Histochem Cell Biol (2003) 120:371–382DOI 10.1007/s00418-003-0576-6

O R I G I N A L P A P E R

Raquel Garc�a-Olivas · Johan Hoebeke ·Susanna Castel · Manuel Reina · Gunnar Fager ·Florentina Lustig · Sen�n Vilar�

Differential binding of platelet-derived growth factor isoformsto glycosaminoglycans

Accepted: 2 September 2003 / Published online: 14 October 2003� Springer-Verlag 2003

Abstract The platelet-derived growth factor (PDGF)family comprises disulfide-bonded dimeric isoforms andplays a key role in the proliferation and migration ofmesenchymal cells. Traditionally, it consists of homo-and heterodimers of A and B polypeptide chains thatoccur as long (AL and BL) or short (AS and BS) isoforms.Short isoforms lack the basic C-terminal extension thatmediates binding to heparin. In the present study, weshow that certain PDGF isoforms bind in a specificmanner to glycosaminoglycans (GAGs). Experimentsperformed with wild-type and mutant Chinese hamsterovary cells deficient in the synthesis of GAGs revealedthat PDGF long isoforms bind to heparan sulfate andchondroitin sulfate, while PDGF short isoforms only bindto heparan sulfate. This was confirmed by digestion ofcell surface GAGs with heparitinase and chondroitinaseABC and by incubation with sodium chloride to preventGAG sulfation. Furthermore, exogenous GAGs inhibitedthe binding of long isoforms to the cell membrane moreefficiently than that of short isoforms. Additionally, weperformed surface plasmon resonance experiments tostudy the inhibition of PDGF isoforms binding to lowmolecular weight heparin by GAGs. These experimentsshowed that PDGF-AAL and PDGF-BBS isoforms boundto GAGs with the highest affinity. In conclusion, PDGFactivity at the cell surface may depend on the expressionof various cellular GAG species.

Keywords PDGF · LPL · Heparan sulfate · Chondroitinsulfate · Dermatan sulfate · Heparin · CHO

Introduction

Platelet-derived growth factor (PDGF) is a mitogen formesenchymal cells such as fibroblasts and smooth musclecells (SMCs) and has a key role in embryonic develop-ment, angiogenesis, and wound healing. It has also beenimplicated in malignant processes such as autocrinetransformation, human oncogenesis, and atherosclerosis(Heldin and Westermark 1999).

PDGF is formed by homo- or heterodimers of disul-fide-linked A and B polypeptide chains encoded by twodistinct genes. These polypeptides share 60% amino acidsequence identity. Recently, two new members of thePDGF family that form disulphide-linked homodimershave been identified: PDGF-C (Li et al. 2000) and PDGF-D (Bergsten et al. 2001). The PDGF-A chain is present aslong (PDGF-AL) or short (PDGF-AS) isoforms resultingfrom the alternative splicing of exon 6, which encodes thebasic carboxyl-terminal sequence. Thus, PDGF-AL con-tains a basic amino acid carboxy-terminal extension witha high proportion of lysine and arginine (Andersson et al.1994; Lustig et al. 1996). Unlike PDGF-A, the PDGF-Bchain is always synthesized as the long isoform (PDGF-BL), which contains a polybasic sequence similar, but notidentical, to PDGF-AL (Schilling et al. 1998). Theproteolytic processing of PDGF-BL during protein mat-uration gives rise to the short PDGF-B isoform (PDGF-BS), lacking this basic extension (Heldin 1992). Theextensions present in PDGF-AL and PDGF-BL, alsoreferred to as retention motifs, are responsible for theirinteraction with the cell surface and with the surroundingextracellular matrix, whereas short isoforms are moreeasily released and active at some distance from theproducer cell (Pollock and Richardson 1992; Raines andRoss 1992; Andersson et al. 1994).

PDGF signaling requires PDGF binding to two highaffinity tyrosine kinase receptors, a and b. The former

R. Garc�a-Olivas · S. Castel · M. Reina · S. Vilar� ())Department of Cellular Biology,Faculty of Biology, University of Barcelona,Avenida Diagonal 645, 08028 Barcelona, Spaine-mail: svilaro@ub.eduTel.: +34-93-4021550Fax: +34-93-4034607

J. HoebekeUPR9021 of CNRS,Institut de Biologie Mol�culaire et Cellulaire,15 rue R. Descartes, 67084 Strasbourg, France

G. Fager · F. LustigWallenberg Laboratory for Cardiovascular Research,Sahlgren’s University Hospital, 41345 Gothenburg, Sweden

binds to A-, B-, and C-chains, while the latter binds boththe B- and D-chains (Li and Eriksson 2003). Furthermore,PDGF interacts with glycosaminoglycans (GAGs), whichare the side chains of proteoglycans (PGs; Fager et al.1995; Feyzi et al. 1997; Lustig et al. 1999). Given thehigh sulfate and carboxyl group content in their GAGs,PGs are the most negatively charged polymers in livingtissues. They interact with proteins containing clusters ofpositively charged amino acids. The binding of growthfactors like the basic fibroblast growth factor (bFGF) toheparan sulfate proteoglycans (HSPGs) enhances theirbinding to high affinity receptors (Rapraeger et al. 1991;Yayon et al. 1991). In the case of lipolytic enzymes suchas lipoprotein lipase (LPL), this binding promotes thecellular uptake of lipoproteins (Nielsen et al. 1997;Casaroli-Marano et al. 1998). Growth factors may alsointeract with PGs via their core protein moieties. Thus,transforming growth factor (TGF-b) binds to coreproteins of PGs, for example, type-III TGF-b receptorbetaglycan and decorin, a small interstitial extracellularmatrix dermatan sulfate proteoglycan (Hildebrand et al.1994). The binding of bFGF and PDGF-AA to the coreprotein of NG2, a chondroitin sulfate proteoglycan, hasbeen described elsewhere (Goretzki et al. 1999).

