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(The FASEB Journal. 2005;19:211-221.)
© 2005 FASEB

Heparanase accelerates wound angiogenesis and wound healing in mouse and rat models

Eyal Zcharia*,1, Rachel Zilka{dagger},{ddagger},1, Alon Yaar{dagger}, Oron Yacoby-Zeevi{dagger}, Anna Zetser§, Shula Metzger||,, Ronit Sarid{ddagger}, Annamaria Naggi||,, Benito Casu||,, Neta Ilan{dagger},§,2, Israel Vlodavsky§,2 and Rinat Abramovitch{dagger}{dagger}

* Departments of Oncology,
|| Medicine and
{dagger}{dagger} Gene Therapy, Hadassah-Hebrew University Medical Center, Jerusalem, Israel;
{dagger} InSight Biopharmaceuticals, Rabin Science Park, Rehovot, Israel;
{ddagger} Faculty of Life Sciences, Bar Ilan University, Ramat-Gan, Israel;
§ Cancer and Vascular Biology Research Center, the Bruce Rappaport Faculty of Medicine, Technion, Haifa, Israel; and
|| G. Ronzoni Institute for Chemical and Biochemical Research, Milan, Italy

2Correspondence: Cancer and Vascular Biology Research Center, Faculty of Medicine, Technion, P.O. Box 9649, Haifa 31096, Israel. E-mail: vlodavsk{at}cc.huji.ac.il; netailan{at}tx.technion.ac.il


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Orchestration of the rapid formation and reorganization of new tissue observed in wound healing involves not only cells and polypeptides but also the extracellular matrix (ECM) microenvironment. The ability of heparan sulfate (HS) to interact with major components of the ECM suggests a key role for HS in maintaining the structural integrity of the ECM. Heparanase, an endoglycosidase-degrading HS in the ECM and cell surface, is involved in the enzymatic machinery that enables cellular invasion and release of HS-bound polypeptides residing in the ECM. Bioavailabilty and activation of multitude mediators capable of promoting cell migration, proliferation, and neovascularization are of particular importance in the complex setting of wound healing. We provide evidence that heparanase is normally expressed in skin and in the wound granulation tissue. Heparanase stimulated keratinocyte cell migration and wound closure in vitro. Topical application of recombinant heparanase significantly accelerated wound healing in a flap/punch model and markedly improved flap survival. These heparanase effects were associated with enhanced wound epithelialization and blood vessel maturation. Similarly, a marked elevation in wound angiogenesis, evaluated by MRI analysis and histological analyses, was observed in heparanase-overexpressing transgenic mice. This effect was blocked by a novel, newly developed, heparanase-inhibiting glycol-split fragment of heparin. These results clearly indicate that elevation of heparanase levels in healing wounds markedly accelerates tissue repair and skin survival that are mediated primarily by an enhanced angiogenic response.—Zcharia, E., Zilka, R., Yaar, A., Yacoby-Zeevi, O., Zetser, A., Metzger, S., Sarid, R., Naggi, A., Casu, B., Ilan, N., Vlodavsky, I., Abramovitch, R. Heparanase accelerates wound angiogenesis and wound healing in mouse and rat models.


Key Words: heparan sulfate • keratinocytes • blood vessel maturation • extracellular matrix • MRI • tissue repair • wound fluid


