The American Journal of Pathology, Vol. 185, No. 6, June 2015

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MUSCULOSKELETAL PATHOLOGY

Heparanase Stimulates Chondrogenesis and IsUp-Regulated in Human Ectopic Cartilage

A Mechanism Possibly Involved in Hereditary MultipleExostosesJulianne Huegel, Motomi Enomoto-Iwamoto, Federica Sgariglia, Eiki Koyama, and Maurizio Pacifici

From the Translational Research Program in Pediatric Orthopaedics, Division of Orthopaedic Surgery, The Children’s Hospital of Philadelphia,Philadelphia, Pennsylvania

Accepted for publication

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February 10, 2015.

Address correspondence toMaurizio Pacifici, Ph.D., Divi-sion of Orthopaedic Surgery,The Children’s Hospital ofPhiladelphia, 3615 Civic CenterBlvd., ARC Ste. 902, Philadel-phia, PA 19104. E-mail:pacificm@email.chop.edu.

opyright ª 2015 American Society for Inve

ublished by Elsevier Inc. All rights reserved

ttp://dx.doi.org/10.1016/j.ajpath.2015.02.014

Hereditary multiple exostoses is a pediatric skeletal disorder characterized by benign cartilaginoustumors called exostoses that form next to growing skeletal elements. Hereditary multiple exostosespatients carry heterozygous mutations in the heparan sulfate (HS)-synthesizing enzymes EXT1 or EXT2,but studies suggest that EXT haploinsufficiency and ensuing partial HS deficiency are insufficient forexostosis formation. Searching for additional pathways, we analyzed presence and distribution ofheparanase in human exostoses. Heparanase was readily detectable in most chondrocytes, particularlyin cell clusters. In control growth plates from unaffected persons, however, heparanase was detectableonly in hypertrophic zone. Treatment of mouse embryo limb mesenchymal micromass cultures withexogenous heparanase greatly stimulated chondrogenesis and bone morphogenetic protein signaling asrevealed by Smad1/5/8 phosphorylation. It also stimulated cell migration and proliferation. Interferingwith HS function both with the chemical antagonist Surfen or treatment with bacterial heparitinase up-regulated endogenous heparanase gene expression, suggesting a counterintuitive feedback mechanismthat would result in further HS reduction and increased signaling. Thus, we tested a potent heparanaseinhibitor (SST0001), which strongly inhibited chondrogenesis. Our data clearly indicate that heparanaseis able to stimulate chondrogenesis, bone morphogenetic protein signaling, cell migration, and cellproliferation in chondrogenic cells. These properties may allow heparanase to play a role in exostosisgenesis and pathogenesis, thus making it a conceivable therapeutic target in hereditary multipleexostoses. (Am J Pathol 2015, 185: 1676e1685; http://dx.doi.org/10.1016/j.ajpath.2015.02.014)

Supported by the NIH National Institute of Arthritis and Musculoskeletaland Skin Diseases grant R01AR061758 (M.P. and E.K.). J.H. was therecipient of the National Research Service Awards predoctoral awardF31DE022204 from the NIH National Institute of Dental and CraniofacialResearch during the course of the study.Disclosures: SST0001 used in this study was provided by Sigma-Tau

Pharmaceuticals, Inc. (Gaithersburg, MD).Current address for J.H., McKay Orthopaedic Research Laboratory, Depart-

ment of Orthopaedic Surgery, University of Pennsylvania, Philadelphia, PA.

Heparanase is a multifunctional protein that is involved in avariety of physiologic and pathologic processes and repre-sents the only entity of its kind encoded in the mammaliangenome.1,2 The enzyme can cleave heparan sulfate (HS)chains present in syndecans, glypicans, and other HS-richproteoglycans and, in so doing, affects proteoglycan homeo-stasis, function,mobility, and internalization and can influencevarious processes, including cell spreading, migration, andproliferation.3,4 Importantly, cleavage of HS chains can alsorelease HS-bound cytokines, growth factors, and signalingproteins, thus enhancing their bioavailability, range of action,and effects on target cells.5,6 Latent heparanase on the cellsurface can interact with syndecans, and this interaction leads

stigative Pathology.