The interaction of PDGF with various matrix- and cellsurface-associated GAGs [heparan sulfate (HS), chondro-itin sulfate (CS), dermatan sulfate (DS)] differentiallyregulates its mitogenic function (Fager et al. 1995).Accordingly, while heparin (Lustig et al. 1996) and HSand DS (Fager et al. 1995) inhibit PDGF-inducedproliferation in human SMCs, CS does not. Amongnaturally occurring GAG species, HS shows the highestbinding affinity to the PDGF-AL homodimer (PDGF-AAL) in vitro (Lustig et al. 1999). The strong bindingaffinity of PDGF-AAL to GAGs requires anR(X)4K(X)8T-motif in the basic carboxyl-terminal aminoacid extension. Surface plasmon resonance (SPR) analysishas revealed that the affinity of the binding of PDGF-AASto heparin is 100-fold lower than that of PDGF-AAL(Lustig et al. 1999). The binding affinity of PDGF-BBL toheparin is 10-fold lower than that of PDGF-AAL becausea distinct binding mechanism is involved (Lustig et al.1999). Therefore, the presence of the carboxyl-terminalextension in PDGF and the polybasic sequence in thePDGF-A or -B chains may be crucial for the bindingaffinity to distinct GAGs.

The accumulation and distribution of PDGF isoformsin tissues may thus depend on their differential binding tothe GAGs produced by the cells. The aim of this studywas to evaluate the binding of several PDGF isoforms tovarious GAGs both by SPR analyses and in cell cultures.To this end, we used Chinese hamster ovary (CHO)mutants defective at various sites in glycosaminoglycanbiosynthesis (Esko et al. 1988). Evidence was obtainedthat PDGF isoforms display distinct binding affinities toindividual GAG species.

Materials and methods

Materials

The four recombinant PDGF homodimers (PDGF-AAL, PDGF-BBL, PDGF-AAS, and PDGF-BBS) were constructed, expressed,and produced as described elsewhere (Lustig et al. 1996, 1999).Bovine LPL (bLPL) was purified from milk as reported elsewhere(Bengtsson-Olivecrona and Olivecrona 1991). Nitrocellulose mem-branes were from Schleicher and Schuell (Dassel, Germany). TheECL system was from Amersham (Aylesbury, UK). Ham’s F12growth medium, fetal calf serum, and Dulbecco’s phosphate-buffered saline (DPBS) with Ca2+ and Mg2+ were from Gibco (LifeTechnologies, Paisley, UK). Penicillin G, streptomycin, glutamine,sodium chlorate, HEPES, BSA (fraction V, essentially fatty acid-free), sucrose, glycine, biotinylated concanavalin A, and lowmolecular weight heparin (LMWH; average Mr=3,000) were fromSigma Chemicals (St. Louis, MO, USA). Heparitinase I(EC 4.2.2.8; cat. no. 100704), chondroitinase ABC (EC 4.2.2.4;cat. no. 100330), HS (from bovine kidney, cat. no. 400700), CS(from shark cartilage, cat. no. 400675), and DS (chondroitinsulfate B from pig skin, cat. no. 400660) were from SeikagakuKogyo (Tokyo, Japan). Paraformaldehyde was from Merck (Darm-stadt, Germany). Mowiol mounting medium was from Calbiochem(La Jolla, CA, USA; cat. no. 475904). PDGF recombinanthomodimers were detected with rabbit primary antibodies anti-human PDGF-AA (anti-PDGF-AA) and anti-human PDGF-BB(anti-PDGF-BB) from Genzyme Diagnostics (Cambridge, MA,USA). bLPL was detected with the monoclonal 5D2 antibody fromOncogene (Uniondale, NY, USA). Secondary FITC-conjugatedantibodies and secondary HRP-conjugated swine anti-rabbit anti-body were from Dakopatts (Glostrup, Denmark). StreptavidinTexas red was from Amersham (Arlington Heights, IL, USA).

Slot-blots

Slot-blot assays were performed to determine the specificity of theanti-PDGF-AA and anti-PDGF-BB primary antibodies. Nitrocellu-lose membranes were prewet in deionized water and then in0.02 mM TRIS/HCl buffer containing 1 M NaCl (pH 7.5). Aconcentration of 25 mg/ml of PDGF in 1 M acetic acid was selectedafter initial tests of 12.5–200 mg/ml (not shown). After drying, themembranes were incubated in block solution (BS; 0.02 mM TRIS/HCl, 1 M NaCl, 2% BSA, 0.1% Tween 20) for 30 min at roomtemperature. The blots were further incubated for 1 h with eitherprimary anti-PDGF-AA or anti-PDGF-BB antibody diluted in BS(1:500). The blots were then washed five times in wash solution(BS without BSA) and incubated for 30 min with secondary HRP-conjugated swine anti-rabbit antibody diluted in BS (1:1,000). Afterfive washes in wash solution, the blots were developed with theECL system. The blots were then scanned and bands werequantified with IMAT software (Serveis Cient�fico-T�cnics, Uni-versity of Barcelona).

Cell culture

Wild-type CHO (CHO K1) and mutant CHO 745 cells were kindlyprovided by Dr. J. Esko (Department of Cellular and MolecularMedicine, University of California, San Diego). Mutant CHO 745cells are defective in both HS and CS as a result of alteredxylosyltransferase activity (Esko et al. 1985, 1988). The mutantCHO 677 was from the American Type Culture Collection. It lacksboth N-acetylglucosaminyltransferase and glucuronyltransferaseactivities, which are required for the synthesis of HS and producethree to four times more CS than the wild-type (Esko et al. 1988;Lidholt et al. 1992).

Wild-type and mutant CHO cells were grown in 5% CO2 in airand 100% relative humidity in Ham’s F12 growth mediumsupplemented with 10% (v/v) fetal calf serum, 100 U/ml penicil-lin G, 10 mg/ml streptomycin, and 2 mM glutamine.

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Binding experiments

For binding experiments, wild-type and mutant CHO cells weregrown to 70% confluence on coverslips and used the second dayafter seeding. In some experiments, CHO K1 or CHO 677 cellswere first seeded in the presence of 30 mM sodium chlorate orpretreated with heparitinase I (0.1 U/ml) or chondroitinase ABC(0.01 U/ml) in DPBS with Ca2+ and Mg2+, 0.2% (w/v) BSA for 2 hat 37�C before binding experiments. Cells were prechilled for30 min at 4�C, and incubated for 30 min at 4�C with bLPL or eitherlong or short PDGF-A/-B homodimers in Ham’s F12–20 mMHEPES, pH 7.4, containing 1% (w/v) BSA. bLPL was added to thecells at a protein concentration of 2.5 mg/ml (43.1 nM) and PDGFisoforms were added at 10 mg/ml (270–400 nM) unless otherwiseindicated. In other experiments, cells were incubated with bLPL orPDGF isoforms in the presence of LMWH, HS, CS, or DS atconcentrations ranging from 10 ng/ml to 1 mg/ml. Cells were thenwashed in Ham’s F12–20 mM HEPES and fixed in 3% (v/v)paraformaldehyde–2% (w/v) sucrose in 100 mM phosphate buffer,pH 7.4, for 15 min at room temperature.

