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Published as doi: 10.1096/fj.07-8866com.
 (The FASEB Journal. 2007;21:3986-3993.)
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Anti-heparanase monoclonal antibody enhances heparanase enzymatic activity and facilitates wound healing

Svetlana Gingis-Velitski*, Rivka Ishai-Michaeli{dagger}, Israel Vlodavsky*,1 and Neta Ilan*

* Cancer and Vascular Biology Research Center, The Bruce Rappaport Faculty of Medicine, Technion, Israel; and

{dagger} Department of Oncology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel.

1Correspondence: Cancer and Vascular Biology Research Center, The Bruce Rappaport Faculty of Medicine, Technion, Haifa 31096, Israel. E-mail: vlodavsk{at}cc.huji.ac.il


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Heparanase is a mammalian endo-β-D-glucuronidase capable of cleaving HS side chains at a limited number of sites, activity that is strongly implicated in tumor metastasis, neovascularization, inflammation, and autoimmunity. Clinically, up-regulation of heparanase mRNA and protein expression has been documented in a variety of primary human tumors, correlating with reduced postoperative survival and increased lymph node and distant metastasis, thus providing strong clinical support for the prometastatic feature of the enzyme and making it an attractive target for the development of anticancer and anti-inflammatory drugs. Screening a panel of monoclonal antibodies for their ability to inhibit heparanase enzymatic activity, we noted that one hybridoma, 6F8, exhibited the opposite effect and significantly enhanced heparanase activity. Here, we provide evidence that antibody 6F8 enhances the activity of recombinant and cellular heparanase, facilitates invasion of tumor-derived cells in vitro, and improves wound healing in a mouse punch model in vivo. These results support a role of heparanase in the course of wound healing and, moreover, suggest that monoclonal antibodies can be applied clinically for the enhancement, rather than inhibition, of certain enzymes.—Gingis-Velitski, S., Ishai-Michaeli, R., Vlodavsky, I., Ilan, N. Anti-heparanase monoclonal antibody enhances heparanase enzymatic activity and facilitates wound healing.


Key Words: heparan sulfate • cell invasion • cell migration • stimulatory antibody


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HEPARANASE IS A MAMMALIAN endo-β-D-glucuronidase capable of cleaving HS side chains at a limited number of sites, yielding HS fragments of appreciable size (~5–7 kDa) and biological activity (1 , 2) . Heparanase activity has long been detected in a number of cell types and tissues (2) and has been shown to relate to several cell behaviors, including tumor metastasis and inflammation. Heparanase activity’s correlation with the metastatic potential of tumor-derived cells is attributed to enhanced cell dissemination as a consequence of HS cleavage and remodeling of the extracellular matrix (ECM) barrier (3) . As noted, heparanase activity is implicated in neovascularization, inflammation, and autoimmunity, facilitating migration and invasion of vascular endothelial cells and activated cells of the immune system (3) . A proof-of-concept for this notion has been provided by applying siRNA and ribozyme technologies, demonstrating a causal involvement of heparanase in tumor metastasis (4) , angiogenesis (4) , and inflammation (5) . Clinically, up-regulation of heparanase mRNA and protein expression has been documented in a variety of primary human tumors, correlating with reduced postoperative survival and increased lymph node and distant metastasis, thus providing strong clinical support for the prometastatic feature of the enzyme (3 , 6 7 8) . Heparanase induction was also noted in pathological disorders other than human neoplasm (5 , 9 , 10) , making the enzyme an attractive target for the development of anticancer and anti-inflammatory drugs.

