Polymer International Polym Int 48 :313–319 (1999)

Sulphated polysaccharides derived fromdextran: biomaterials for vascular therapyFre� de� ric Chaubet,* Re� mi Huynh, Jacqueline Champion, JacquelineJozefonvicz and Didier LetourneurLaboratoire de Recherches s ur les CNRS UMR 7540, Ins titut Paris 13 , avenue JB 93430Macromole� cules , Galile� e,Univers ite� Cle� ment ,Villetaneus e , France

Abstract : CMDBS are synthetic dextran derivatives randomly substituted with carboxymethyl (CM),

benzylamide (B), sulphonate and sulphate groups (S). Depending on their overall composition, these

compounds are endowed with heparin-like properties such as anticoagulant activity. Indeed, some

CMDBS with high CM and S contents delay blood coagulation, whilst some derivatized dextrans

without signiücant anticoagulant capacity are potent antiproliferative agents for rat smooth muscle

cells (SMCs) in vitro as well as heparin. The growth inhibition is dose dependent, reversible and non-

toxic. This result is of prime interest for medical use because proliferation of vascular SMCs is postu-

lated to be a key step in the pathogenesis of atherosclerosis or restenosis after vascular surgery such as

angioplasty. By varying the overall composition in the diþ erent substituents, we have also prepared

CMDBS exhibiting a stimulatory eþ ect on the in vitro growth of human endothelial cells (EC).

Heparin, under similar experimental conditions, slightly inhibited EC growth. The data indicate a

synergistic role of all substituents grafted onto the dextran backbone without considering that any can

be responsible alone for this eþ ect. We conclude that a suitable distribution of CM, B and S groups on

dextran can mimic heparin activity in terms of anticoagulant activity and antiproliferative capacity

on SMC growth. Moreover, some CMDBS are also endowed with a stimulatory eþ ect on EC growth.

These properties confer great interest to these synthetic polysaccharides for vascular therapy.

1999 Society of Chemical Industry(

Keywords: vascular therapy ; anticoagulant activity ; smooth muscle cell growth inhibition; endothelial cellgrowth stimulation; derivatized dextrans ; heparin

INTRODUCTION

Over the last twenty years, heparin as an anticoagu-lant agent has stimulated great interest in the prep-aration of sulphated forms of polysaccharidesexhibiting heparin-like properties. Widely studiedexamples are glycosaminoglycans,1 natural or semi-synthetic bioactive polysaccharides such as pentosanpolysulphate,2 derivatized dextrans3,4 and fucansextracted from seaweeds.5 In our laboratory, we havedeveloped anticoagulant heparin-like polymers thatcould be synthesized by random substitution onselected substrates,6 ie soluble or crosslinkeddextran,3,7h11 and crosslinked polystyrene.12h14These polymers are able to interact with biologicalmacromolecules through selected binding sites. Con-cerning anticoagulant activity, the speciücity of thesesites depends upon short saccharide sequences alongthe polymer backbone.15 These sequences mimicactive sites such as the heparin pentasaccharideresponsible for the anticoagulant activity of thenatural macromolecule.16

Among the polysaccharides studied for theirheparin-like properties, a family of derivatized dex-trans called CMDBS delay the coagulation ofplasma. They catalyse the inactivation of thrombin, aprocoagulant enzyme, by natural inhibitors such asantithrombin (AT) or heparin cofactor II(HCII)3,17,18 and by direct inhibition of thrombin.They are also endowed with anticomplementaryactivity in vitro19 and in vivo,20 and can modulate theproliferation of vascular cells.21h23

The CMDBS synthesis involves three sequentialreactions : (i) random carboxymethylation (CM) ofhydroxyl groups on D-glucose units leading to carbo-xymethyl dextran (CMD); (ii) derivatization of someof the carboxylic groups as benzylamides (B) givingCMDB polymers ; (iii) partial sulphonation of phenylrings and sulphation of free hydroxyls (S).4,24 Theanticoagulant activity of CMDBS is mainly depen-dent on the CM and S contents, on the relative pro-portions of these groups and on the molecular weightof the polymer.3,4,17,24 Interestingly, neither CMD

* Corres pondence to Laboratoire deFre� de� ric Chaubet,

Recherches s ur les Macromole� cules , CNRS UMR 7540, Ins titutGalile� e, Univers ite� Paris 13, avenue JB Cle� ment, 93430 Villeta-neus e, France.

