staining was present in tumor vessel endothelial cells. D and H, glioma vessels stained with irrelevant control antibody. Some background staining was present in the tumor cells, but not in the blood vessels. I and M, normal brain vessels stained for syndecan-2. Only weak staining was present in endothelial cells. J and N, normal brain vessels stained for syndecan-3. Moderate-to-strong staining was present in endothelial cells. K and O, normal brain vessels stained for glypican-1. Only very weak staining was present in endothelial cells. L and P, normal brain vessels stained with irrelevant control antibody. Magnification is ×200 in the main panels and approximately ×600 in the insets. TV, tumor vessel; NV, normal vessel.

Glypican-1 Overexpression Stimulates Endothelial Cell Growth and Sensitizes Endothelial Cells to FGF2 Stimulation-- MBE cells expressed low levels of glypican-1, mirroring normal brain vessel endothelial cells in vivo. Nevertheless, MBE cell glypican-1 was capable of supporting sustained binding of FGF2 to FGFR1 (Fig. 2C). To determine how glypican-1 up-regulation as it occurs in tumor vessels affects FGF2 signaling in the same cell, we measured FGF2-induced proliferation in MBE cells transfected with glypican-1. Modest glypican-1 overexpression did not affect the levels of other HSPG core proteins (Fig. 7A); and importantly, even in the transfected cells, glypican-1 remained a minor constituent of the total HSPG spectrum. Yet cells transfected with glypican-1 displayed a dramatically elevated base-line proliferation rate (Fig. 7B) and an increased sensitivity to FGF2 stimulation, resulting in a leftward shift of the dose-response curve by at least an order of magnitude (Fig. 7C). Estimated half-maximal stimulation occurred at 23.6 pM FGF2 in the mock-transfected cells and at 0.97 pM in the glypican-1-transfected cells.


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Fig. 7.   Effect of glypican-1 and syndecan-1 overexpression on FGF2-induced proliferation of brain endothelial cells. A-C, stable transfection of MBE cells with glypican-1 (Gpc1). A, HSPGs extracted from MBE cells stably transfected with mouse glypican-1 cDNA and from mock-transfected cells were analyzed by SDS-PAGE. The membrane was blotted with anti-Delta HS antibody 3G10 to detect all HSPGs produced by these cells. B, BrdUrd incorporation was determined as a measure of proliferation. Base-line growth was measured in the absence of FGF2. C, proliferation was measured in response to increasing concentrations of FGF2. Response curves were normalized by defining base-line growth without added FGF2 as 0% and maximal growth at the highest FGF2 concentration (100 pM) as 100%. Each data point represents the mean of triplicates. Error bars indicate S.E. The asterisk indicates a significant difference by the Wilcoxon rank sum test. D-F, stable transfection of MBE cells with syndecan-1 (Sdc1). D, HSPGs extracted from MBE cells stably transfected with mouse syndecan-1 cDNA and from mock-transfected cells were analyzed by SDS-PAGE. The membrane was blotted with anti-Delta HS antibody 3G10 to detect all HSPGs produced by these cells. E, base-line growth was recorded in the absence of FGF2 as described for B. F, FGF2 dose response was measured as described for C.

To determine whether the effect of glypican-1 on MBE cell growth and FGF2 response is specific to this core protein, MBE cells were also transfected with syndecan-1. This HSPG was chosen because it represents a different class of cell-surface HSPGs (transmembrane versus lipid anchor), is present at similarly low base-line levels as glypican-1, and is not up-regulated in glioma vessels. Similar to glypican-1-transfected cells, syndecan-1 was overexpressed at modest levels in the transfected cell population (Fig. 7D). In contrast to glypican-1-transfected cells, syndecan-1-overexpressing cells demonstrated a slightly decreased growth rate (Fig. 7E), and their sensitivity to FGF2-induced mitogenesis was unchanged compared with mock-transfected controls (Fig. 7F). These results suggest a specific role for the glypican-1 core protein in endothelial cell growth and FGF2-induced mitogenesis independent of HS chain composition.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In summary, we have shown that all endothelial cell HSPG core proteins carry HS chains similarly capable of binding FGF2, stabilizing the ternary receptor signaling complex with FGFR1c, and activating this receptor tyrosine kinase. Nevertheless, we found that glypican-1, which is overexpressed in glioma vessels, has a specific ability to sensitize brain endothelial cells to mitogenic FGF2 stimulation.

Vascular endothelial growth factor (VEGF) has received most of the attention recently as a mediator of tumor angiogenesis because of its target cell specificity, but FGF2 is similarly potent. In fact, VEGF and FGF2 can act synergistically (29) or have distinct roles in tumor development by exerting their angiogenic activity at different tumor growth stages and in different tumor regions. FGF2 expression predominates in small tumors and at the tumor periphery, whereas VEGF expression is most prominently observed in larger tumors and in the tumor center (30, 31), consistent with transcriptional regulation of VEGF via hypoxia-inducible factors.

Our observation that different HSPGs produced by one cell type have similar activities is novel and in apparent conflict with the presently prevailing view that HSPGs are specific FGF co-receptors. Yet most studies to date have focused on individual HSPGs and not compared binding characteristics of the entire HSPG spectrum produced by a single cell type. Using an affinity chromatography approach similar to ours, investigators recently identified syndecan-1 as the HSPG responsible for FGF receptor binding in prostate epithelial cells, suggesting that this core protein is specifically endowed with HS chains particularly capable of interacting with the receptor tyrosine kinase (32). However, the findings of that study could also be explained by a predominance of syndecan-1 in this cell type and therefore in the starting material. Our data suggest that the enzyme machinery in the Golgi responsible for HS synthesis and modification does not distinguish between different core proteins. This does not imply that HS chain composition is static. HS binding activities have been shown to change dramatically over time during development and malignant transformation (7). Also, different cell types can decorate the same HSPG core protein with very diverse HS chains (33).