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
MOST SKIN LESIONS heal rapidly and efficiently within 1 or 2 wk. Nevertheless, 35 million cutaneous wounds that require major intervention occur yearly in the U.S. alone (1) . Although these acute wounds generally heal, the end product is neither esthetically nor functionally perfect. In contrast to acute wounds, chronic wounds such as diabetic foot ulcers do not heal well. According to a recent survey, ~86,000 lower limbs are amputated annually due to wound complications emerging from diabetes, resulting in severe morbidity and loss of quality of life. Wound healing orchestrates multiple cell types (i.e., neutrophils, macrophages, fibroblasts, keratinocytes, endothelial cells), soluble (i.e., growth factors, cytokines, chemokines), and insoluble (extracellular matrix components) mediators in a complex sequence of events divided into three overlapping phases: inflammatory, proliferative, and remodeling (2 3 4) . Orchestration and regulation of the rapid new tissue development observed in wound healing depend not only on cells and bioavailable polypeptides, but also on the extracellular matrix (ECM) microenvironment. The ECM of a healing wound undergoes rapid changes as the fibrin clot is replaced by fibronectin and hyaluronan and subsequently by type I and III collagen (1) . Important components of the ECM are proteoglycans (5) . These integral components consist of a number of core proteins with covalently bound glycoaminoglycan side chains. Glycosaminoglycans are linear long chain, high molecular weight carbohydrates composed of alternating hexuronic acid and hexosamine residues that are usually highly sulfated. The ability of heparan sulfate proteoglycans (HSPG) to interact with major ECM constituents such as fibronectin, collagen type IV, and laminin suggests a key role for HSPG in the self-assembly, insolubility, and integrity of the ECM (6 7 8) . Apart from their structural function, HSPG associate with a multitude of polypeptides that are intimately involved in regulating each and every aspect of wound healing. These include inflammatory mediators (interleukins-1, 8, and 10, SDF-1, MIP-1, MCP-1, RANTES), angiogenic factors (bFGF, VEGF), and growth-promoting factors (PDGF-BB, KGF, HB-EGF) that induce proliferation and migration of keratinocytes and fibroblasts (5 , 9 10 11) . While intensive research has focused on enzymes capable of degrading and remodeling protein components in the ECM and their role in wound healing (3 , 12) , less attention has been paid to enzymes cleaving glycosaminoglycan side chains. Heparanase is an endo-ß-D-glucuronidase that is capable of cleaving heparan sulfate (HS) side chains at a limited number of sites, yielding HS fragments of appreciable size (~5–7 kDa; refs 13 , 14 ). Heparanase-mediated ECM degradation and remodeling traditionally have been correlated with the metastatic potential of tumor-derived cell types (15 16 17 18) . Similarly, heparanase has been shown to facilitate cell invasion associated with autoimmunity, inflammation and angiogenesis (16 , 18 19 20) . In addition to being directly involved in the enzymatic machinery that orchestrates in cellular invasion, heparanase activity releases ECM resident, HS-bound polypeptides, converting them into bioactive molecules (18 , 21) . The multitude of potential mediators capable of being released by heparanase is of particular importance in the complex setting of wound healing. We have now tested this hypothesis in an ischemic model of wound healing in the rat. We provide evidence that heparanase is normally expressed in the skin and wound granulation tissue. Topical application of recombinant heparanase accelerated wound healing in a flap/punch model and significantly improved flap survival. These effects correlated with enhanced wound epithelialization and blood vessel formation and maturation. Similarly, a marked elevation in wound angiogenesis was observed in heparanase-overexpressing transgenic mice, and this effect was completely blocked by heparanase-inhibiting, glycol-split modified heparin. These results clearly indicate that heparanase normally is present in healing wounds and that elevated levels of heparanase facilitate tissue repair and skin survival, most likely through an enhanced angiogenic response.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Heparanase purification
Recombinant enzymatically active heparanase was purified from heparanase-transfected CHO cells (17 , 22) . CHO cells were harvested by trypsin, centrifuged, and the cell pellet was suspended in 20 mM citrate phosphate buffer pH 5.4. The suspension was subjected to four cycles of freeze and thaw (liquid nitrogen/37°C, 5 min each), the cell extract was centrifuged (18,000 rpm, 15 min, 2–8°C) and the supernatant collected and filtered through a 0.45 µM filter. The filtrate was applied onto a Source 15 S column (Pharmacia, Piscataway, NJ, USA) equilibrated with 20 mM phosphate buffer (pH 6.8). The column was washed with 20 mM phosphate buffer, pH 6.8, followed by 20 mM phosphate buffer, pH 8.0, and heparanase was eluted with a linear gradient (0 to 35%) of 8 column volumes of 1.5 M NaCl in 20 mM phosphate buffer, pH 8.0. Active fractions were pooled and applied onto Fractogel EMD SO3– (Merck, Rahway, NJ, USA) column equilibrated with 20 mM citrate phosphate buffer (pH 5.4). The column was washed with 20 mM citrate phosphate buffer (pH 5.4), followed by 20 mM phosphate buffer (pH 8.0). Heparanase was eluted with a linear gradient (0 to 22%) of 1 column volume, 10 column volumes (22–25%), and 15 column volumes (25–100%) of 1.5 M NaCl in 20 mM phosphate buffer (pH 8.0). Finally, heparanase eluted from the Fractogel column was applied onto a HiTrap heparin column (Pharmacia) equilibrated with 20 mM phosphate buffer, pH 8.0, and eluted with a linear gradient of 1 column volume (0–20%) and 15 column volumes (20–80%) of 1.5 M NaCl in 20 mM phosphate buffer (pH 8.0). Eluted fractions were analyzed by gradient SDS-PAGE, stained with Gelcode® (Pierce, Rockford, IL, USA), and pooled according to their purity. At least 90% pure, highly active heparanase preparation was obtained. The gel electrophoresis pattern showing the 65 kDa pro-heparanase and the active 50 and 8 kDa heparanase subunits (23) is presented in Fig. 3B .



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  		Figure 3. Heparanase accelerates wound healing in a flap/punch model. A) Schematic diagram of the flap/punch model. Punch wounds, including the flap incisions, were harvested 7 days after wounding and processed for histological analysis by cutting each sample (2 punch wounds and incisions from each side) in the center of the punch. B) Gelcode®-stained SDS-PAGE of purified heparanase. Recombinant enzymatically active heparanase was purified from heparanase-transfected CHO cells, as described in Materials and Methods, and subjected to SDS-PAGE. Lane 1: molecular weight markers. Lane 2: recombinant heparanase. Arrows mark the 65 kDa heparanase proenzyme and the 50 and 8 kDa subunits of the active heparanase heterodimer. C) H&E staining of untreated (Con) and heparanase-treated (Hepa) wounds. Arrows mark the wound edges. Original magnification: x4. D) Wounds were treated with saline (Con), heparanase (Hepa), as described in Materials and Methods. Wound diameter (mean ±SE) measured 7 days after wounding. Bars represent wounds in 5 rats, 2 wounds/flap, each measured twice.