Contract/grant s pons or : Centre National de la Recherche Scienti-fique.

(Received 27 May 1998; revis ed vers ion received 7 September

1998; accepted 14 December 1998)

( 1999 Society of Chemical Industry. Polym Int 0959-8103/99/$17.50 313

F Chaubet et al

nor CMDB exhibit anticoagulant activity. Thus, it isclearly established that the anticoagulant activity ofCMDBS is closely dependent on the distribution ofthe substituents along the polymer chain.3,6 In addi-tion, the structure of CMD precursor strongly inýu-ences the distribution of B and S groups. In thisrespect, the correlations between individual CMDBSstructures and their coresponding anticoagulantactivities have been partially clariüed by studying thedistribution of CM groups, using UV spectroscopy,1H (1D and 2D) and 13C NMR spectroscopy andMonte Carlo simulations.15,25

The cellular and molecular mechanisms regulatingendothelial cell (EC) and smooth muscle cell (SMC)growth are essential for maintaining blood vesselintegrity and tissue repair during wound healing.During the past two decades much work has beenfocused on the eþects of heparin and heparan sul-phates.26 These closely related sulphated glycosa-minoglycans modulate vascular cell proliferation invitro and in vivo.27 Furthermore, heparin-like sub-stances inhibiting aortic SMCs28 were identiüed inconditioned media from bovine endothelial cells.Antiproliferative species of heparin sulphates are alsoproduced by SMCs.29 Structure–function studiessuggest that heparin antiproliferative activity is cor-related to oligosaccharide size and charge.30 More-over, the anticoagulant and antiproliferativeproperties of heparin are clearly separate.30 Somechemically modiüed heparin molecules or analogues,devoid of anticoagulant activity that retain anti-proliferative activity on SMC, will be useful in theclinical management of SMC hyperplasia aftervascular surgery.31 In contrast, stimulation of ECgrowth oþers clinical applications for wound healingand endothelialization of human vascular protheses.

In this paper, we present examples of derivatizeddextrans endowed with biological activities at threediþerent levels, and intended for vascular therapy(anticoagulant activity), growth stimulation of endo-thelial cells, and inhibition of smooth muscle cellproliferation.

MATERIALS AND METHODS

Dextran T40 (batch 32202; M1 W \ 43900g mol~1 ;from Leuconostoc mesenteroidesM1 n \ 26200g mol~1)

was purchased from Pharmacia. All reagents werepurchased in analytical grade from Fluka (StQuentin Fallavier, France), Merck (Nogent surMarne, France), Sigma (St Quentin Fallavier,France) and Carlo Erba (Nanterre, France). Dicho-romethane was distilled over just(CH2Cl2) P2O5before use. Hog intestine heparin (H410) with spe-ciüc anticoagulant activity of 170IU mg~1 wasobtained from Sanoü Recherche (Gentilly, France).Elemental analyses of nitrogen and sulphur wereperformed by the Service de Microanalyse duCNRS, Gif sur Yvette, France.

Preparation of derivatized dextrans

The CMDB and both CMDBS JCk and JC29 wereprepared as previously described.4,24 In brief, carbo-xymethylations of starting dextran were performedwith monochloroacetic acid in alkaline medium at60¡C for 1h. Then CMDB were obtained quantitat-ively by coupling benzylamine to carboxylic groupsof CMD using N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoleine as coupling agent in water :ethanol (1 : 1v/v) mixture at room temperature.Finally, sulphonation of the phenyl ring and sul-phation of free OH of the CMDB were carried out inone step by the action of chlorosulphonic acid on theCMDB precursor in dried dichloromethane.