Immunofluorescence experiments

Fixed cells were rinsed twice in PBS (10 mM phosphate, 150 mMNaCl, pH 7.4)–20 mM glycine and blocked with PBS–20 mMglycine–1% (w/v) BSA for 10 min at room temperature. To detectthe cells incubated with bLPL/PDGF isoforms, cells were doublelabeled with concanavalin A that recognizes sugar residues at thecell membrane. In particular, concanavalin A has affinity forterminal a-d-mannosyl and a-d-glucosyl residues. For the immun-odetection of bound bLPL, primary monoclonal 5D2 antibody (at10 mg/ml) was used and for bound PDGF isoforms, rabbit primaryantibodies anti-PDGF-AA for both PDGF-AAL and PDGF-AAS,and anti-PDGF-BB for both PDGF-BBL and PDGF-BBS (at 10 mg/ml) were used. The primary antibodies were co-incubated withbiotinylated concanavalin A (at 16.6 mg/ml) for 45 min at 37�C. Tovisualize 5D2, we added FITC-conjugated rabbit anti-mouse (at1:50 dilution) and to visualize rabbit antibodies, FITC-conjugatedswine anti-rabbit (at 1:50 dilution) was used. To detect biotinylatedconcanavalin A, streptavidin Texas red (at 1:150 dilution) wasemployed. Secondary antibodies were incubated for 30 min at 37�Cin the dark. Both primary and secondary antibodies were diluted inblocking solution. After successive washes in PBS, the coverslipswere mounted upside-down on a glass slide with 5 ml Mowiolmounting medium.

Confocal scanning laser microscopy

For the acquisition of digital images at two fluorescence emissionwavelengths, a Leica TCS 4D (Leica Lasertechnik, Heidelberg,Germany) confocal scanning laser microscope was adapted to aninverted Leitz DMIRBE microscope. Images were taken using a63� (numerical aperture 1.4, oil) Leitz Plan-Apochromatic objec-tive. FITC and Texas red were sequentially excited at the 488-nmand 568-nm lines of a 75-mW argon–krypton laser. Image size was512�512. Three-dimensional projection images in the extendedfocus mode (Wilson 1990) were obtained from nine serial opticalsections (z-step=0.75 mm) at the confocal microscope normal scanrate.

Fluorescence quantification and statistical analysis

Fluorescence intensity was quantified by a Metamorph ImageAnalysis System v. 4.0 (Universal Imaging, West Chest, PA, USA)and expressed in arbitrary units, as defined by the mean fluores-cence intensity (MFI). The MFI of the PDGF bound to the cellsurface was obtained by dividing total gray values by the total pixelarea of each cell, which had been deduced from concanavalin Astaining. Fluorescence from four fields (at least 50 cells) wasquantified for each experimental condition. Background fluores-cence values including autofluorescence and non-specific fluores-cence were obtained by imaging cells in the same conditions. Tofacilitate comparison between experiments, MFI values werenormalized to 100% in control conditions. MFI data werestatistically analyzed using SSPS for Windows v. 9 and ANOVA.Post hoc multiple comparison statistical analysis was carried out(C-Dunnet), with significance at P<0.05.

Surface plasmon resonance analyses

All experiments were performed in a BIAcore 2000 (BIAcore,Uppsala, Sweden) SPR equipment at 25�C. Streptavidin wasimmobilized on F1 chips following standard procedures andbiotinylated LMWH was fixed on the streptavidin chip as describedelsewhere (Lustig et al. 1996). The PDGF isoforms at concentra-tions between 75 and 100 nM were preincubated with increasingamounts of the various GAGs before interaction with the immo-bilized LMWH at a flux of 5 ml/min.

The initial velocity (k0) of the interaction with the immobilizedPDGF-AAL at increasing concentrations of GAGs was assessed bya linear fit using the BIAevaluation 3.1 software. Since k0 isdirectly correlated with the active PDGF isoform concentration, thedecrease in this parameter reflects the decrease in available (free)PDGF during the adsorption phase. The percentage of inhibitionwas calculated as (1�k0i/k0)�100, where k0i is the initial velocity inthe presence of inhibitor and k0 the initial velocity in the absence ofinhibitor. The IC50 values were determined graphically by interpo-lation on a plot of the logarithmic concentration of inhibitors inmicrograms per milliliter over the percentage of inhibition.

Results

Specificity of the anti-PDGF-AAand anti-PDGF-BB antibodies

In order to verify that the antibodies anti-PDGF-AA andanti-PDGF-BB qualitatively and specifically detect bothlong and short isoforms, slot-blot assays were performed(Table 1). Slot-blots using 20 mg of the anti-PDGF-AA oranti-PDGF-BB antibodies and 0.6 mg of PDGF isoformswere scanned and bands were quantified. The resultsshow that anti-PDGF-AA recognizes both PDGF-AALand PDGF-AAS isoforms equally well, while anti-PDGF-BB recognizes both PDGF-BBL and PDGF-BBS isoformsin a similar way. Unexpectedly, anti-PDGF-AA also

Table 1 Slot-blots estimatingthe binding of antibodies anti-PDGF-AA and anti-PDGF-BBto different platelet-derivedgrowth factor (PDGF) isoforms.The blots were scanned andquantified. Data are presentedas total intensity values of thebands

Antibody PDGF

AAL AAS BBL BBS

Anti-PDGF-AA 20.3�10E4 18�10E4 14�10E4 3.1�10E4Anti-PDGF-BB 7.6�10E4 5.4�10E4 101.5�10E4 83.6�10E4

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recognizes PDGF-BBL isoform. Similar results wereobtained with other commercial antibodies specific forPDGF-AA isoforms (not shown). However, the binding ofPDGF-BBL on the CHO cell surface was not detectedwith anti-PDGF-AA antibodies (data not shown).