Antibodies and reagents
Polyclonal anti-heparanase antibodies (Ab-p9), directed against a synthetic peptide corresponding to residues 335-353 of the human heparanase protein, and monoclonal anti-human heparanase antibody (mAb 130) were kindly provided by InSight Pharmaceuticals (Rehovot, Israel) (17 , 24) . Anti-smooth muscle actin monoclonal antibody was purchased from Sigma (St. Louis, MO, USA) and anti-PCNA was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Recombinant rat PDGF-BB was purchased from R&D Systems (Minneapolis, MN, USA).

Compound ST1514 was kindly provided by Dr. Claudio Pisano (Sigma-Tau, Research Department, Pomezia, Rome, Italy). Briefly, heparin was subjected to controlled alkali-catalyzed removal of sulfate groups of iduronic acid 2-O-sulfate residues, giving rise to the corresponding epoxide derivative. The epoxide rings were opened, followed by oxidative glycol-splitting of the newly formed (and the preexisting) nonsulfated uronic acid residues (25) . The ST1514 compound is 50% glycol-split modified heparin (H50gs; MW=11,200) (25) .

Animal housing and wound healing protocols
All procedures were conducted using facilities and protocols approved by the Animal Care and Use Committee of the Hadassah-Hebrew University School of Medicine. Male Sprague-Dawley rats (250–280 g; Harlan, Jerusalem, Israel) were anesthetized by intraperitoneal (i.p.) injection of ketamine (50 mg/kg) and xylazine (5 mg/kg), shaved, and an ischemic wound model was created as described (26) . Two longitudinal incisions, each 7 cm in length and 3 cm from the dorsal midline, were made, then the two incisions were connected at the caudal end with a third incision across the midline (see diagram in Fig. 3A ). The flap was elevated to the base of the cranial pedicle, then replaced onto its bed and secured with sutures. Two 8 mm-diameter excisions were created with a sterile biopsy punch ~1.5 cm from the dorsal midline; the perimeter of each excision was 0.5 cm from the longitudinal incision. Wounds were left undressed and rats were housed separately after wounding. Heparanase (1 µg), PDGF (0.5 µg), or control saline were applied topically three times a day for 3 days postwounding. For flap survival studies, skin flaps were created as above, albeit without any excision, and 1 mL of heparanase (50 µg) was uniformly applied before flap replacement and suturing. Wound healing was examined by histological analysis 14 days postwounding. Flap survival was evaluated microscopically by measuring the area of the dark necrotic skin tissue and by histological analysis. For calculation of flap healing index, necrosis of less than 10% of the flap was considered to be healed.

Heparanase transgenic mice
Generation and characterization of heparanase-overexpressing transgenic mice were described (24) . Control and heparanase transgenic mice were anesthetized (pentobarbital, 30 mg/kg, i.p.), shaved, and 1 cm-long, full-thickness incisions were made on the mouse back skin. Incisions were closed by cyanoacrylate glue and examined on days 1, 3, and 7 postwounding by MRI analysis (27 28 29) or fixed and analyzed by histology.

MRI analysis of blood vessel density
MRI experiments were performed on a horizontal 4.7T Biospec spectrometer (Bruker, Germany), using an actively RF decoupled surface coil 2 cm in diameter and a bird cage transmission coil, as described previously (28 , 29) . Briefly, mice were anesthetized (pentobarbital, 30 mg/kg i.p.) and placed supine with the wound area located at the center of the surface coil. Wound vascularity was reflected by a decrease in the mean signal intensity surrounding the wound in gradient echo T2* weighted images (repetition time=100 ms; echo time=10 ms). Data are reported as the apparent vessel density {AVD=–ln(s(t)/s(0))}, in which s(t) is the mean intensity around the wound and s(0) is the mean intensity of a distant muscle, as described (27) .

Histology
Wound samples were fixed with 4% formaldehyde in PBS, embedded in paraffin, and sectioned. After deparaffinization and rehydration, 5 µm sections were washed (3x) with PBS and stained with hematoxyline/eosine or Mason-Trichrom, as described (24) . Tissue sections were then washed, mounted, and visualized with a Zeiss axioscope microscope. Wound healing was calculated by measuring the distance between the epithelial edges at the wound diameter.

Immunohistochemistry
Immunohistochemistry was performed as described previously, with minor modifications (30) . Briefly, 5 µm sections were deparaffinized and rehydrated. The tissue was then denatured for 3 min in a microwave oven in citrate buffer (0.01 M, pH 6.0). Blocking steps included successive incubations with 3% H2O2 in methanol and 5% goat serum. Tissue sections were incubated with the respective primary antibody or with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 3.3% horse serum as control, followed by incubation with the respective HRP-conjugated antibodies (Jackson Laboratories, Bar Harbor, ME, USA). Color was developed using AEC substrate kit (Zymed, South San Francisco, CA, USA), followed by counterstaining with Mayer’s hematoxylin.

Wound fluid analysis
To obtain wound fluid, ~5 mm3 polyvinyl sponge was inserted into each wound and the wound was sealed (n=6). One day after incision, the sponges were removed and wound fluid was extracted by centrifugation. Fluid samples, equilibrated to similar protein content, were subjected to immunoblot analysis and determination of heparanase activity.