Glypican-1 overexpression in endothelial cells results in significantly enhanced growth and improved FGF2 response despite the fact that its HS chains are apparently not unique and the total HSPG content is not substantially increased. This observation suggests a specific role for the glypican-1 core protein in angiogenic FGF2 signaling and raises the question of what distinguishes glypican-1, the sole endothelial glypican (34), from other cell-surface HSPGs. Glypican HS chain attachment sites are located near the cell membrane, whereas HS attachment sites in syndecans are near the N terminus of the variably sized ectodomain. The proximity of the HS chains to the cell surface may support the formation of a ternary receptor complex with FGFR. Glypicans also distinguish themselves from syndecans by their cell membrane anchorage via glycosylphosphatidylinositol. The lack of a transmembrane domain and the presence of a lipid tether likely endow glypican-1 with increased mobility within the lipid bilayer, favoring FGFR complex formation and oligomerization possibly within cholesterol-rich lipid rafts. Interestingly, tethering FGF3 directly to the cell surface via a glycosylphosphatidylinositol anchor potentiates its transforming activity, apparently enabling this FGF to bypass glypican binding (35).

Volk and co-workers have identified a specific role for the syndecan-4 core protein in FGF2 signaling (14). Glypican-1 could not substitute for syndecan-4 in the HSPG-deficient cells used in this study. Experiments with HSPG core protein chimeras and dominant-negative approaches ascribed a unique function and possibly direct signaling role to the syndecan-4 cytoplasmic domain. The MBE cells used in our study produce ample endogenous HSPGs, including syndecan-4, thereby meeting the requirement for this crucial signal. Endogenous MBE cell HSPGs together with FGFR1 are sufficient to mediate an FGF2 response, but apparently the signal is not optimal, as glypican-1 overexpression further sensitizes MBE cells to respond to lower FGF2 concentrations. The "optimal" HSPG co-receptor glypican-1 may compensate in brain endothelial cells for suboptimal FGF2 supply and/or HSPG composition during active angiogenesis (36). Angiogenesis modulation by HSPGs apparently varies in different organ sites, as we observed primarily syndecan-4 overexpression in breast carcinoma microvessels and both glypican-1 and syndecan-4 up-regulation in skin wound granulation tissue.2 This finding re-enforces the well established concept of endothelial heterogeneity.

Both activation and inhibition of angiogenesis by glypican-1 apart from FGF2 signaling have been described. Shed glypican-1 can protect VEGF165 from oxidative damage in a chaperone-like function (34). Glypican-1 has also been identified as an essential low-affinity co-receptor for the potent angiogenesis inhibitor endostatin (37). The roles for glypican-1 in neoplasia extend beyond angiogenesis. Glypican-1 is overexpressed in pancreatic carcinomas and is required for FGF2 signaling in this tumor cell type (38). Recently, glypican-1 overexpression has been described for breast carcinomas as well (39). Although regulation of syndecan expression has been well studied (40), relatively little is known about the regulation of glypican expression. Gene silencing by promoter methylation has been reported to result in suppression of glypican-3 expression (41). Further work will be needed to address the signals involved in inducing glypican-1 expression in glioma vessels.

Glypican-1-mediated sensitization of endothelial cells to FGF2 likely contributes to the active angiogenic phenotype characteristic for high-grade gliomas. Overexpression of this HSPG may have another deleterious effect in this grim disease. Radiation tissue injury is mediated primarily by apoptosis of vascular endothelial cells (42). FGF2 is a potent endothelial cell survival factor during irradiation (42). Potentiation of this survival signal by glypican-1 may be partially responsible for radiation resistance inevitably developing in gliomas. The central role of glypican-1 in signaling of both FGF2 and VEGF may, on the other hand, present an attractive opportunity for therapeutic intervention.

    ACKNOWLEDGEMENT

We thank Jens Eickhoff for statistical analyses of experimental data.

    FOOTNOTES

* This work was supported by American Cancer Society Research Scholar Grant RSG-01-068-01 (to A. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Dept. of Obstetrics and Gynecology, University of Kiel, Michaelisstr. 16, D-24105 Kiel, Germany.

§ To whom correspondence should be addressed: Dept. of Pathology and Laboratory Medicine, University of Wisconsin, Clinical Sciences Center K4/850, Madison, WI 53562-8550. Tel.: 608-265-9283; Fax: 608-265-6215; E-mail: afriedl@facstaff.wisc.edu.

Published, JBC Papers in Press, February 18, 2003, DOI 10.1074/jbc.M211259200

2 D. Qiao and A. Friedl, unpublished data.

    ABBREVIATIONS

The abbreviations used are: FGF2, fibroblast growth factor-2; FGFR, fibroblast growth factor receptor; HS, heparan sulfate; HSPG, heparan sulfate proteoglycan; vWF, von Willebrand factor; MBE, mouse brain endothelial; HMVEC, human microvascular endothelial cell; BrdUrd, bromodeoxyuridine; AP, alkaline phosphatase; VEGF, vascular endothelial growth factor.

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