Binding of bLPL and PDGF isoformson the CHO K1 cell surface

CHO cells were used as a model for analyzing the bindingof PDGF isoforms to cell-derived GAGs because theyexpress HS and CS. The binding of PDGF isoforms wascompared to that of bLPL, a well-known HS-bindingprotein. The cell surface distribution of binding sites forbLPL and PDGF isoforms in wild-type CHO K1 cells wasdetermined by incubating the cells for 30 min at 4�C witheither bLPL (2.5 mg/ml) or one of the four recombinantPDGF isoforms (10 mg/ml) (Fig. 1). Immunofluorescencedetection of cell surface-bound molecules was thenperformed with anti-LPL, anti-PDGF-AA for the PDGF-AAL and PDGF-AAS isoforms, and anti-PDGF-BB for thePDGF-BBL and PDGF-BBS isoforms. In order to identifyindividual cells in the quantitative studies, cells were alsostained with concanavalin A which stains the cellmembrane (Fig. 1). The cells presented a fine punctuatestaining pattern consisting of small fluorescent clusters.Both PDGF-BB isoforms showed high binding to thesubstrate in addition to their binding to the cell surface.Since concanavalin A staining defines cell perimeter, thisenables the quantification of fluorescence intensity ofPDGF binding on CHO cell surface. The distribution ofbinding sites for bLPL, characteristic of a classicalheparin-binding protein, was similar to that for PDGFisoforms.

To quantify the cell surface binding of bLPL andPDGF isoforms to CHO K1 cells, binding of bLPL andPDGF isoforms at increasing concentrations (0–10 mg/ml)and subsequent immunodetection were performed. Three-dimensional projection images were obtained from nineserial optical sections at the confocal microscope. The cellsurface-associated fluorescence was measured accordingto the area defined by concanavalin staining and ex-pressed in MFI units (Fig. 2). Linear plots of MFI valuesof the concentrations ranging from 0 to 5 mg/ml of bLPLand PDGF isoforms were obtained for each binding curveand slope values were calculated.

Binding of bLPL to the cell membrane increased in aconcentration-dependent manner and reached saturationat the highest concentrations (5–10 mg/ml). Binding datayielded a high slope value (6.19; r=0.97), pointing to thehigh affinity of bLPL to the cells. The binding of thePDGF-AAL isoform to the cell membrane also increasedin a concentration-dependent manner, with a significantincrease above 1.2 mg/ml. In contrast, the binding of thePDGF-AAS isoform to the cell surface was low up to5 mg/ml and then significantly increased without reachingsaturation in the concentration range tested. The linearregression analysis of these data yielded a slope 15.8-fold

Fig. 1 Cell surface distribution of bovine lipoprotein lipase (bLPL)and platelet-derived growth factor (PDGF) isoforms on wild-typeChinese hamster ovary (CHO) cells. CHO K1 cells were incubatedfor 30 min at 4�C with bLPL (2.5 mg/ml) or PDGF isoforms (10 mg/ml). Coverslips were fixed, rinsed with PBS, and processed forimmunocytochemistry. Double labeling of antibodies and conca-navalin A, which stains cell surface, was performed to detect thecells. Three-dimensional projections of nine horizontal sectionswere obtained by confocal microscopy. The binding of bLPL/PDGF isoforms was detected with specific antibodies followed byFITC-conjugated secondary antibodies and the biotinylated conca-navalin A was detected with streptavidin Texas red. Bar 10 mm

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higher for PDGF-AAL (3.96; r=0.99) than for PDGF-AAS(0.25; r=0.81), suggesting that PDGF-AAL has a higheraffinity for the cells than PDGF-AAS.

Like PDGF-AAL, the PDGF-BBL isoform also boundto the cell surface in a concentration-dependent manner,showing a significant increase above 1.2 mg/ml. However,the binding of the PDGF-BBS isoform did not signifi-cantly differ from that of PDGF-BBL at low concentra-tions and increased above 5 mg/ml without reachingsaturation in the concentration range tested. Binding datashowed a slope 2.4-fold larger for PDGF-BBL (2.97;r=0.97) than for PDGF-BBS (1.21; r=0.74), suggestingthat PDGF-BBL shows higher affinity for the cells than

PDGF-BBS, although these differences were not asmarked as those found for PDGF-AA isoforms.

Altogether, these results indicate that bLPL and bothlong PDGF isoforms bind to the surface of CHO K1 cellsfollowing a concentration-dependent pattern. PDGF-AALbinds to the cells with much higher affinity than PDGF-AAS, while there are only minor differences in the cellbinding affinity between long and short PDGF-BBisoforms.

Identification of GAGs on the CHOcell surface participating in bLPL and PDGF binding

Binding to heparan sulfate

Binding of bLPL and PDGF isoforms to cell surface HSwas assessed by several approaches. First, binding ofthese molecules to mutant CHO clones deficient in GAGbiosynthesis was compared with their binding to the wild-type clone, which presents HS and CS on the cell surface.Two CHO mutants were used: the CHO 745 clone, with adefect in the synthesis of all cellular GAGs, and theCHO 677 clone, which does not synthesize HS butproduces about three times as much CS as wild-typeCHO K1 cells (Fig. 3; summarized results in Table 2).Second, CHO K1 cells were treated with heparitinase I tospecifically remove HS from the cell surface or withsodium chlorate to prevent GAG sulfation. The binding ofbLPL (2.5 mg/ml) and PDGF isoforms (10 mg/ml) inheparitinase I-treated and sodium chlorate-treatedCHO K1 cells was compared with that in untreated cells(Fig. 4; summarized results in Table 2).

Binding of bLPL in CHO 745 and CHO 677 cells wasstrongly reduced as compared to the binding in wild-typeCHO K1 cells (by 84% and 81%, respectively; Fig. 3;Table 2). Similar patterns were obtained after hepariti-nase I and sodium chlorate treatments in CHO K1 cells.Treatment with heparitinase I and sodium chloratereduced bLPL binding by 83% and 87%, respectively,as compared to control cells (Fig. 4; Table 2).