Attempts to inhibit heparanase enzymatic activity were initiated at the early days of heparanase research, in parallel with the emerging clinical relevance of this activity (11 , 12) . More recently, with the availability of recombinant heparanase and the establishment of high-throughput screen methods, a variety of inhibitory molecules have been developed, including peptides, small molecules, modified nonanticoagulant species of heparin, as well as several other polyanionic molecules such as laminaran sulfate, suramin, and PI-88 (3 , 8 , 13) . Similarly, anti-heparanase polyclonal antibodies were developed and demonstrated to neutralize heparanase enzymatic activity and to inhibit cell invasion (14) , proteinuria (15) , and neointima formation (16) . Neutralizing anti-heparanase monoclonal antibodies, however, have not been reported so far. In an attempt to develop such antibodies, we have screened a panel of monoclonal antibodies for their ability to inhibit heparanase enzymatic activity. None of the antibodies tested was capable of heparanase inhibition. One hybridoma, 6F8, exhibited the opposite effect and significantly enhanced heparanase activity. Here, we provide evidence that antibody 6F8 enhances the activity of recombinant and cellular heparanase, facilitates invasion of tumor-derived cells in vitro, and improves wound healing in a mouse punch model in vivo. These results further support a role of heparanase in the course of wound healing and suggest that monoclonal antibodies can be applied clinically to enhance, rather than inhibit, certain enzymes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies and reagents
Monoclonal anti-heparanase antibodies were generated by immunizing Balb/C mice with the entire 65 kDa heparanase protein. Hybridomas were obtained by routine procedure and were selected by ELISA using the 65 kDa heparanase for coating (17) . Several hybridomas that reacted positively with heparanase were selected for further characterization. Hybridoma subclass was determined by isotyping kit according to the manufacturer’s (Serotec, Oxford, UK) instructions. Mouse IgG (Sigma, St. Louis, MO, USA) was used as control for all experiments.

Heparanase activity assay
Preparation of ECM-coated 35 mm dishes and determination of heparanase activity were performed as described in detail elsewhere (18 19 20) . To evaluate the effect of hybridomas on heparanase activity, purified active heparanase (40 ng) or cell lysate prepared from 2 x 106 cells was preincubated (2 h, 4°C) with protein A-purified monoclonal antibody (1 µg) or control IgG in 1 ml serum-free RPMI medium. Subsequently, the incubation medium was applied onto 35S-labeled ECM (2 h, 37°C) and the reaction mixture (1 ml) containing sulfate-labeled degradation fragments was subjected to gel filtration on a Sepharose CL-6B column. Fractions (0.2 ml) were eluted with PBS and their radioactivity was counted in a β-scintillation counter. Degradation fragments of HS side chains are eluted at 0.5Kav < 0.8 (fractions 15–30) and represent heparanase generated degradation products. RPMI medium conditioned by 2 x 106 cells was similarly evaluated.

Cells and cell culture
HEK 293, U87-MG human glioma, and MDA-MB-231 human breast carcinoma cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were grown in Dulbecco’s modified Eagle’s medium (Biological Industries, Beit Haemek, Israel) supplemented with 10% fetal calf serum and antibiotics. Subconfluent U87, MDA-231, and HEK 293 cells were stably transfected with human heparanase gene constructs using FuGENE 6 reagent according to the manufacturer’s (Roche Applied Science, Indianapolis, IL, USA) instructions, as described (21 22 23 24) . Transfection proceeded for 48 h, followed by selection with Zeocin (Invitrogen, Carlsbad, CA, USA) for 2 wk. Stable transfectant pools were further expanded and analyzed.

Cell migration assay
The human keratinocytes cell line HACAT was kindly provided by Dr. Norbert E. Fusenig (DKFZ, Heidelberg, Germany) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% calf serum. Cell migration was evaluated applying the in vitro scratch assay essentially as described (25 , 26) . Briefly, cells were allowed to grow in tissue culture plates until confluence followed by creating a "scratch" along the cell monolayer diameter with the wide end of a 1 ml tip (time 0). Plates were washed twice with PBS to remove detached cells, incubated with complete growth medium, and cell migration into the wounded area was examined for 4 days in the presence of antibody 6F8 (1 µg/ml) or control mouse IgG.