Synthesis of CMDSu

CMDSu was prepared by carboxymethylation ofdextran followed by sulphation with SO3–pyridinecomplex in dry DMF. 2.2g of a carboxymethylateddextran with a degree of substitution (ds) in CMgroups of 1.0 was dissolved in 100ml of distilledwater. The solution was eluted through an amberliteIR 120 H` column (50ml) and the eluate was neu-tralized with tributylamine (10% in ethanol). Afterlyophilization, the salt was dissolved in 50ml of dryDMF. A solution of 0.06mol of SO3–pyridinecomplex in 6 ml of dry DMF was slowly addedunder stirring. After 2h under argon, the reactionwas stopped by adding 70ml of distilled water at 4¡Cand the mixture was neutralized with 2M NaOH.Then the solution was puriüed with an ultraültrationdevice (Filtron) (membrane cut-oþ 10000g mol~1)using successively 2 l of carbonate buþer 0.05 M (pH9.6) and 2] 2 l of distilled water. After concentra-tion and lyophilization, 2.17g of puriüed CMDSuwas obtained.

Reduction of carboxylic acid of CMDSu

300mg of CMDSu was dissolved in 30ml of 0.2MMES buþer, pH 4.75. Then 4.54g of CMC ((N-cyclohexyl-N@(2-morpholinoethyl) carbodiimide)methyl p-toluenesulphonate) was added in small por-tions ([CMS]/[COOH]\ 10). The solution waskept at room temperature for 1h under stirring, thepH being maintained at 4.75 using 1M HCl. Thenthe pH was increased to 7 with 2M Tris buþer and9g of solid were added. After 2h at 30¡C,NaBH4the solution was ultraültered as described above(membrane cut-oþ 5000g mol~1) and the reducedproduct was obtained quantitatively in a yield ofreduction of 92%.

Characterization of the samples

The CM contents were determined using acidimetrictitration in a water : acetone mixture (1 : 1v/v). Theamounts of B- and S-containing groups were estab-lished using elemental analysis of nitrogen andsulphur, respectively. The chromatographic molecu-lar weight of functionalized dextrans (except(Msec)CMDSu and the reduced derivative) and heparinwere determined by high performance steric exclu-

314 Polym Int 48 :313–319 (1999)

Sulphated polysaccharides derived from dextran

sion chromatography (SEC) in 0.15M sodium chlo-ride buþered at pH 7 with 0.05M using aNaH2PO4Lichrospher Si3000 Diol column Merck (Nogent surMarne, France) connected with a HEMA Sec Bio40column (Altech). The relative SEC molecularweights of the CMDSu derivatives were determinedusing a Zorbax GF450 column and the same eluentas above. The columns were calibrated with pullulanstandards (Polymer Laboratories Ltd). For nativedextran a of 35000g mol~1 was obtained, inMsecagreement with the and values reportedM1 w M1 nabove. The of H410 heparin obtained in theMsecsame conditions was 20000g mol~1.

Coagulation assays

All assays were performed using normal humanplatelet-poor pooled plasma. The anticoagulantactivity of each sample was obtained by measuringthe activated partial thromboplastin time (APTT)according to the manufacturer’s speciücations(Stago, France). The anticoagulant activity of eachdextran derivative was calculated (IU mg~1) withrespect to heparin standard (170IU mg~1).4

Cell culture

Eagle’s minimal essential medium (MEM) and Dul-belcco’s modiüed Eagle medium (DMEM) were pur-chased from Gibco BRL (Cergy Pontoise, France).Penicillin, streptomycin, L-glutamine and trypsin–EDTA were from Flow Laboratories (Les Ulis,France), collagenase from Seromed (Berlin,Germany). Foetal calf serum (FCS) was obtainedfrom Eurobio (Les Ulis, France). Twenty four-welltissue culture dishes from Costar were used.