Regarding PDGF-AA isoforms, the binding of PDGF-AAL detected in HS-deficient CHO 677 cells was onlyreduced by 42% as compared to the binding in CHO K1wild-type cells. However, the binding of PDGF-AAL inthe GAG-deficient CHO 745 cells was reduced by 73%.On the other hand, the binding of PDGF-AAS dropped toa similar extent in both mutant clones (by 76% inCHO 677 and by 75% in CHO 745; Fig. 3; Table 2).Similar results were obtained for PDGF-AA isoformsafter heparitinase I and sodium chlorate treatments.Treatment with heparitinase I decreased the binding ofPDGF-AAL by 49%, similar to the reduction in PDGF-AAL binding observed in the HS-deficient clone, whereassodium chlorate led to a 72% reduction, similar to thatobserved in the GAG-deficient clone. In contrast, thebinding of PDGF-AAS decreased by 74% after digestionwith heparitinase I, similar to the reduction in both mutant

Fig. 2 Binding of bLPL and PDGF isoforms on CHO wild-typecells. CHO K1 cells were incubated for 30 min at 4�C at increasingconcentrations of bLPL or PDGF isoforms and then processed forimmunocytochemistry. The fluorescence associated with the cellsurface was quantified by Metamorph Image Analysis System andexpressed as mean fluorescence intensity (MFI) units. Each pointrepresents data from a single coverslip (n=50 cells). Values are themean € SEM from one representative of three independentexperiments performed in duplicate

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clones, and by 87% after sodium chlorate treatment(Fig. 4; Table 2).

Regarding PDGF-BB isoforms, the binding of PDGF-BBL and PDGF-BBS was only partially reduced in bothCHO mutants as compared to the binding in the wild-typeCHO K1 clone (Fig. 3; Table 2). Thus, the binding ofPDGF-BBL in the CHO 677 and CHO 745 clones wasreduced by 24% and by 38%, respectively. The binding of

PDGF-BBS in both CHO mutants also dropped to asimilar extent (by 43% and by 37%, respectively).Heparitinase I and sodium chlorate treatments alsopartially inhibited the binding of both PDGF-BB isoformsin CHO K1 cells. Treatment with heparitinase I dimin-ished PDGF-BBL and PDGF-BBS binding by 37% and45%, respectively, and treatment with sodium chloratedecreased PDGF-BBL and PDGF-BBS binding by 41%and 26%, respectively (Fig. 4; Table 2).

Fig. 3 Binding of bLPL and PDGF isoforms on wild-type (WT) andglycosaminoglycan (GAG)-defective CHO cells. CHO K1 andmutant clones CHO 677 [heparan sulfate (HS)-defective] andCHO 745 (GAG-defective) were incubated for 30 min at 4�C withbLPL (2.5 mg/ml) or PDGF isoforms (10 mg/ml) and processed forimmunocytochemistry. Fluorescence associated with cells wasquantified. Results are presented as percentages (%) of the bindingversus CHO K1 cells. Values are the average of three independentexperiments performed in duplicate. Asterisks Significant differ-ences versus CHO K1 (P<0.05). The statistical significance ofdifferences relative to controls was determined by post hoc multiplecomparison (C-Dunnet) and performed with MFI data

Fig. 4 Binding of bLPL and PDGF isoforms in heparitinase- andsodium chlorate-treated CHO K1 cells. CHO K1 cells were treatedfor 2 h with heparitinase I (0.1 U/ml) to eliminate cell surface HS orseeded in the presence of 30 mM sodium chlorate for 48 h toprevent GAG sulfation. Control cells were run in parallel. The cellswere then incubated for 30 min at 4�C with bLPL (2.5 mg/ml) orPDGF isoforms (10 mg/ml) and processed for immunocytochem-istry. The fluorescence associated with cells was quantified. Resultsare given as percentages of the binding in untreated cells. Valuesare the average of three independent experiments performed induplicate. Asterisks Significant differences versus control (P<0.05).The statistical significance of differences relative to controls wasdetermined by post hoc multiple comparison (C-Dunnet) andprocessed with MFI data

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These results suggest that the binding of bLPL in CHOcells is about 80% dependent on the presence of HS,while HS accounts for approximately 45% of PDGF-AALand for approximately 75% of PDGF-AAS binding to thecells. The binding of PDGF-BBL is approximately 30%and that of PDGF-BBS is approximately 45% dependenton HS. In addition, both PDGF-BB isoforms bind largelyon the CHO 745 cell surface and their binding to cellsurface GAGs in CHO K1 cells only accounts for about50% of their total binding.

Binding to chondroitin sulfate

To evaluate the binding of PDGF isoforms to cell surfaceCS, treatments with chondroitinase ABC and sodiumchlorate were applied in the HS-defective and CS-overexpressing CHO 677 cells. Chondroitinase ABCand sodium chlorate treatments significantly reduced, toa similar extent, the binding of long PDGF isoforms(Fig. 5; summarized results in Table 2). Chondroiti-nase ABC and sodium chlorate decreased PDGF-AALbinding by 56% and 57%, respectively, as compared tothe binding in CHO 677-untreated cells. The binding ofPDGF-BBL dropped quite similarly after chondroiti-nase ABC and sodium chlorate treatments (35% reductionin chondroitinase ABC-treated CHO 677 cells and 46%reduction in sodium chlorate-treated CHO 677 cells).However, the binding of short isoforms in CHO 677 cells,which is comparable to the binding in GAG-deficientCHO 745 cells (Fig. 3), did not drop significantly afterchondroitinase ABC or sodium chlorate treatment.

In summary, PDGF-AA and -BB long isoforms boundto CS while PDGF-AA and -BB short isoforms did not.Likewise, digestion with chondroitinase ABC in CHO K1cells inhibited PDGF-AAL and PDGF-BBL binding by45% and 20%, respectively, whereas the binding ofPDGF-AA and -BB short isoforms was not affected (datanot shown). Taken together, these results indicate that CSaccounts for about 50% of PDGF-AAL binding and forabout 30% of PDGF-BBL binding in CHO cells.