Matrigel invasion assay
Invasion assay was performed using modified Boyden chambers with Matrigel-coated polycarbonate nucleopore membrane (Corning, Corning, NY, USA), essentially as described (27 , 28) . Briefly, cells were serum-starved for 20 h, then detached with trypsin-EDTA solution. Cells (2x105/0.2 ml) were added to the upper chamber in the presence of 6F8 or control mouse IgG antibodies (1 µg/ml), and invading cells adhering to the lower side of the membrane were visualized after 6 h by crystal violate staining and counted, as described (28) .

Animal housing and wound healing protocol
All procedures were conducted using facilities and protocols approved by the Animal Care and Use Committee of the Technion School of Medicine. Male C57BL mice (25–30 g, Harlan, Jerusalem, Israel) were anesthetized by intraperitoneal (i.p.) injection of ketamine (50 mg/kg) and xylazine (5 mg/kg), shaved, and two 8 mm diameter, full-thickness excisions were created with a sterile biopsy punch on the mouse back (n=5), yielding 10 wounds in each group. Wounds were left undressed and monoclonal 6F8 antibody or control mouse IgG was injected i.p. before and 3 and 5 days after wounding. Since wound healing is less efficient in older animals, relatively old mice (8–10 months of age) were employed for the wound healing experiments.

Histology
Wounds were harvested 7 days postwounding, fixed with 4% formaldehyde in PBS, embedded in paraffin, and sectioned. After deparaffinization and rehydration, 5 µm sections were washed three times with PBS and stained with hematoxyline/eosine or Masson’s trichrome, as described (26 , 29) . Tissue sections were then mounted and visualized with a Zeiss axioscope microscope. Wound healing was calculated by measuring the distance between the epithelial edges at the wound diameter (26) .

Statistics
Data are presented as mean ± SE. Statistical significance was analyzed by a 2-tailed Student’s t test. The value of P < 0.05 is considered significant. All experiments were repeated at least twice with similar results. Experiments evaluating heparanase activity were repeated at least five times.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Monoclonal antibody 6F8 enhances heparanase enzymatic activity
We tested the ability of several monoclonal antibodies to inhibit the enzymatic activity of recombinant heparanase. These studies were carried out at physiological pH (~7.2), optimal for the interaction between antibodies and their antigens. Although the heparanase enzyme performs best under acidic conditions (30) , activity was readily detected under these experimental settings (Fig. 1 ). Nevertheless, none of the antibodies tested (i.e., 1E1, 44C4) inhibited heparanase activity (Fig. 1A, B ). On the contrary, one monoclonal antibody, 6F8 (IgG1) exhibited the opposite trend and significantly enhanced heparanase activity, whereas control mouse IgG had no stimulatory or inhibitory effect (Fig. 1C , square). As a control, antibody 6F8 was applied to the 35S-labeled ECM without prior incubation with recombinant heparanase. No heparanase activity was detected under these conditions (Fig. 1C , circle), confirming that heparanase activity is not copurified with the 6F8 antibody and ruling out the possibility that it is a catalytic antibody.


Figure 1
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  		Figure 1. Antibody 6F8 enhances heparanase enzymatic activity. Purified recombinant active heparanase (40 ng) was left untreated (rectangles, panel C) or was preincubated with 1E1 (A), 44C4 (B), 6F8 (C) anti-heparanase monoclonal antibodies (diamonds), or control mouse IgG (1 µg; square) for 2 h in serum-free RPMI medium on ice. The mixture was then applied onto 35S-labeled ECM-coated dishes and heparanase activity was determined as described under Materials and Methods. Antibody 6F8 was similarly applied without prior incubation with recombinant heparanase (circles, panel C), yielding no detectable heparanase activity.