Heparanase activity assay
Preparation of ECM-coated dishes and determination of heparanase activity were performed as described in detail elsewhere (17 , 23) . Wound fluids containing equal amounts of protein were incubated (16 h, 37°C) with sulfate-labeled ECM in 1 mL heparanase reaction buffer. The incubation medium containing sulfate-labeled degradation fragments released from the ECM was subjected to gel filtration on a Sepharose CL-6B column. Intact HSPG are eluted just after the void volume (fractions 1–10) whereas HS degradation fragments are eluted toward the Vt of the column (fractions 15–35; 0.5<Kav<0.8) (23 , 24) .

Immunoblot analysis
Aliquots of wound fluids were mixed with heparin-Sepharose Fast Flow beads (Amersham Bioscience, Little Chalfont, UK). After incubation (1 h, 4°C), the beads were washed with PBS and boiled in sample buffer. Proteins were separated by electrophoresis and transferred to Immobilon-P membrane (Millipore, Bedford, MA, USA). Heparanase was detected by anti-heparanase monoclonal (mAb130) or polyclonal (Ab-p9) antibodies, followed by HRP-conjugated antibodies (Jackson Laboratories) and enhanced chemiluminescence (Pierce), as described (30) .

Cell culture and cell migration assay
The human keratinocytes cell line HACAT (31) was kindly provided by Dr. Norbert E. Fusenig (DKFZ, Heidelberg, Germany) and cultured in DMEM supplemented with 10% calf serum. Cells were allowed to grow in tissue culture plates until confluence, then scraped 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 followed for 4 days in the presence or absence of exogenously added heparanase, as described (31 , 32) .

Statistics
Data are presented as mean ± SE. Statistical significance was analyzed by two-tailed Student’s t test. The value of P < 0.05 is considered significant.

All experiments were repeated at least twice, with similar results.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Heparanase expression in the skin and wound granulation tissues
Heparanase expression under normal physiological conditions is restricted to the placenta and some blood-borne cells including platelets, neutrophils, mast cells, and lymphocytes (16 , 18 , 19) . More recently, relatively high heparanase expression levels were noted in skin tissue, possibly correlating with keratinocyte differentiation (33) . To evaluate whether our anti-heparanase antibodies can be applied in a rat model, formalin-fixed, paraffin-embedded rat skin sections were immunostained with the p9 anti-heparanase antibody. As shown in Fig. 1 , heparanase expression was mainly detected in the stratum granulosum and the first layer of the stratum corneum in the upper part of the epidermis (Fig. 1A, B ). This staining pattern agrees with heparanase staining in human skin biopsies (33) . As a control, sections were similarly stained for PCNA, a commonly used marker of cell proliferation. As expected and in contrast to heparanase staining, PCNA reactivity was mainly confined to the basal cell layer of the epidermis (Fig. 1C, D ). Heparanase expression was also detected in hair follicles (Fig. 1A, B ) and in sebaceous glands (Fig. 1E ). In the latter, strong heparanase reactivity was observed in the cell nucleus. Nuclear localization of heparanase agrees with recent findings demonstrating heparanase nuclear localization in tumor-derived cells (34) . Next, we examined heparanase expression and localization in the skin tissue after wounding. Away from the wound, heparanase expression was restricted to the stratum granulosum (Fig. 2 A), as demonstrated in Fig. 1A, B . The newly formed multilayer epithelium adjacent to the wound edge exhibited a high level of heparanase in most cells with the exception of the basal cell layer (Fig. 2B ), apparently localizing to areas of cell-cell junctions. The most intense staining was detected in migrating keratinocytes at the leading edge of the wound (Fig. 2C ). In these cells, high heparanase expression was detected in the tip of the migration front. This staining pattern suggests that heparanase may play a role in keratinocyte migration during wound healing. To test this hypothesis, human keratinocytes (HACAT) were grown to confluency and the monolayer was scratched with a pipette tip. Cells were left untreated (Fig. 2D ) or treated with exogenous heparanase at 1 (Fig. 2E ) or 5 µg/mL (Fig. 2F ), and cell migration into the wounded area was examined over 4 days. The addition of heparanase stimulated a significant, dose-dependent migratory response compared with untreated cell cultures (Fig. 2D-F ), supporting a promigratory effect of heparanase during wound healing.



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  		Figure 1. Heparanase expression in rat skin tissue. Skin samples harvested from the rat back were formalin-fixed and embedded in paraffin. Sections (5 µm) were immunostained with anti-heparanase p9 (A, B, E) or anti-PCNA (C, D) antibodies. Negative control for the staining procedure (no primary antibody) is shown in panel F. Note heparanase expression in the upper layers of the epidermis, whereas PCNA expression is mainly detected in the basal keratinocyte layer. Original magnifications: A, C, F) x20; B, D, E) x40.



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  		Figure 2. Heparanase expression in wound tissue. Wounds were generated with 8 mm sterile biopsy punch and wound tissue was harvested 7 days later, fixed, and embedded in paraffin. A–C) Heparanase expression in resting, proliferating, and migrating keratinocytes. Sections (5 µm) were stained with the p9 anti-heparanase antibody. A) Healthy skin tissue away from the wound. Proliferating (B) and migrating (C) keratinocytes. D–F) Migration assay. HACAT cells were grown to confluency and scraped with a pipette tip. After 2 washes, cells were grown in growth medium without (D) or with exogenously added heparanase at 1 (E) or 5 (F) µg/mL and cell migration was evaluated after 4 days. G–L) Heparanase expression in the wound granulation tissue. Sections of wound granulation tissue stained with the p9 anti-heparanase antibody (G–I) or double stained with anti-SMA (J) and anti-heparanase (K) antibodies. Merge image is shown in panel L. Original magnifications: A) x40; B, C) x100; D–F) x20; G) x4; H) x20; I–L) x40.