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to rapid internalization of the complex and delivery to lyso-somes where the enzyme is activated by cathepsin L.7 Hep-aranase has additional important functions. It can lead tosyndecan clustering and activation of downstream effector

Heparanase and Exostosis Formation

pathways that involve protein kinaseC,Src, andRac14 and canactivate b1-integrin,8 mechanisms all facilitating cellspreading and migration.9 Because some of these actions donot appear to require enzymatic activity and can be elicited byinactive heparanase as well, they are likely to involve and relyon the nonenzymatic domains of the protein.10 As a reflectionof its multiple and potent biological activities, heparanase isoften up-regulated in human cancers and is closely associatedwith, and may lead to, neoplastic cell behavior and metas-tasis.2,8,11 The protein is believed to facilitate the invasivebehavior of cancer cells and tumor growth by release ofextracellular matrix-bound angiogenic factors, includingvascular endothelial growth factor and by up-regulatingexpression of important genes such as HGF, MMP9, andVEGFA itself.1,6,12 Indeed, inhibitors of heparanase adminis-tered systemically were found to reduce progression of tumorxenografts in mice.13 These and other studies have led to thecurrent notion that heparanase inhibition by pharmacologicstrategies may represent a promising and effective cancertherapy.14

Benign ectopic cartilaginous/bony tumors called exostosescharacterize the pediatric skeletal disorder hereditary multi-ple exostoses (HME).15,16 The exostoses are growth plate-like structures that form next to, but never within, thegrowth plates of long bones, ribs, pelvis, and other skeletalelements. Because of size and location, the exostoses cancause a variety of health problems, including skeletal growthretardation and deformities, chronic pain, compression ofnerves and blood vessels, and psychological concerns.15,17,18

In the majority of HME patients the exostoses remain benignthrough life, but in approximately 2% to 5% of them theexostoses progress to malignancy, turn into osteosarcomas orchondrosarcomas, and thus become life threatening.19 MostHME patients carry heterozygous loss-of-function mutationsin EXT1 or EXT2 that encode glycosyltransferases respon-sible for HS synthesis.20,21 EXT1 and EXT2 form proteincomplexes in the Golgi and are both required for HS syn-thesis.20 HME patients thus have reduced levels, but not lack,of HS in their tissues. Surprisingly however, the cartilaginousportions of human exostoses display barely detectable levelsof HS,22 indicating that exostosis formation may require asevere loss of HS beyondwhat would be caused bymere EXThaploinsufficiency. This requirement was confirmed intransgenic mouse studies that involved conditional Ext1 and/or Ext2 ablation.23e25 Studies have suggested mechanismsthat could account for a severe drop of HS in human exos-toses, including EXT loss-of-heterozygosity, large andencompassing genomic deletions, a second hit in anothergene, and background genetic traits such as modifiers.21,26,27

However, there are still no clear answers nor obviousgenotype-phenotype correlations in HME, despite that thesyndrome can vary significantly in severity within affectedfamily members and among persons from different families.28

We, therefore, sought to investigate possible additionalpathogenic pathways in exostosis formation and progressionin HME and focused on heparanase. We do find that the

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protein is widespread in the growth plate-like cartilaginousportions of human exostoses but displays a restricted dis-tribution in normal growth plates from unaffected controlpatients. We show also that the enzyme greatly stimulateschondrogenesis, a mechanism that could promote andfacilitate inception and growth of exostoses.

Materials and Methods

Human Exostosis Samples

Exostosis tissue samples from four consenting HME pa-tients were taken after surgical removal and were processedby the centralized Children’s Hospital of Philadelphia Pa-thology Laboratory. Control growth plate tissue was har-vested from surgically removed toes of children who wereundergoing treatment for polydactyly. Fixed tissue wasembedded in paraffin and sectioned (6 mm). After specimenswere used for clinical diagnosis, extra slides were de-identified and provided to us for the present study. Thispractice was reviewed and approved by Children’s Hospitalof Philadelphia’s Institutional Review Board.

Monolayer and Micromass Cell Culture

ATDC5 cells, derived from mouse embryonal carcinoma,were cultured in 10-cm dishes with a 1:1 mixture ofDulbecco’s modified Eagle’s medium and Ham’s F-12 (LifeTechnologies, Carlsbad, CA) supplemented with 5% fetalbovine serum (ATCC, Manassas, VA), 10 mg/mL humantransferrin (Mediatech, Herndon, VA), 3 � 10�8 mg/L so-dium selenite (Mediatech), and 20 mg/mL insulin at 37�Cunder 5% CO2. Experiments were conducted once cellsreached confluence.

Micromass cultures were prepared from the mesenchymalcells of embryonic day 11.5 mouse embryo limb buds.29

Dissociated cells were suspended at a concentration of5� 106 cells/mL in Dulbecco’s modified Eagle’s medium thatcontained 3% fetal bovine serum and antibiotics. Micromasscultures were initiated by spotting 20 mL of the cell suspensions(1.5 � 105 cells) onto the surface of 24-well tissue culturedishes. After a 90-minute incubation at 37�C in a humidifiedcarbon dioxide incubator to allow for cell attachment, the cul-tures were supplied with 0.25 mL of medium. After 24 hours,medium supplemented with the indicated concentrations of bis-2-methyl-4-aminoquinolyl-6-carbamide (Surfen; obtained fromthe Open Chemical Repository in the Developmental Thera-peutic Program at the National Cancer Institute, NSC12155),bacterial heparitinase III (Sigma-Aldrich, St. Louis, MO), re-combinant human bone morphogenetic protein (BMP)2 (R&DSystems, Minneapolis, MN), recombinant human heparanase(kindly provided by Drs. Israel Vlodavsky, Technion, Haifa,Israel, and Ralph Sanderson, University of Alabama at Bir-mingham, Birmingham, AL), or SST0001 (Sigma-Tau Phar-maceuticals Inc., Gaithersburg, MD). Fresh reagents (drug and/or protein) were given with medium change every other day.