We next confirmed and validated the ability of antibody 6F8 to enhance the activity of cellular heparanase. Conditioned medium was collected from heparanase-transfected HEK 293 (Fig. 2 A) and MDA-231 (Fig. 2C ) cells, applied to 35S-labeled ECM-coated dishes in the presence of antibody 6F8 or control mouse IgG (1 µg/ml), and heparanase activity was evaluated (Fig. 2) . In addition, we examined heparanase activity in the corresponding cell lysates (Fig. 2B, D ). We observed a consistent 2- to 3-fold increase in heparanase activity by antibody 6F8 (Fig. 2) , suggesting that this antibody can be utilized to enhance heparanase activity in cellular models. Similar results were obtained with U87 glioma and HT-29 colon carcinoma cells (data not shown).


Figure 2
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  		Figure 2. Antibody 6F8 enhances the activity of cellular heparanase. Heparanase-transfected HEK-293 (B) and MDA-231 (D) cells were suspended (2x106/ml) in phosphate/citrate buffer (pH 6.8) and subjected to three freeze/thaw cycles. The resultant lysates were incubated with antibody 6F8 (1 µg) or control mouse IgG for 2 h on ice, applied onto 35S-labeled ECM-coated dishes, and heparanase activity was determined as above. Conditioned medium was collected from heparanase-transfected HEK-293 (A) and MDA-231 (C) cells; 1 ml was incubated with control mouse IgG or antibody 6F8, followed by determination of heparanase activity described above.

Antibody 6F8 facilitates cellular invasion
Heparanase activity is well correlated with the metastatic potential of tumor-derived cells, a feature best recapitulated in vitro by cellular invasion through a reconstituted-basement membrane matrix (Matrigel). We therefore examined the invasive capacity of heparanase-transfected MDA-231 (Fig. 3 , top panels) and U87 cells (Fig. 3 , bottom panels) in the presence of antibody 6F8 or control mouse IgG utilizing Matrigel-coated transwell inserts. Matrigel invasion by 231 and U87 cells was increased 2-fold in the presence of antibody 6F8 (Fig. 3) , consistent with the observed enhancement of heparanase activity (Fig. 2) . Unlike the migration of individual tumor cells through 8 µm pore transwell filters, epithelial cells exhibit characteristic sheet migration on a solid support. We previously reported that exogenous addition of heparanase stimulates sheet migration of human HACAT keratinocytes in a wound scratch assay (26) . Similarly, sheet migration of HACAT cells and wound closure in vitro was significantly enhanced by antibody 6F8 (Fig. 4 , middle) compared with control mouse IgG (Fig. 4 , left panel) and anti-heparanase 1E1 monoclonal antibody (Fig. 4 , right panel), which does not affect heparanase activity (Fig. 1) . These results imply that antibody 6F8 is capable of enhancing the activity of endogenous cellular heparanase and thus may be utilized in vivo.


Figure 3
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  		Figure 3. Antibody 6F8 facilitates cellular invasion. Heparanase-transfected MDA-231 (A, B) and U87 (C, D) cells (2x105) were plated onto Matrigel-coated 8 µm Transwell filters in the presence of mouse IgG or antibody 6F8 (1 µg). Invading cells adhering to the lower side of the membrane were visualized (A, C) and counted (B, D) after 6 h.


Figure 4
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  		Figure 4. Antibody 6F8 enhances keratinocyte migration. HACAT cells were allowed to grow in tissue culture plates until confluence, followed by a scratch made along the cell monolayer with the wide end of a 1 ml tip (time 0). Plates were washed twice with PBS to remove detached cells, incubated with complete growth medium, and cell migration into the wounded area was examined for 4 days in the presence of control mouse IgG (left), anti-heparanase 6F8 (middle) or 1E1 (right) monoclonal antibodies (1 µg/ml).