To further explore heparanase expression in the wound granulation tissue, punch wounds were harvested 7 days after wounding and stained with the p9 anti-heparanase antibody. Examination of the wound granulation tissue at low magnification revealed a gradient of heparanase staining, being most intense at the newly formed tissue and gradually declining toward the base of the wound (Fig. 2G ). Examining the granulation tissue at higher magnifications revealed a high heparanase expression in some, but not all, newly formed blood vessels (Fig. 2H, I ). Heparanase expression by endothelial cells was further confirmed by using double staining with anti-heparanase and anti-smooth muscle actin (SMA) antibodies as a marker for pericytes. Recruitment of pericytes was detected in a relatively small number of blood vessels (see below) and was characterized by a thin, single layer of SMA-positive cells (Fig. 2J ). Clearly, heparanase expression was not detected in the SMA-positive cells, but rather in the endothelial cell layer, facing the vessel lumen (Fig. 2K, L ).

Heparanase accelerates wound healing
Expression of high heparanase levels by migrating keratinocytes (Fig. 2B, C ) and in the granulation tissue (Fig. 2G-I ) suggests that heparanase may play a role in the course of wound healing. To test this, a flap/punch model of wound healing under ischemic conditions was used (Fig. 3 A) (26) . This model system was chosen because wound healing is known to be impaired in ischemic tissue environment and treatments that stimulate neovascularization are thought to accelerate healing of ischemic wounds (35 , 36) . The potent proangiogenic effect attributed to heparanase under clinical (37 , 38) and experimental (21 , 29 , 30 , 32) settings further suggests heparanase relevance to ischemic wound healing. Highly purified active recombinant heparanase (Fig. 3B , 1 µg/wound) was applied topically three times a day for 3 days postwounding, when a blood clot was generated and wounds were practically sealed. As a control, wounds were similarly treated with PDGF (0.5 µg/wound), the only growth factor approved for acceleration of wound healing (39) . Saline was used as control for the heparanase and PDGF treatments. The wound area was harvested 7 days postwounding and analyzed by histology. Heparanase-treated wounds appeared to have healed better, leaving smaller areas of uncovered granulation tissue (Fig. 3C ) than in untreated wounds. Analysis of 10 wounds in 5 rats revealed an average wound diameter of 2775± 215 µm for the control saline-treated wounds compared with 1850±365 µm for the heparanase-treated wounds (Fig. 3D ), representing a 40% reduction in wound diameter. These differences are statistically significant (P<0.01). Surprisingly, PDGF was less effective in this model system and wound diameters were not statistically different from those of the control ones (Fig. 3D ).

A single application of heparanase on the flap incisions before its replacement improved the survival of the flap skin. To further examine this effect, flap wounds were created without any punch. Heparanase was topically applied (50 µg/flap) once on the open flap and the incision wounds before replacing the flaps onto their bed. Flap appearance was evaluated macroscopically 2 wk later. As shown in Fig. 4 , control saline-treated flaps developed massive necrosis that appeared as large dark areas, apparently resulting from the lack of an appropriate blood supply. In striking contrast, most heparanase-treated flaps appeared vital without any signs or with marginal signs of necrosis (Fig. 4) . When the healing index (level of necrosis as percentage of the total flap area) was calculated, 62.5% of the heparanase-treated flaps were considered healed vs. 16.5% of the control, saline-treated flaps (P=0.0659). These results suggest that heparanase beneficial effect is not restricted to wound healing (Fig. 3) , but may improve wound survival under different pathological settings such as skin grafting.



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  		Figure 4. Heparanase improves flap tissue survival. Flap wounds were created as described in Materials and Methods without any punch made. 1 mL saline (Con) or heparanase (Hepa, 50 µg) was applied topically once on top of the entire flap bed and incisions before replacing the flaps onto their bed and suturing. Gross flap morphology was examined and evaluated after 2 wk. A significant improvement of flap appearance was noted in response to heparanase treatment.

Heparanase enhances wound epithelialization and blood vessel maturation
To define mechanisms that would account for the improvement of wound healing and flap survival upon heparanase treatment, we examined several parameters critical for wound healing. Careful measurements of granulation tissue thickness did not reveal a significant improvement in response to heparanase treatment of the punch wounds (data not shown). In contrast, epithelium thickness was significantly affected (Fig. 5 ). Measurements taken in the area of flap incisions revealed an average epithelium thickness of 121±13 µm for the control saline-treated incisions compared with 193±18 µm for the heparanase-treated ones (Fig. 5C, P <0.01). We previously demonstrated that topical heparanase application resulted in a strong angiogenic response in mouse punch wounds (21) . Close examination of the rat punch wounds did not reveal a consistent increase in blood vessel number, but rather in blood vessel maturation (Fig. 5D-F ). Immunostaining of wound sections with anti-SMA antibody could barely detect pericytes recruited to the newly formed vasculature in the granulation tissue of control, saline-treated wounds (Fig. 5D ). In striking contrast, SMA-positive blood vessels were readily detected in the heparanase-treated wounds, often appearing in a dense, multilayer manner (Fig. 5E ). Counting 4 random fields in each of 10 wounds revealed a 7-fold increase in the number of SMA-positive blood vessels after heparanase treatment (Fig. 5F ), a highly significant difference (P<0.0001).