Table 2 Identification of gly-cosaminoglycans (GAGs) onChinese hamster ovary (CHO)cell surface participating in bo-vine lipoprotein lipase (bLPL)and PDGF isoform binding.Summary of the results ofFigs. 3, 4 and 5. Data are givenas percentages (%) of the re-duction of binding compared tocontrol cells. Values presentedare representative of two inde-pendent experiments. (– No in-hibition detected, HS heparansulfate, CS chondroitin sulfate)

bLPL -AAL -AAS -BBL -BBS

Binding to HS; % reduction of binding compared to control CHO K1 cells

CHO 677(HS-deficient) 81 42 76 24 43CHO 745(GAG-deficient) 84 73 75 38 37Heparitinase I 83 49 74 37 45Chlorate 87 72 87 41 26

Binding to CS; % reduction of binding compared to control CHO 677 (HS-defective) cells

Chondroitinase ABC 56 13* 35 –Chlorate 57 11* 46 –

* No significant differences over the control observed

Fig. 5 Binding of PDGF isoforms in chondroitinase- and sodiumchlorate-treated CHO 677 cells. CHO 677 cells were seeded for48 h in the presence of 30 mM sodium chlorate to prevent GAGsulfation or treated for 2 h with chondroitinase ABC (0.01 U/ml) toeliminate cell surface chondroitin sulfate. Control cells were run inparallel. The cells were then incubated for 30 min at 4�C withPDGF isoforms (10 mg/ml) and processed for immunocytochem-istry. The fluorescence associated with cells was quantified. Dataare given as percentages of the binding in untreated cells. Valuesare the average of three independent experiments performed induplicate. Asterisks Significant differences versus control (P<0.05).The statistical significance of differences relative to controls wasdetermined by post hoc multiple comparison (C-Dunnet) andprocessed with MFI data

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Effect of exogenous GAGs on the binding of bLPLand PDGF isoforms

The inhibition of bLPL and PDGF isoform binding toCHO K1 cell surface by exogenous GAGs was evaluatedby fluorescence quantification (Tables 3, 4, 5). In theseexperiments, bLPL (2.5 mg/ml) and PDGF isoforms(10 mg/ml) were mixed with increasing concentrations(10 ng/ml to 1 mg/ml) of exogenous GAGs (LMWH, HS,CS, or DS) and added to the cells. After 30 minincubation at 4�C, cells were processed for immunocy-tochemistry. The binding of bLPL was decreased byLMWH in a dose-dependent manner (by 82% at 1 mg/ml). DS halved the binding of bLPL at 10 mg/ml andreduced it by 94% at 1 mg/ml. HS diminished bLPLbinding by 91% at 1 mg/ml but CS did not affect bLPLbinding at all. Thus, bLPL binding on the cell surface wasmore efficiently reduced by LMWH, DS, and to a lesserextent by HS.

The binding of PDGF-AAL on the CHO K1 cellsurface was inhibited at low concentrations of LMWH,CS, and HS. LMWH decreased PDGF-AAL binding by90% at 1 mg/ml. HS and CS partially inhibited PDGF-AAL binding at 1 mg/ml and DS was also an effectiveinhibitor of PDGF-AAL binding, although at higher

concentrations (73% at 1 mg/ml). On the other hand,the binding of PDGF-AAS was not affected by LMWH,HS, or CS. DS was the only GAG that displaced thebinding of PDGF-AAS (52% at 1 mg/ml).

As for PDGF-BB isoforms, the binding of PDGF-BBLon CHO K1 cell surface was largely diminished byLMWH and by DS (by 98% and 94% at 1 mg/ml,respectively). CS and HS partially reduced the binding ofPDGF-BBL. However, the binding of PDGF-BBS wasslightly decreased by LMWH and by DS (by approxi-mately 30% at 1 mg/ml). Neither CS nor HS affectedPDGF-BBS binding. Accordingly, these data indicate thatthe binding of long PDGF isoforms was displaced furtherfrom the cell membrane by GAGs than that of shortPDGF isoforms.

The inhibition of PDGF isoform binding to LMWH inthe presence of exogenous GAGs was estimated by SPRanalysis (Table 6). PDGF isoforms were preincubatedwith increasing concentrations of GAGs (LMWH, HS,CS, or DS) before interaction with LMWH and IC50values (mg/ml) were calculated (Table 6). The IC50 values(mg/ml) for the inhibition of PDGF-AAL binding toLMWH were very low, pointing to its close affinity forGAGs, especially HS and DS. However, PDGF-AASbinding to LMWH was inhibited by LMWH and DS

Table 6 GAG inhibition of PDGF interaction with LMWH bysurface plasmon resonance analyses. PDGF isoforms at concentra-tions between 75 and 100 nM were preincubated at increasingamounts of LMWH, DS, HS, and CS before interaction withimmobilized LMWH. Values presented are representative of twoindependent experiments. IC50 values were calculated by interpo-lation on a plot of the logarithmic concentration of inhibitors inmicrograms per milliliter over the percentage of inhibition. (– Noinhibition detected)

LMWH HS CS DS

-AAL 0.394 0.0783 0.116 0.0906-AAS 1.07 – – 4.74-BBL 2.86 – >100 42.5-BBS 2.09 3.16 0.0164 0.109

Table 5 Effect of exogenous GAGs on the cell surface binding of PDGF-BB isoforms. Details as for Table 3

GAG LMWH HS CS DS

PDGF-BBL PDGF-BBS PDGF-BBL PDGF-BBS PDGF-BBL PDGF-BBS PDGF-BBL PDGF-BBS

10 ng/ml 6 – 10 – 8 5 – 471 mg/ml 20 31 22 – 51 – 12 291 mg/ml 98 31 37 – 51 – 94 –

Table 3 Effect of exogenous GAGs on the cell surface binding ofbLPL. CHO K1 cells were incubated with bLPL or PDGF isoformsfor 30 min at 4�C at the indicated concentrations of low molecularweight heparin (LMWH), dermatan sulfate (DS), HS, and CS andprocessed for immunocytochemistry. Fluorescence associated withcells was quantified. Data are given as percentages (%) of reductionof binding compared to the binding in control cells without GAGs.Values are representative of two independent experiments. (– Noinhibition detected)

GAG bLPL

LMWH HS CS DS

100 ng/ml 40 – – –10 mg/ml 76 – – 451 mg/ml 82 91 – 94

Table 4 Effect of exogenous GAGs on the cell surface binding of PDGF-AA isoforms. Details as for Table 3

GAG LMWH HS CS DS

PDGF-AAL PDGF-AAS PDGF-AAL PDGF-AAS PDGF-AAL PDGF-AAS PDGF-AAL PDGF-AAS

10 ng/ml 42 – 25 – 27 11 – –1 mg/ml 75 – 45 – 60 – 53 311 mg/ml 90 – – – – – 73 52

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only at higher concentrations, indicating that it binds toDS and LMWH with less affinity than PDGF-AAL.Neither HS nor CS inhibited the binding of PDGF-AAS toLMWH. Therefore, PDGF-AAL binds to GAGs withhigher affinity than PDGF-AAS.