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Equivalent amounts of vehicle were added to control cultures.Cultures were stained with Alcian Blue (pH 1.0) after 4 and 6days to monitor chondrogenic cell differentiation by standardmethods. Micromass analysis was performed with ImageJversion 1.47 (NIH, Bethesda, MD). Images were made binaryunder an RGB threshold, and particle analysis was used tomeasure nodule size, number, and Alcian Blueepositive area.

Cell Proliferation and Migration Analysis

ATDC5 cells were treated at day 0with human heparanase (400ng/mL) or a vehicle control. At this time, one culture dish washarvested for DNA. DNA was also isolated from control andtreated cells at day 3, 7, and 14. DNA isolation was performedwithTRIzol reagent (Invitrogen,Carlsbad, CA) according to themanufacturer’s protocols and measured with a NanoDrop(ThermoScientific, Marietta, OH). ATDC5 migration wasanalyzed via a scratch test. Cells were replated on a six-well dishand allowed to reach confluence before treatment. After 24hours of treatment, a straight line scratch was made with a P10pipette tip. Reference points were made with a fine-tippedmarker on the bottom of the dish. Images at time 0 were takenwith a phase-contrast microscope (Leica, Wetzlar, Germany).Cells were cultured at 37�C in a humidified carbon dioxideincubator for 4 hours, with images in the same field of viewtaken at 2 and4hours. The open scratch areawas quantifiedwithImageJ (n Z 6 per treatment group; NIH). In micromass cul-tures, migration was determined by expansion of themicromassitself from day 0 to day 4 or 6. The diameters of untreatedmicromasses (n Z 10) were measured and averaged for abaseline size. This baseline was subtracted from the averagediameter of treated cultures (nZ 6) to estimate cell migration.

Semiquantitative PCR Analyses

Total RNA was extracted from cells by the guanidine phenolmethod with the use of TRIzol reagent (Invitrogen) accordingto the manufacturer’s protocols and was measured with aNanoDrop. One microgram of total RNA was reverse tran-scribed with the SuperScript III First-Strand Synthesis System(Life Technologies). Band intensities for semiquantitativePCR were normalized to glyceraldehyde-3-phosphate dehy-drogenase and compared, whereas band intensities and cyclenumbers were linear. The following primer sets were used:Gapdh forward primer (50-CGTCCCGTAGACAAAATG-GT-30) and reverse primer (50-TTGATGGCAACAATCTC-CAC-30); Runx2 forward primer (50-CGCACGACAACC-GCACCAT-30) and reverse primer (50-AACTTCCTGTG-CTCCGTGCTG-30); Agg forward primer (50-GGAGCGA-GTCCAACTCTTCA-30) and reverse primer (50-CGCTCA-GTGAGTTGTCATGG-30); Col2a1 forward primer (50-CT-ACGGTGTCAGGGCCAG-30) and reverse primer (50-GT-GTCACACACACAGATGCG-30); BMP2 forward primer(50-TCTTCCGGGAACAGATACAGG-30) and reverse primer(50-TCTCCTCTAAATGGGCCACTT-30); BMPRI forwardprimer (50-GCTTGCGGCAAtCGTGTCTAA-30) and reverse

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primer (50-GCAGCCTGTGAAGATGTAGAGG-30); andBMPRII forward primer (50-CACACCAGCCTTATACTC-TAGATA-30) and reverse primer (50-CACATATCTGTTA-TGAAACTTGAG-30). Band intensities were quantified bycomputer-assisted image analysis (ImageQuant TL; GEHealthcare, Chalfont St. Giles, UK).

Protein Analysis

ATDC5 cells were grown to 100% confluence in six-wellplates and treated with indicated concentrations of Surfen.Micromass cultures were treated with control vehicle, Surfen,human heparanase, SST0001, or recombinant human BMP2.At specified time points, total cellular proteins were har-vested in SDS-PAGE sample buffer, electrophoresed on 4%to 15% SDS-Bis-Tris gels (40 mg per lane), and transferred topolyvinylidene difluoride membranes (Invitrogen). Mem-branes were incubated overnight at 4�C with dilutions ofantibodies against phospho-Smad1/5/8 (dilution 1:1000;9511; Cell Signaling Technology Inc., Danvers, MA),Smad1 (dilution 1:500; 63439; Abcam Inc., Cambridge,MA), or human heparanase (dilution 1:750; 59787; AbcamInc.). Enhanced chemiluminescent immunoblotting detectionsystem (ThermoScientific) was used to detect the antigeneantibody complexes. The membranes were re-blotted withantibodies to a-tubulin (dilution 1:2000; T-5168; Sigma-Aldrich) for normalization, and band intensities were quan-tified by computer-assisted image analysis.