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  		Figure 5. Heparanase improves incision wound epithelialization and blood vessel maturation. A, B) H&E staining and epithelialization analysis of incision wounds. Punch wounds were harvested and processed for histology as described in Materials and Methods, and control (A) and heparanase-treated (B) incisions were evaluated by H&E staining. C) Graphical presentation (mean±SE) of epithelium thickness measured in 10 incision wounds created in 5 rats. D–F) Enhanced pericyte recruitment upon heparanase treatment. Punch wounds were harvested and 5 µm sections of saline- (D) and heparanase- (E) treated wound tissue were stained with anti-SMA antibody. F) The number of SMA-positive blood vessels was calculated by counting 4 random fields in sections taken from 5 rats (mean±SE).

Elevated heparanase levels in wound fluids of heparanase transgenic mice
We recently characterized a transgenic mouse that overexpresses heparanase in various tissues (24) , including the skin (data not shown). To quantitate heparanase levels in wound fluids, polyvinyl sponges were implanted into incision wounds and wound fluids were extracted from the sponges after 1 day (n=6). Heparanase activity was detected in the wound fluids extracted from control wounds (Fig. 6 A, Con), in agreement with heparanase expression in the wound tissue (Fig. 2) . A marked 8-fold increase in heparanase activity was measured in the fluids extracted from wounds created in the heparanase transgenic (hpa-tg) mice (Fig. 6A , Tg). Elevated levels of heparanase in the wound fluids of the hpa-tg mice was further confirmed by immunoblotting (Fig. 6B , upper panel), clearly indicating heparanase overexpression in the hpa-tg mouse model. Next, we evaluated the effect of heparanase overexpression on the bioavailability of growth factors known to be involved in tissue repair. Elevated bFGF levels (7-fold increase) were determined in the wound fluid of the hpa-tg vs. control mice (Fig. 6B , lower panel), supporting the notion that heparanase activity not only facilitates cell invasion but is also responsible for release of biologically active HS-bound polypeptides.



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  		Figure 6. Heparanase protein and activity in wound fluids of control and heparanase-transgenic mice. Full-thickness dermal incisions were made in the back of control (Con) and heparanase transgenic (Tg) mice (n=6) and polyvinyl sponges were implanted in the wound area before its closure with cyanoacrylate. Sponges were collected 24 h later and wound fluids were extracted by centrifugation. Samples equilibrated for protein content were analyzed for A) heparanase activity, using sulfate-labeled ECM as a substrate. Presented are the gel filtration (Sepharose 6B) patterns of HS degradation fragments released from ECM by wound fluids derived from control (•) and hpa-tg ({square}) mice. B) Heparanase and bFGF proteins detected in wound fluids of control (con) and hpa-tg (Tg) mice by immunoblotting, applying p9 (upper panel) and mAb130 (middle panel) anti-heparanase antibodies, or anti-bFGF antibodies (lower panel). Recombinant heparanase and bFGF were applied as positive (Pos) controls.

Acceleration of wound angiogenesis in heparanase transgenic mice
To further explore heparanase contribution to wound angiogenesis, 1 cm-long, full-thickness incision was created on the back skin of control and heparanase transgenic (hpa-tg) mice. We used a noninvasive MRI method to analyze the wound vascular density as a function of time (27 , 29) . Control mice exhibited an increase in apparent vessel density (AVD) that peaked 3 days after wounding, followed by a gradual decrease (Fig. 7 A, control). In contrast, a marked 4-fold increase in AVD was already evident in the hpa-tg mice 1 day after wounding (Fig. 7A ) and the high AVD level persisted until the end of the experiment on day 7 (Fig. 7A , Tg). The elevation of AVD characterized by MRI analysis was confirmed by gross examination of the wound areas on day 7, when the experiment was terminated. Indeed, wounds in the hpa-tg mice appeared red compared with control wounds (Fig. 7B , upper panel). Blood vessel maturation, evaluated by staining of wound sections with anti-SMA antibody, revealed a 5- to 6-fold increase in pericyte recruitment to the newly formed wound vasculature of the hpa-tg mice (Fig. 7B , lower panel). Thus, after topical application (Fig. 5D-F ) or upon constitutive overexpression, heparanase stimulated pericyte recruitment and blood vessel maturation.



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  		Figure 7. Heparanase overexpression induces wound angiogenesis. A) MRI analysis. 1 cm-long, full-thickness dermal incisions were made in the back of control (n=10) and hpa-tg (Tg) (n=12) mice. The extent of neovascularization was evaluated by MRI analysis of the apparent vessel density (AVD) in the wound area, on days 1, 3, and 7 after incision, as described in Materials and Methods (mean ±SE; P<0.001). B) Gross and histological examination of wounds. Seven days after the initial incision, mice were killed and the wound area was subjected to morphological (upper panels) and histological examination. Tissue sections (5 µm) were stained with hematoxylin and eosin (H&E, middle panel) or immunostained with anti-SMA antibodies (SMA, lower panel). Gross examination of the wound area revealed a markedly increased angiogenic response in the skin of the transgenic (right) vs. control (left) mice. Original magnification: upper panels: x4; middle panels: x20; lower panels: x40.