The IC50 values for the inhibition of PDGF-BB bindingto LMWH (Table 6) differ from those obtained for PDGF-AA isoforms and GAG inhibition of PDGF-BB binding towild-type CHO K1 cells. PDGF-BBL showed high IC50values, suggesting it was hardly affected by GAGs. It hadpoor affinity for DS and CS and did not bind to HS. Onthe other hand, PDGF-BBS showed high affinity for CSand DS and poor affinity for HS. LMWH inhibited thebinding of both PDGF-BB isoforms to a similar extent.Overall, SPR data indicate that PDGF-BBS is a morespecific ligand of GAGs than PDGF-BBL.

Discussion

Cell surface GAGs play a key role in growth factorsignaling, since they behave as low affinity receptors thatmodulate the binding to high-affinity tyrosine kinasereceptors. Furthermore, growth factors and cytokines canbe stored in the tissue through binding to GAGs. Here, wecompared the binding of PDGF isoforms to cell surfaceGAGs with the binding of bLPL, which is a well-knownligand for cell surface HSPGs (Sehayek et al. 1995;Lookene et al. 1996). To this end, we used the wild-typeand mutant CHO clones deficient in the synthesis ofGAGs to distinguish differential binding of PDGFisoforms. Similarly, CHO cell mutants have been usedto study low-affinity binding sites for bFGF (Yayon et al.1991). CHO K1 cells are a suitable model because theglycosaminoglycan pool in wild-type cells is composed of40–70% HS and 30–55% chondroitin 4-sulfate (Esko etal. 1985, 1987). CHO cells transfected with PDGFisoforms have been used to study PDGF interaction withmatrix- and cell surface-associated GAGs (Kelly et al.1993). In another set of experiments, we compared theGAG inhibition of binding of PDGF isoforms to LMWHby SPR analyses. Our data demonstrate that PDGFisoforms display a variety of binding efficiencies todistinct GAG species.

Both PDGF-AA isoforms bind to the cell surface ofCHO K1 cells following a pattern similar to that obtainedwith bLPL. Most of the cell surface binding of bLPL andPDGF-AA isoforms was inhibited by sodium chlorate,indicating that sulfated GAGs are the main cell surfacereceptors on CHO K1 cells. It is well established thatbLPL binds to the CHO cell surface through PGs(Berryman and Bensadoun 1995; Nielsen et al. 1997).The similarity between the immunofluorescence stainingpattern of bLPL and PDGF-AA suggests that sulfated PGsare cell surface receptors for both PDGF-AA isoforms.Furthermore, cell surface binding experiments indicatethat PDGF-AAL binds with higher affinity than PDGF-AAS. This correlates with experiments in vitro, whichhave revealed that PDGF-AAL binds to cell-derived

GAGs and to LMWH with higher affinity than PDGF-AAS. The high affinity of PDGF-AAL for cell surfaceGAGs is due to the carboxyl-terminal extension region ofPDGF-AAL, which is absent in the PDGF-AAS isoform(Lustig et al. 1999). The differential affinity of PDGF-AAisoforms may have relevant physiological consequences,since PDGF-AAL may have mostly an autocrine action,while PDGF-AAS may act on non-producer cells (Khachi-gian et al. 1992; Raines and Ross 1992). Transfectionstudies in COS and 3T3 cells have shown that secretedPDGF-AAL remains associated with the cell surface ofexpressing cells (LaRochelle et al. 1991) or at the cellboundaries in CHO cells (Kelly et al. 1993), while PDGF-AAS is mostly released to the medium (stman et al.1991; Pollock and Richardson 1992; Kelly et al. 1993).Although PDGF-AAL is produced by most PDGF-AA-expressing cells, this only accounts for about 10% of thetotal PDGF-AA mRNA (Matoskova et al. 1989; Young etal. 1990). However, the differentiation of macrophagesincreases the production of PDGF-AAL mRNA from 10%to 40% and endothelial cells also synthesize 40% of totalPDGF-AA mRNA as the long isoform (Krettek et al.1997). The upregulation of PDGF-AAL expression hasalso been described in the pathogenesis of cardiacallograft vasculopathy (Zhao et al. 1995) and in activelyhealing human wounds treated with recombinant PDGF-BB (Pierce et al. 1995). Therefore, although PDGF-AALis only a minor PDGF isoform in certain cells with aspecific phenotypic state, it may be easily retained on thecell surface. PDGF-AAS, despite its lower affinity for cellsurface GAGs, may bind to the cell surface GAGs if localconcentrations are high enough.

The binding to GAG species varies between long andshort PDGF-AA isoforms. The carboxyl-terminal exten-sion of PDGF-AAL binds to HS because PDGF-AAL canbe partially released from the cell surface and the matrixby heparitinase digestion (Andersson et al. 1994) and withexogenously added heparin (Kelly et al. 1993). Treatmentwith chondroitinase releases some PDGF-AAL from theextracellular matrix (Kelly et al. 1993), suggesting thatPDGF-AAL also binds to CS. Digestion with glycosidasesand studies with mutant clones confirm that both HS andCS contribute similarly to cell surface binding of PDGF-AAL and that PDGF-AAS binds mainly to HS. GAGcompetition for the binding to LMWH and to the cellsurface indicates that the binding of PDGF-AAL is moreeffectively displaced by all GAG species than that ofPDGF-AAS. Previous SPR results have shown that HS isthe GAG with the highest affinity for PDGF-AAL (Lustiget al. 1996). Here we report that DS and CS are alsoeffective inhibitors of the PDGF-AAL binding to LMWHand to the cell surface. Therefore, PDGF-AAL may bindto both cell surface HS and CS with similar affinitydepending on the GAG composition, whereas PDGF-AASbinds mainly to cell surface HS with low affinity in CHOcells. These data point to the presence of other heparin orHS binding sites with lower affinity than the carboxyl-terminal extension.