Immunohistochemistry

Immunostaining for heparanase was performed with paraffinsections that were deparaffinized and de-masked with 5 mg/mL hyaluronidase for 1 hour at 37�C to de-mask. Sectionswere incubated with anti-heparanase polyclonal antibodies(59787; Abcam Inc.) at 1:200 dilution in 3% normal goatserum in phosphate-buffered saline overnight at 4�C. Afterrinsing, sections were then incubated with biotinylated goatanti-rabbit secondary antibody, and the signal was visualizedwith a horseradish peroxidase/diaminobenzidine detectionimmunohistochemistry kit according to the manufacturer’sinstructions (Abcam Inc.). For collagen X staining, com-panion serial sections were treated with 0.1% pepsin in 0.02NHCl for 10 minutes at 37�C, incubated with 1:1000 dilutionof rabbit collagen X antibodies (Cosmo Bio, Carlsbad, CA)in 10% normal goat serum for 1 hour, followed by treatmentwith ABC reagent (Vector Laboratories, Burlingame, CA)for 1 hour for antibody detection. Bright-field images weretaken with a SPOT insight camera (Diagnostic Instruments,Inc., Sterling Heights, MI) operated with SPOT softwareversion 4.0 (Diagnostic Instruments, Inc.).

Statistical Analysis

Data were validated by two-tailed Student’s t-tests. Thethreshold for significance for all tests was set as P < 0.05.

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Figure 1 Heparanase is broadly distributed in human exostoses butrestricted in control growth plate. AeD: Immunohistochemical stainingof longitudinal sections of control human growth plate shows thatheparanase is detectable in hcs at the coj and is also prominent inperichondrium (C, arrowheads). Strong staining is appreciable in ablood vessel present in the SOC as expected (D). EeH: Sections fromhuman exostoses were stained in parallel. Heparanase staining is clearin nearly every chondrocyte regardless of location within tissue andapparent phenotypic maturation status (E and F) and also in neigh-boring perichondrium-like tissue (G, arrowheads). Clusters of largehypertrophying chondrocytes display strong staining, particularly intheir pericellular compartment (H). I: Continuous sections from theabove exostosis specimen were stained with collagen X antibodies. Notethat only mature and hcs stain positively. B and F are higher magnifi-cation images of areas in A and E, respectively. Scale bars: 250 mm (Aand E); 75 mm (B, D, F, G, H, and I); 300 mm (C). Coj, chondro-osseousjunction; hc, hypertrophic chondrocyte; pc, perichondrium; phc, pre-hypertrophic cartilage; rc/pl, resting-proliferating cartilage; SOC, sec-ondary ossification center.

Heparanase and Exostosis Formation

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Results

Broad Heparanase Distribution in Human Exostoses

Mature and symptomatic exostoses removed at surgery aretypically composed of a cartilaginous cap and an underlyingbony stem that connects with the affected skeletal element,be it a long bone or a rib. The cartilaginous cap often dis-plays a growth plate-like structure and organization, withsmall chondrocytes located at its distal end and surroundedby a connective/perichondrium-like tissue layer and withlarge hypertrophying chondrocytes located more proximallynear the bony portion.15,16 To determine whether hepar-anase presence and distribution were altered in exostoses,we obtained exostoses from consenting HME patients un-dergoing surgical treatment and processed them for sectionimmunostaining with rabbit antibodies against human hep-aranase. For comparison, we used longitudinal sections ofgrowth plates from control nonaffected persons who hadundergone surgical treatment for polydactyly. In thesecontrol specimens, heparanase staining was clearly andstrongly detected in the hypertrophic chondrocytes at thechondro-osseous junction (Figure 1, A and B) and in theperichondrium (Figure 1C), but little to no staining wasobserved in resting and proliferative zones (Figure 1, AeC).Blood vessels that invaded the secondary ossification centerdisplayed high heparanase staining as expected,30 thusacting as an internal positive control of staining specificityand sensitivity (Figure 1D). In contrast, heparanase stainingwas found in all chondrocytes within the exostoses,regardless of location within the tissue and apparent matu-ration stage (Figure 1, EeG). Tissue portions that containedenlarged and hypertrophying cells in clusters were evenmore strongly stained, particularly in their pericellularcompartment (Figure 1H). However, staining for collagen Xin companion serial sections showed that it was restricted tomature and hypertrophic chondrocytes (Figure 1I).