Heparanase inhibitor inhibits wound angiogenesis
Analysis of a series of modified species of heparin led to the identification of several nonanticoagulant compounds that efficiently inhibit heparanase enzymatic activity. The synthesis and biochemical nature of these compounds were described (25) and their heparanase-inhibiting properties are being investigated (40 ; A. Naggi, et al., unpublished results). A promising heparanase inhibitor is a glycol-split fragment of heparin (ST1514), which inhibits the enzyme at nanomolar concentrations (0.02–0.1 nM) in vitro (Fig. 8 A). Administration of this heparanase inhibitor (i.p., 100 µg/mouse, n=4 mice/group) before wounding and each consecutive day thereafter resulted in reduced AVD in the control nontransgenic mice, inhibition that was most pronounced on day 3 after wounding, as indicated by MRI analysis of the wound area (Fig. 8B ). Similarly, the heparanase inhibitor significantly inhibited wound angiogenesis in the hpa-tg mice, as indicated by a marked decrease in AVD to levels measured in control mice subjected to wounding and treatment with the same inhibitor (Fig. 8B ). These results indicate that heparanase activity promotes wound angiogenesis under normal conditions, even more so in heparanase-overexpressing mice, leading to an increased vascular density in the wound area.



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  		Figure 8. Glycol-split modified heparin inhibits heparanase activity and wound angiogenesis. A) Heparanase activity. Purified recombinant heparanase (20 ng) was incubated (4 h, 37°C) with sulfate-labeled ECM in the absence ({blacksquare}) and presence ({square}) of 1 µg/mL compound ST 1514 (Hepa+ST1514). Sulfate-labeled material released into the incubation medium was subjected to gel filtration on Sepharose 6B, as described in Materials and Methods. HS degradation fragments are eluted in fractions 20-40 (peak II). B) MRI analysis. Control (•) and heparanase transgenic ({blacksquare}) mice (n=6) were wounded (see legend to Fig. 7 ) and treated with control vehicle (PBS) or compound ST 1514 (O and {square}, 100 µg/mouse) administered i.p. before wounding and every other day thereafter. MRI analysis was performed on days 1, 3, and 7 postwounding to calculate the AVD, as described in Materials and Methods. Total of 9 fields per wound (3 sectionsx3 fields) were analyzed and the standard error did not exceed ±25% of the mean. A marked reduction in wound vascularity is noted in response to treatment with compound ST 1514.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Heparanase activity has long been correlated with pathologies associated with cell invasion such as cancer metastasis and autoimmunity (16 , 17) . The data presented here suggest that under certain pathological conditions heparanase may possess beneficial capabilities rather than indicating a poor prognosis. Heparan sulfate is the most abundant glycosaminoglycan present in the epidermis (41) . The exact function of HSPG in the epidermis is largely unknown; nevertheless, they are thought to be involved in a variety of processes such as cell-cell interaction, cell adhesion, proliferation, differentiation, morphogenesis, and ECM structural integrity (41 , 42) . Moreover, HSPG are implicated in wound healing, as syndecan-1-deficient mice exhibit defects in keratinocyte activation (43) . Modulation of the physiological functions of HSPG is attributed in part to the activity of the endo-ß-D-glucuronidase enzyme, heparanase (17) . Heparanase expression was noted to be restricted to the plantar stratum corneum of human (33) and rat (Fig. 1A, B ) epidermis. Expression of HS is essentially confined to the basement membrane between the dermis and epidermis (33) , suggesting that under resting conditions heparanase may modulate membrane-bound rather than basement membrane HS. Heparanase localization was significantly altered during wound healing and appeared in most keratinocytes adjacent to the wound margins (Fig. 2B ) and in the migrating tip of the wound (Fig. 2C ). Thus, heparanase may play a role in the wound healing process by enhancing keratinocyte cell migration (Fig. 2D-F ). Topical heparanase application accelerated wound healing in the flap/punch model (Fig. 3) , resulting in an average wound diameter as low as 60% of control wounds (Fig. 3D ). We recently reported that heparanase overexpression enhances adhesion and migration of human glioma cells (30) . Exogenously added heparanase stimulated endothelial cell migration (32) , indicating that heparanase can modulate migratory responses in different cell types.

Since the late 1970s there has been considerable interest in evaluating the therapeutic potential of growth factors in the treatment of patients with impaired wound healing (4 , 39) , yet the only growth factor currently approved for clinical use is PDGF (39) . In the flap/punch model, heparanase seems to be even more efficient than PDGF in accelerating wound healing (Fig. 3D ). It is conceivable that such high effectiveness is due to the release of a combination of HS-bound factors that may act cooperatively or synergistically to promote cell migration, proliferation, and tissue vascularization and thereby exert a very potent effect. Release of HS-bound growth factors is exemplified by the high levels of bFGF found in the wound fluid of heparanase transgenic mice (Fig. 6B ) correlated with a marked elevation of heparanase activity (Fig. 6A ) and expression levels (Fig. 6B ). Growth factors other than bFGF are likely to become more readily bioavailable upon heparanase application or overexpression. Although the release of such factors is not demonstrated here directly, some indirect evidence supports this possibility, as outlined below.