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The binding pattern of both PDGF-BB isoforms differsfrom that of PDGF-AA isoforms. The cell surface bindingpattern of PDGF-BB isoforms is reminiscent of that ofbLPL and of PDGF-AA isoforms. However, PDGF-BBisoforms also bind to the substrate to a high extent, whilePDGF-AA isoforms bind almost exclusively to the cellsurface. PDGF-BB isoform binding to the substrate maybe associated with the extracellular matrix, since inhibi-tion studies of PDGF-BB binding with high concentra-tions of GAGs reduced the fluorescence associated withthe substrate. Moreover, both PDGF-BB isoforms bind tothe surface of the glycosaminoglycan-deficient CHO 745clone. Sodium chlorate partially inhibits the binding ofPDGF-BB isoforms in CHO K1 cells, whereas it almostabolishes that of PDGF-AA isoforms. Sodium chlorate-treated fibroblasts can be mitogenically stimulated byPDGF-BB but not by bFGF, suggesting that sulfation ofGAGs is not essential to PDGF-BB activity (Rapraeger etal. 1991). Altogether, these results suggest that PDGF-BBisoforms bind to GAGs and to cell surface proteins inCHO cells.

Our results indicate that PDGF-BBL binds to the cellsurface of wild-type CHO K1 cells with higher affinitythan PDGF-BBS, but with lower affinity than PDGF-AAL.Previous biochemical SPR data have shown that PDGF-BBL binds to LMWH with fourfold higher affinity thanPDGF-BBS (Lustig et al. 1999). The binding to GAGspecies also differs between long and short PDGF-BBisoforms. Like PDGF-AA isoforms, PDGF-BBL binds toboth HS and CS, as confirmed by digestion withglycosidases and experiments with mutant clones, where-as PDGF-BBS binds to HS in CHO cells. Moreover,GAGs displace PDGF-BBL binding to the cell surfacemore easily than PDGF-BBS. In contrast, PDGF-BBSbinding to LMWH is more efficiently inhibited by GAGsthan PDGF-BBL binding as shown by SPR experiments.The binding of PDGF-BB isoforms to cells is largely dueto interactions with non-GAG proteins as indicated by thesignificant binding of PDGF-BB isoforms on theCHO 745 GAG-deficient cells. Indeed, the existence ofnon-GAG binding sites on cells could have a biologicalfunction which is not revealed in SPR experiments.Inhibition by GAGs may thus reflect non-specific elec-trostatic interactions that hinder the binding of PDGFisoforms to negatively charged cell membrane proteins.

Inhibition experiments of PDGF binding to CHO K1cell surface by exogenous GAGs revealed that someGAGs show dose dependence at low concentrations butno inhibition at higher dose. This can be explained by themonovalent binding of the PDGFs in an excess of GAGsinto solution. The second binding site is thus free forinteraction with the cell GAGs. A similar phenomenon iswell known in immunology where immunoprecipitationhas a bell-like form as a function of antigen concentration.At low antigen concentration, bivalent interaction withthe antibody is possible, which allows the formation ofinsoluble immune complexes. At high concentrations ofantigen, the antibodies bind monovalently thus preventingthe formation of an insoluble immune complex.

The physiological relevance of PDGF binding toGAGs suggests that this interaction facilitates PDGFstorage in tissues, which ensures tissue homeostasis andprevents losses by diffusion or inactivation. While PDGFis expressed at low levels in arteries from healthy adults,increased levels of both PDGF-A and -B chain mRNAand protein have been detected in a variety of humanatherosclerotic tissues (Ross 1993; Rekhter and Gordon1994). In addition, both PDGF-A and -B chain mRNAs(Ueda et al. 1996) and protein (Caplice et al. 1997) areexpressed in human restenotic tissues after angioplasty.

Several studies evidence the contribution of PGs to thedevelopment of arterial disease. Thus, HSPGs areactivated in injured rat arteries and may be involved inintimal thickening (Nikkari et al. 1994; Cizmeci-Smith etal. 1997). The accumulation of major extracellular PGs isimplicated in the pathogenesis of atherosclerosis due tothe retention of cholesterol-rich lipoproteins (Williams2001) and the regulation of PDGF and TGF-b distribu-tion. Thus, specific PG deposition that parallels PDGF-and/or TGF-b1-expressing cells is observed duringdevelopment of atherosclerosis and is consistent withspecific in vitro activities of PDGF and TGF-b1 in SMCs(Evanko et al. 1998). Importantly, the addition of HSmimetics or heparin/HS fragments, which presumablybind to growth factors, are described to prevent vascularproliferation (Castellot et al. 1986; Schmidt et al. 1992;Benezra et al. 1994). Similarly, HSPGs isolated from therat arterial wall suppresses the expansion of the neointimawhen introduced into injured arteries (Bingley et al.1997). The enzymatic removal of binding sites on HS canalso have the same effect in vivo (Silver et al. 1998). Ourstudies also suggest that DS binds to PDGF isoforms.Moreover, bLPL which binds HS, is more displaced fromthe cell surface by DS than by HS. DS is also described tohave high affinity for other molecules previously thoughtto interact only with HS, such as FGF-2 (Bashkin et al.1989) and hepatocyte growth factor/scatter factor (Lyonet al. 1998). SMC-derived DS is described to bind PDGFand low-density lipoproteins and to inhibit cellular DNAsynthesis in SMCs (Fager et al. 1995).

In summary, our data indicate that long PDGFisoforms bind to both HS and CS, whereas short PDGFisoforms bind to HS in CHO cells. SPR analyses showthat PDGF-AAL and PDGF-BBS isoforms bind to GAGswith higher affinity. This study may provide evidence thatPDGF activity at the cell membrane may depend on theGAG species expressed by the cells and on the presenceof long or short PDGF isoforms. Further studies arerequired to investigate how the binding to these low-affinity sites affects PDGF function at the cell surface.

Acknowledgements The authors are grateful to Dr. ChristineBerndt for helpful comments in revising the manuscript and toRobin Rycroft for his expert editorial help and critical reading ofthe manuscript. This study was supported by the BIOMED programof the European Community (grant BMH4-CT98–3289).

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