Heparanase Stimulates Cell Migration and Proliferationand Chondrogenesis

What could be the significance and possible pathologicimplications of the findings in the previous section? Exos-toses are ectopic cartilaginous outgrowths and as such, mustdepend on local cell proliferation and migration and onchondrogenic differentiation to initiate, sustain, and propeltheir development and growth.31 Thus, we asked whetherheparanase would influence such processes. To analyze cellproliferation, we grew ATDC5 chondrogenic cells inmonolayer culture in control medium or medium that con-tained 400 ng/mL human recombinant heparanase, a con-centration used in previous studies and elicited maximalresponses.32 DNA quantification at three time points over a2-week culture period showed that heparanase had stimu-lated cell growth by nearly threefold by day 14 over controlvalues (Figure 2A). To examine cell migration, we used the

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Figure 2 Treatmentwith human recombinant heparanase stimulates cell proliferation,migration, and chondrogenesis.A:DNAquantification shows that heparanasestimulates proliferation of ATDC5 cells inmonolayer as early as 3 days after treatment comparedwith vehicle-treated controls.B: Scratchwound-healing assays show thatcell migration is increased by heparanase treatment. Wound closure was monitored by sequential phase contrast imaging up to 3 hours. C: Representative images ofstained limb bud cell micromass cultures on day 4 and 6 treated with recombinant heparanase or vehicle. Note the substantial increase in the number and stainingintensity of cartilage nodules in treated versus control cultures.D: Image-based quantification of Alcian Blueepositive areas showsmore than twofold increase in treatedcultures over control set at 1. E:Measurement of overall micromass diameter on day 4 and 6 indicates that proliferation/migration of peripheral cells was stimulated byheparanase treatment. Increase in diameter for eachmicromasswas set relative to that at 24 hours after plating and is described in arbitrary units. F: Immunoblot imagesshow that heparanase treatment had increased Smad1/5/8 phosphorylation protein levels in day 4 and 6 micromass cultures relative to respective controls. Membraneswere re-blottedwitha-tub antibodies for sample loading normalization.G:Normalized band intensity quantifications of blots in Fwith controls set at 1.nZ 3 (A);nZ6(B, D, and E). *P < 0.05, **P < 0.01. Scale barZ 1.5 mm. Cntl, control; Hep’ase, heparanase protein; a-tub, a-tubulin.

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scratch and wound healing system.32 Accordingly, confluentmonolayers were scratched in the middle with a pipette tip,and the repopulation of the scratch area by migrating cellswas determined over a 3-hour period in the absence orpresence of exogenous heparanase. Clearly, cultures treatedwith heparanase repopulated the wound area more rapidlyand more extensively than untreated control cells by 3 hours(Figure 2B).

To examine chondrogenic cell differentiation, we usedmicromass cultures.29,33 In this popular experimental system,progenitor chondrogenic cells are isolated from embryonic limbbuds and are grown at high cell density in spot cultures in whichthey resume their differentiation program and give rise tocartilaginous nodules over time. For these experiments, cellsisolated from mouse embryo limb buds were plated in micro-mass and grown in medium lacking or containing exogenousheparanase as in the previous paragraph. Staining with AlcianBlue on day 4 and 6 showed that control cultures displayedseveral positive cartilage nodules scattered in their centralportion as expected (Figure 2C), but more numerous and morestrongly staining nodules were present in companionheparanase-treated cultures (Figure 2C). Computer-assistedimaging showed that the Alcian Blueestaining area wasmore than twofold higher in treated than in control cultures(P< 0.05) (Figure 2D). We obtained similar results previously

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by using exogenous bacterial heparitinase III that cleaves HS infar smaller fragments than mammalian enzyme,34 indicatingthat chondrogenesis is stimulated regardless of HS fragmentsize. The peripheral portion of micromass cultures normallycontain variously shaped fibroblastic cells that actively prolif-erate and migrate away from the micromass over time.29 Thus,we measured the overall maximal diameter of the cultures andfound that the heparanase-treated cultures had achieved asignificantly larger diameter at both day 4 and day 6 thancontrols (Figure 2E), reaffirming that heparanase promotes cellproliferation and/or migration.BMP signaling is a main regulator and stimulator of

chondrogenesis,35 and we previously showed that it is ectop-ically activated during the early stages of exostosis-like tissueformation in HME mouse models that involved conditionalExt1 ablation.33 Thus, we tested whether human heparanasetreatment would stimulate BMP signaling along with itsstimulation of chondrogenesis as seen in the previous para-graph. Immunoblot analysis with antibodies to phosphorylatedSmad1/5/8 by using whole micromass cell extracts showedthat pSmad levels were increased after heparanase treatment atboth day 4 and day 6 of culture compared with controls(Figure 2, F and G), likely signifying that heparanase hadstimulated chondrogenesis by increasing bioavailability and/orsignaling activity of endogenous cell/matrix-associated BMPs.