1) Topical heparanase application (Fig. 5D, E ) or constitutive overexpression (Fig. 7B ) resulted in a marked elevation of pericyte recruitment and blood vessel maturation. Pericytes are thought to stabilize the newly formed vessels and may be held responsible for the minimal regression observed in the transgenic wound vasculature (Fig. 7A ). Although this effect is not necessarily beneficial for wound healing in rodents, it may be of great significance for chronic wound healing in humans. Genetic studies have so far identified two mediators responsible for pericyte recruitment: PDGF-BB/PDGFR-ß and angiopoietin (Ang)-1/Tie 2 receptor (44 , 45) . PDGF-BB (46) and presumably Ang-1 (47) are both heparin binding proteins and therefore are expected to be released by heparanase activity, accounting for blood vessel maturation. 2) We have used a novel, noninvasive MRI technology to study angiogenic parameters after wounding. Previous MRI analysis revealed enhanced tumor vascular density and maturation upon heparanase overexpression (29) . MRI analysis already revealed a marked elevation in vascular density in the wounds of hpa-tg mice 1 day after wounding, with minimal regression thereafter (Fig. 7A ). Such a rapid increase in vascular function is not likely to result solely from newly formed blood vessels, but may reflect alternations in the surrounding vascular tone. Both bFGF and PDGF exhibit vasodilatation activity (48 49 50 51) that may contribute to the elevated vascular density observed in wounds of the hpa-tg mice shortly after wounding. 3) Successful wound healing does not rely only on a proper angiogenic response, but also requires efficient migration and proliferation of keratinocytes. Exogenously added heparanase or its constitutive overexpression resulted in a 2- to 3-fold increased epithelium thickness. While bFGF and PDGF are mitogens for fibroblasts and endothelial cells (3) , activation of keratinocytes may not be mediated by these factors but instead be due to release of HS-bound KGF and/or HB-EGF, which stimulate keratinocyte proliferation and migration (4) . Thus, the coordinate, simultaneous release of a combination of HS-bound growth factors such as bFGF, PDGF/Ang-1 (vascular maturation), and HB-EGF/KGF (epithelium thickness) is unique to heparanase and may account for its efficient wound healing-promoting effect. The efficient rescue of the flap tissue from necrosis implies that heparanase may be effective not only in the healing of chronic vasculopathy wounds, but in acute wounds such as burns and in skin grafting procedures.

Specific heparanase inhibitors are expected to provide pivotal tools for studying heparanase functions under normal and pathological conditions. Currently available heparanase inhibitors are various sulfated poly- and oligosaccharides such as heparin fragments, laminaran sulfate, and PI-88 (52 53 54) . These compounds were shown to inhibit heparanase activity and elicit anti-metastatic and anti-angiogenic effects (52 53 54) . Nevertheless, laminaran sulfate and species of heparin affect bFGF binding to its receptor, endothelial cell proliferation (55) , and angiogenesis (56) . Species of heparin also inhibit selectin-mediated cell adhesion (57) . The lack of specificity makes interpretation regarding heparanase function questionable when using these and other polysulfated inhibitors of the enzyme (58) . Introduction of glycol-splits along the heparin molecule yields a more flexible structure and, thereby, a more potent and specific heparanase inhibitor, effective at nanomolar concentrations and lacking an anti-coagulant activity (25 , 40 ; A. Naggi et al., unpublished results). Mice treated with this newly developed heparanase inhibitor exhibited a marked reduction in vascular density in the wound area (Fig. 8B ), suggesting that heparanase plays a significant role in the course of normal wound angiogenesis. Moreover, hpa-tg mice treated with the glycol-split inhibitor (ST1514) responded by a pronounced decrease in the wound vascular density (Fig. 8B ), reaching values similar to those observed in similarly treated control mice.

Our results indicate that heparanase activity is critically involved in wound angiogenesis and that elevation of heparanase levels by either topical administration or constitutive overexpression would enhance wound vascularization and wound healing. This effect may represent a more general involvement of heparanase in tissue regeneration as implied by heparanase up-regulation in bone fractures (59) and in response to liver hepatectomy (60) , as well as in tissue morphogenesis, indicated by overbranching, widening, and vascularization of ducts in the mammary glands of heparanase transgenic mice (24) .


   ACKNOWLEDGMENTS
 
We thank Drs. Claudio Pisano and Sergio Penco (Sigma-Tau Research Department, Pomezia, Rome, Italy) for providing the ST1514 glycol-split heparin and for their continuous support and assistance. This work was supported by grants from the Israel Science Foundation (#532/02), NIH (CA106456), Center for the Study of Emerging Diseases (CSED), the European Commission (5th Framework program, contract #QLK-CT-2002-02049), and the Rappaport Family Institute Fund.


   FOOTNOTES
 
1 These authors contributed equally to this work.

Received for publication April 1, 2004. Accepted for publication September 23, 2004.


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