Antibody 6F8 improves wound healing in a mouse punch model
Relatively high levels of heparanase have been detected in skin tissue (31) , and further elevation in heparanase expression has been observed in the leading edge of migrating keratinocytes and in wound granulation tissue (26) . Moreover, elevated levels of bFGF have been found in the wound fluid of heparanase transgenic mice compared with control mice, likely due to enhanced heparanase activity and the associated release of HS-bound polypeptides (26) . Therefore, we examined the ability of antibody 6F8 to enhance wound healing in a mouse punch model. Histological examination of 8 mm punch wounds revealed a significant improvement of wound healing in mice that were injected with antibody 6F8 (10 mg/kg; Fig. 5 A, f–j) compared with mice injected with control mouse IgG (Fig. 5A, a –e). Thus, in two separate experiments 37.5 and 60% of wounds treated with antibody 6F8 appeared completely closed, whereas only 0 and 37.5% of control wounds were healed, respectively (Fig. 5A, B ). Moreover, significant improvement of wound closure was measured in wounds that were not completely healed. Wound diameter (i.e., granulation tissue lacking epidermal keratinocytes) of control wounds was 1104 ± 189 and 1355 ± 139 µm compared with 493±169 and 381+139 µm of wounds treated with antibody 6F8 (Fig. 5B ; Table 1 ), differences that are statistically highly significant (P=0.02). In addition, granulation tissue of wounds treated with antibody 6F8 appeared thicker and denser than control wounds (Fig. 5A ), in agreement with the improved wound healing. In fact, wounds treated with antibody 6F8 appeared to undergo remodeling and maturation, replacing the wound granulation tissue with dense collagen matrix. This is best demonstrated by Masson’s trichrome staining, clearly revealing enhanced collagen deposition in 6F8 (Fig. 5C , 6F8) vs. IgG (Fig. 5C , Con) -treated wounds.


Figure 5
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  		Figure 5. Antibody 6F8 improves wound healing in a mouse punch model. C57/Bl mice (n=5) were anesthetized and full-thickness 8 mm punch wounds were created on the mouse back (two wounds/mouse). Mice were injected i.p. with control mouse IgG (Con) or antibody 6F8 (10 mg/kg) prior to as well as 3 and 5 days after wounding. The wound tissue was harvested 7 days postwounding, fixed with paraformaldehyde, dehydrated, embedded in paraffin, and sectioned. Five micrometer sections were rehydrated and stained with hematoxylin-eosin (A, representative sections corresponding to each control (a–e) and 6F8-treated (f–j) mouse) or Masson’s trichrome (C). Wound healing was calculated by measuring the distance between the epithelial edges (arrows) at the wound diameter (B; see Table 1 ).


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  		Table 1. Wound diameter and closure upon antibody 6F8 or mouse IgG treatment


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Heparanase activity has long been correlated with the metastatic potential of tumor cells, a notion that is now supported experimentally (4) and clinically (3 , 6) . Studies documenting up-regulation of heparanase by primary human malignancies are rapidly accumulating (3 , 32 , 33) , urging the development and clinical testing of heparanase inhibitors (3 , 8 , 13) . More recent publications, however, highlight the involvement of heparanase in normal tissue development, regeneration, and morphogenesis, collectively implying that under certain conditions heparanase may prove beneficial rather than predict poor prognosis. For example, increased bone mass, cortical thickness, and bone formation rate were noted in transgenic mice that overexpress heparanase (34) . These mice also exhibited overbranching of mammary ducts and acceleration of hair growth (35) . Moreover, heparanase was up-regulated after liver hepatectomy (36) and during healing of bone fracture (37) , thought to represent a specialized form of wound healing. We previously reported that heparanase is expressed at relatively high levels by wound granulation tissue and have demonstrated that topical application of heparanase facilitates wound healing (26) . Furthermore, heparanase activity was readily detected in wound fluids, and a marked elevation of heparanase activity was measured in wound fluids collected from heparanase transgenic mice (26) . Similarly, the increased levels of bFGF found in wound fluids of heparanase transgenics, likely due to release of HS-bound bFGF by heparanase enzymatic activity, correlate with elevation of wound angiogenesis (26) . Wound angiogenesis was restored to the levels of those of control mice upon inhibition of heparanase activity (26) , supporting the notion that heparanase enzymatic activity plays an important role in the course of wound healing.