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Figure 3 Heparanase expression is responsive to modulation in heparansulfate levels or function. AeD: Immunoblot images and densitometrichistograms indicate that endogenous heparanase protein levels increase bySurfen treatment (þ) in limb bud micromasses (A and B) and ATDC5 cellcultures (C and D) compared with respective untreated controls (�). E and F:Semiquantitative RT-PCR and densitometric analyses show that Surfentreatment increases heparanase gene expression in micromass cultures.G and H: Immunoblot and densitometric quantification analyses showup-regulation of endogenous heparanase protein levels on treatment withrecombinant BMP2. Data are expressed as means� SD. *P< 0.05. BMP, bonemorphogenetic protein; Cntl, control; Hep’ase, heparanase protein; Surfen,bis-2-methyl-4-aminoquinolyl-6-carbamide; a-tub, a-tubulin.

Heparanase and Exostosis Formation

Heparanase Gene Expression Is Responsive to HSLevels

The widespread presence of heparanase in exostoses andits pro-chondrogenic and signaling effects seen in Figure 2raised the question of whether heparanase is an earlyresponse gene, able to be up-regulated soon after or concomi-tantly with declining HS levels and thus perpetuating oramplifying cellular responses. To test this hypothesis, wetreated primary limb mesenchymal cell micromass cultureswith the HS antagonist Surfen. As we and others showed pre-viously, this compound interferes with HS function and elicitsfunctional HS deficiency,36 triggering cellular responses similarto Ext gene ablation, including enhanced chondrogenesis.33

Interestingly, we found that treatment with an optimal con-centration of Surfen (7.5 mmol/L)33 significantly increased theprotein levels of endogenous heparanase as indicated byimmunoblotting (Figure 3A), approximately eightfold at thisdose (Figure 3B). A similar response was observed in ADTC5cell cultures (Figure 3, C and D). Control of this responseappeared to be at the transcriptional and/or RNA stabilizationlevels because the heparanase mRNA amounts were increasedas well in Surfen-treated cultures as indicated by semi-quantitative RT-PCR (Figure 3, E and F).

A related and congruent possibility is that endogenousheparanase levels may be stimulated not only by decliningHSlevels/function but also by concurrent BMP bioavailability.Thus, we treated day 2 micromass cultures with exogenous100 ng/mL recombinant human BMP2, a concentration thatstrongly stimulates BMP signaling and chondrogenesis,33 for24 or 48 hours. Immunoblot analysis showed that endogenousheparanase levels were clearly increased in BMP2-treatedcultures compared with companion untreated cultures(Figure 3G), approximately twofold as revealed by densi-tometry and normalization (Figure 3H).

Chondrogenesis Is Inhibited by a HeparanaseAntagonist

Given our results that link heparanase to the stimulation ofchondrogenesis and BMP signaling (and likely other growthfactor signaling pathways), we asked whether inhibitingendogenous heparanase would have negative repercussionson chondrogenesis. Thus, we used the heparanase inhibitorSST0001, a modified heparin molecule without anticoagu-lant properties and with high affinity for heparanase andstrong inhibitory activity.37 This compound was alreadythoroughly tested for safety and pharmacokinetics in anumber of animal models and was shown to have anti-tumorigenic properties.38 On the basis of this groundwork,we chose three concentrations with which to treat limb budmicromass cultures, at or above the concentration that in-hibits 50% for heparanase inhibition. We found thatSST0001 treatment strongly reduced the formation of AlcianBlueepositive chondrogenic nodules (Figure 4A) in a dose-dependent manner as indicated by image quantification

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(Figure 4B). Counterstaining with hematoxylin showed thatoverall cell number and density had not changed significantlyafter treatment (Figure 4A). Whole cellular RNA and DNAcontent also remained similar in treated and untreated cul-tures at day 4 and day 6, although minor decreases wereobserved by these methods at the maximal 20 mg/mL dose

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Figure 4 Chondrogenesis is inhibited by aheparanase antagonist. A: Images of Alcian Blueestained micromass cultures on day 4 and 6 show adose-dependent reduction in cartilage nodule for-mation on treatment with the heparanase antago-nist SST0001. Panels on the right show images ofcontrol and treated micromass cultures counter-stained with hematoxylin. B: Image-assisted quan-tification of Alcian Blueepositive area in controlversus SST0001-treated cultures. Statistical signifi-cance was reached at every concentration tested.CeE: Histograms depict gene expression levels ofchondrogenic genes ColII, Agg, and Runx2 in con-trols versus SST0001-treaed micromass cultures onday 4 and 6 measured by semiquantitative RT-PCRand quantified by imaging. F: Immunoblot imagesshow decrease in the endogenous Hep’ase levels inmicromass cultures on SST0001 treatment and in-crease on bacterial Hep treatment compared withcontrol. G: Histograms depict the overall absolutemicromass diameter at day 4 and 6 in control versusSST0001-treated cultures. Data are expressed asmeans � SD. n Z 2 (CeE); n Z 6 (B and G).*P < 0.05, **P < 0.01. Scale bar Z 3 mm. Hep,heparitinase; Hep’ase, heparanase protein; SST,SST0001.

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(not shown). Whole cellular RNA samples were then pro-cessed for semiquantitative RT-PCR, and the data showedthat expression of such chondrogenic marker genes ascollagen II (ColII), aggrecan (Agg), and Runx2 was dosedependently inhibited by SST0001 treatment (Figure 4,CeE). Interestingly, SST0001 treatment also reduced thelevels of endogenous heparanase (Figure 4F) compared withuntreated control cultures (Figure 4F), whereas treatmentwith bacterial heparitinase increased them (Figure 4F). Inaddition, SST0001 treatment reduced the overall diameter ofthe micromasses (Figure 4G), indicating that it had inhibitedmigration of peripheral fibroblastic cells.

Discussion

Our data provide evidence that heparanase is detectable andbroadly distributed in the chondrocytes present in benignhuman exostoses, a phenotype quite distinct from that seen innormal growth plate cartilage where the protein is restrictedto the hypertrophic zone and perichondrium, consistent withdata in a previous study.39 We show also that exogenousrecombinant human heparanase is a strong stimulator of cellmigration, proliferation, and chondrogenesis in mouse cellcultures, indicating that one function, and possibly a mainfunction, of heparanase would be to promote cell recruitmentfor the initiation and outgrowth of the exostoses. This

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mechanistic sequel is reinforced by our observations thatheparanase stimulates the pro-chondrogenic BMP signalingpathway and that endogenous heparanase gene expressionand overall protein levels are increased as the HS levelsdecrease. Thus, heparanase appears to be a part of tightlycontrolled mechanisms that link it to HS levels and alsoresponsive to modulations in HS levels. A significant drop inHS levels occurring in local cells in HME patients wouldelicit increased heparanase expression which in turn wouldelicit further HS degradation, stimulate growth factorrelease and signaling pathways, and promote chondro-genesis, all steps converging on and contributing to exos-tosis growth (Figure 5). Although counterintuitive andseemingly paradoxical, similar inverse relations among HSlevels, Ext1 expression and heparanase expression werereported in cancer studies. Cancer cells with higher Ext1expression exhibited lower heparanase expression, whereascancer cells with lower Ext1 expression (and higher meta-static potential) exhibited higher heparanase expression,and Ext1 knockdown with siRNA up-regulated heparanaseexpression and potentiated metastatic capacity.40 Indeed,heparanase expression levels are used to help predict tumorseverity and patient outcome.41e43 These and other studiessuggest that one of the changes occurring during the tran-sition from benign exostoses to osteosarcoma in HME pa-tients could be an even steeper increase in heparanaseexpression.

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Figure 5 Model illustrates a series of regulatory steps that may cause inception and promotion of formation and growth of exostosis. A: The cells of most patientswith hereditary multiple exostoses bear a heterozygous loss-of-function mutation in EXT1 or EXT2 (depicted here at Extþ/� cells in half white and half red). B: At onepoint prenatally or postnatally, some cellsmay undergo a secondgenetic change (depicted here as red cells along perichondriumandcalledmutant in the text), resultingin a more severe loss of overall EXT expression or function and leading to further reduction in local HS production and levels. C: This in turn may up-regulate Hep’aseexpression, increase growth factor availability and cell proliferation, and induce ectopic chondrogenesis near/at perichondrium. D: The incipient exostosis cells mayrecruit surrounding heterozygous cells, induce them into neoplastic behavior, and promote their incorporation into the outgrowing exostosis. E: Cells within theoutgrowing exostosismay assemble into a typical growth plate-like structure that protrudes away from the surface of the skeletal element (depicted here as a long bone)and cover distally by perichondrium. By inhibiting heparanase activity and possibly other processes, SSTmay inhibit chondrogenesis and in turn exostosis formation asindicated. Surfen (not indicated here) may elicit the opposite and stimulate chondrogenesis. Hep’ase, heparanase; HS, heparan sulfate; SST, SST0001; Surfen, bis-2-methyl-4-aminoquinolyl-6-carbamide.