Here, we describe for the first time a monoclonal antibody, 6F8, that is capable of enhancing heparanase activity. Although monoclonal antibodies are widely used to inhibit the activity of enzymes (mostly kinases) and cell surface receptors, and are successfully applied in the clinic (38 , 39) , a monoclonal antibody that enhances enzyme activity has not yet been characterized. To the best of our knowledge, this is the first demonstration of such an antibody and its potential application. Antibody 6F8 recognizes an epitope that is localized at the C-terminal region of the heparanase protein (amino acids 416–543; data not shown), away from the predicted active site (Glu225 and Glu343) (40) or the heparin binding domains (Lys158-Asp171 and Gln270-Lys280) (18) . Thus, antibody 6F8 does not seem to affect the affinity of heparanase to HS, but rather facilitates heparanase activity by stabilizing an active conformation. The antibody significantly enhances the activity of purified and cellular heparanase (Figs. 1 , 2) and facilitates cellular invasion through reconstituted extracellular matrix (Matrigel; Fig. 3 ), the hallmark of heparanase function. Among the few cell types that express heparanase under normal conditions are keratinocytes (31) . Heparanase localization is significantly altered during wound healing in most keratinocytes appearing adjacent to the wound margin and in the migrating tip of the wound (26) , likely supporting keratinocyte adhesion and migration. Indeed, exogenous heparanase was found to enhance HACAT cell migration (26) , a feature that was recapitulated by antibody 6F8 (Fig. 4) . The direct effect of antibody 6F8 on keratinocyte migration led us to examine the ability of this antibody to improve wound healing. Indeed, antibody 6F8 significantly accelerated the healing of mouse punch wounds quantitatively and qualitatively. The majority of wounds treated with antibody 6F8 appeared to be healed, and the rest were significantly smaller than control wounds (Fig. 5 , Table 1 ). This effect was dose dependent and was significantly reduced by lowering the amount of antibody (data not shown). Moreover, granulation tissue of wounds treated with antibody 6F8 appeared significantly thicker and denser during wound maturation and remodeling (Fig. 5A ). This was best demonstrated by Masson’s trichrome staining, indicating dense collagen deposition replacing the wound granulation tissue (Fig. 5C ).

These findings further support the notion that heparanase activity plays an important role in the course of wound healing and suggest that this effect can be modulated and controlled by applying heparanase inhibitors (i.e., glycol-split heparin) (22) or enhancers (i.e., antibody 6F8). Monoclonal antibodies offer several practical advantages over local or systemic application of purified heparanase: they typically exhibit a long half-life (48–72 h), are well tolerated and easy to manage, and are successfully applied clinically.

Monoclonal 6F8 enhances heparanase activity and facilitates wound healing. This antibody is expected to be a valuable tool for exploring heparanase functions under normal and pathological settings, and in distinguishing between enzymatic activity-dependent and independent features of heparanase (22 , 23 , 41 , 42) . The effects of antibody 6F8 on other aspects of heparanase functions, such as adhesion role of the protein or its proteolytic processing, are currently under investigation.


   ACKNOWLEDGMENTS
 
This work was supported by grants from the Israel Science Foundation (grant 549/06), National Cancer Institute, National Institutes of Health (grant RO1-CA106456), the Israel Cancer Research Fund, and the Rappaport Family Institute Fund.

Received for publication April 22, 2007. Accepted for publication June 7, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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M. Gotte, D. Spillmann, G. W. Yip, E. Versteeg, F. G. Echtermeyer, T. H. van Kuppevelt, and L. Kiesel
Changes in heparan sulfate are associated with delayed wound repair, altered cell migration, adhesion and contractility in the galactosyltransferase I (ss4GalT-7) deficient form of Ehlers-Danlos syndrome
Hum. Mol. Genet., April 1, 2008; 17(7): 996 - 1009.
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