Heparanase and Exostosis Formation

Previous studies have shown that EXT haploinsufficiencyper se is not sufficient for multiple exostosis formation inpatients or mice and that a steeper reduction would beneeded.44,45 Current possible and plausible explanations ofhow Ext expression and/or HS production could decreasefurther include EXT loss-of-heterozygosity; large genomicdeletions; a second hit in another gene; background geneticvariability, including gene modifiers; or compound hetero-zygosity in EXT1 and EXT2.21,26e28 Such changes wouldoccur in subsets of cells within the HME patient; the cellswould be prone to exostosis formation and would give riseto exostoses in conjunction with an active growth plate(Figure 5).

Our data now add another potential culprit heparanase tosuch pathogenic scenarios. The protein could contribute todisease in combination with any of the genetic eventsmentioned in the previous paragraph, together leading to asteeper decrease in EXT expression and HS levels less thanthose achieved by mere haploinsufficiency. Heparanase ispreferentially expressed in perichondrium along the growthplates (Figure 1). Previous studies reported similar data inmouse growth plate perichondrium,46 and we provided evi-dence previously that progenitor cells located within peri-chondrium may be a primary contributor to exostosisformation.33 Thus, by possessing basal heparanase expres-sion, perichondrial cells may be particularly sensitive to

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genetic changes and could require a lower threshold to tilttheir homeostatic balance, boost basal heparanase expres-sion, stimulate otherwise largely inactive growth factorsignaling pathways, incite ectopic chondrogenesis, and pro-mote exostosis formation. One additional aspect of exostosisformation stemming from mouse and human studies is that theexostoses themselves appear to be composed by a mixture ofmutant cells (ie, heterozygous Ext cells that have undergone anadditional genetic change) and plain heterozygous cells.33,44

The mutant cells would recruit the heterozygous cells into theincipient exostosis mass and induce them into a neoplasticbehavior (Figure 5). Given that heparanase facilitates cellmigration and could diffuse into the surroundings, it couldcertainly have a role in such recruitment and mobilizationprocesses during exostosis growth.

The heparanase inhibitor SST0001was shown to interferewith the growth of myeloma and sarcoma cells and concom-itant angiogenesis in mouse models,37,38 reaffirming thatheparanase has a main role in pathogenesis and may be aparticularly good target for cancer therapy. Our data withprimary limb cell micromass cultures provide evidence thatSST0001 also inhibits chondrogenesis and does so powerfullyas revealed by major decreases in development of AlcianBlueepositive cartilage nodules and expression of suchchondrogenic marker genes as collagen II, aggrecan, andRunx2. The data correlate well with a previous study using a

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different heparanase inhibitor, PI-88, with the ADC5 chon-drogenic cell line in which, however, only modest reductionsin Alcian Blue staining and cartilage gene expression wereobserved,46 possibly reflecting diverse drug potency.

Previous studies37,38 and our present data do not allow usto clarify exactly how SST0001 elicited such a strong effect.One possibility is that heparanase has a far more importantrole in chondrogenesis than currently realized, possiblybeing essential in mobilizing chondrogenic factors andenhancing their bioavailability and diffusion amongcondensed prechondrogenic cells. Its effective suppressionwould hamper this process and would elicit a strong anti-chondrogenic effect. Similarly, SST0001 could haveinterfered with cell mobilization and cellecell interactionsneeded by the limb mesenchymal cells to initially condenseand then activate the chondrogenic program.29

SST0001 is a heparin modified through glycol splitting andpartial desulfation and N-acetylation47 that is no longer ananticoagulant and cannot dislodge basic fibroblast growthfactor from the extracellular matrix or enhance its mitogenicactivity, traits that native heparins and HS chains have.48,49

However, by still being a partially sulfated polymer,SST0001 could bind other growth factors, including BMPs,and limit their bioactivity (Figure 5). Our data also show that itdecreased the endogenous levels of heparanase in the micro-masses and reduced cell migration. Thus, although it will takemore direct experimentation to clarify precisely how SST0001blocked chondrogenesis so effectively, the present data dosustain the possibility that SST0001 could be a potent inhibitorof exostosis formation in vivo. Ongoing studies with HMEmouse models should provide such evidence in the near future.

Acknowledgments

We thank Drs. Ralph Sanderson (University of Alabama atBirmingham, Birmingham, AL) and Israel Vlodavsky(Technion-Israel Institute of Technology, Haifa, Israel) forsupplying recombinant human heparanase; Drs. MartaGuttenberg and Tricia Bhatti (Children’s Hospital of Phil-adelphia Pathology Laboratory) for patient exostosis tissuediagnostics and preparation; Dr. Alessandro Noseda(Sigma-Tau Pharmaceuticals, Inc., Gaithersburg, MD) foradvice and for providing SST0001; and the MultipleHereditary Exostoses Research Foundation, an organizationdedicated to the support of families and patients with MHEand for advocating MHE public awareness and biomedicalresearch, for their passionate efforts.

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