Endothelial cells

Human endothelial cells, EA.hy 926 line32 werekindly provided by Dr C-J Edgell (University ofNorth Carolina, USA). The permanent cell linemaintains the characteristics of the parent humanumbilical vein endothelial cells, as the übrinolyticproperties and expression of factor VIII-relatedantigen.33,34 They were routinely cultured inDMEM supplemented with 10% FCS, 2mM L-glutamine and HAT (100lM hypoxanthine, 0.4lMaminopterin, 16lM thymidine) at 37¡C in a 5% CO2incubator. The typical cobblestone morphology ofcells at conýuence was observed by conventionallight microscopy (Zeiss (Switzerland), Axiovert 100).The absence of mycoplasma contamination waschecked using the ýuorescent Hoeschst 33258 dye.

Smooth muscle cells

Thoracic aorta smooth muscle cells from maleSprague–Dawley rats (strain OFA ; Iþa-Credo,L’arbresle, France) were isolated as previouslydescribed.22 Cells were cultured at 37¡C in a humidi-üed 5% atmosphere. Cells between the secondCO2and the fourth passage were used in this work. Con-ýuent cells appeared to present the characteristic

‘hills and valleys’ pattern. Indirect immunoýuores-cence revealed speciüc alpha-actin ülaments.

Growth studies with ECs

Endothelial cells were plated into 16mm multiwellplates in DMEM containing 10% FCS. After 24h,they were growth arrested by placing them inmedium with 0.1% serum for 48h. The control ECswere exposed to medium with 2% or 10% FCS.Treated cells were exposed to DMEM with 2% FCScontaining heparin-like solutions. Cell numbers weremeasured in quadruplicate samples and counted witha Coulter counter (Eaubonne, France).

Growth inhibition studies with SMCs

1.2] 104 SMC were plated into 16mm multiwellplates in MEM containing 10% FCS. After 24h,they were growth arrested by placing them inmedium with 0.1% FCS for 48h. The control cellswere exposed to medium with 10% FCS. Treatedcells were exposed to MEM with 10% FCS contain-ing heparin-like solutions. Cell numbers were mea-sured in quadruplicate samples and counted with aCoulter counter. The degree of inhibition was deter-mined after 5 days as described by Castellot et al28according to the relationship

I(%)\A

1[ net growth with polysaccharide

net growth in controls

B] 100

RESULTS

Synthesis and characterization of functionalized

dextrans

Various dextran derivatives bearing CM, B and Sgroups have been synthesized. The schematic struc-ture of these compounds is given in Fig. 1. Data forthe chemical characterizations of the samples are pre-sented in Table 1. Three successive reactions werenecessary to obtain CMD with a degree of substitut-ion (ds) ranging from 0.7 to 1.1 (data not shown).Then CMDB samples with ds(B) ranging from 0.3 to0.6 were obtained quantitatively (w/w) after three Bcouplings. Finally, one sulphonation reaction led toCMDBS. Only the precursors of CMDBS JCk aregiven in Table 1. The CMDSu sample devoid of Band S groups was prepared quantitatively in one stepfrom CMD by action of the complexSO3–pyridinewhich speciücally sulphates the free hydroxyls.

Coagulation

The speciüc anticoagulant activities of all com-pounds are listed in Table 1. Both CMDBS andCMDSu exhibited signiücant anticoagulant activity,although it was fairly low compared with the activityof heparin. However, the CMDSu sample lost mostof its activity after reduction of the CM groups tohydroxyethyl groups.

The diþerences observed between the anticoagu-lant activities of JCk (lower) and JC29 (higher) may

Polym Int 48 :313–319 (1999) 315

F Chaubet et al

Figure 1. Schematic s tructure of the derivatized dextrans .

Hydroxyl units of a-D-glucos e are either uns ubs tituted orrandomly s ubs tituted with carboxymethyl, carboxymethyl

benzylamide, carboxymethyl benzylamide s ulphonate and

s ulphate groups .

be explained in terms of their respective S contents.By comparison, the higher JC29 activity comparedwith that of CMDSu is attributable to steric actionof the B units, CM and S contents and beingMsecsimilar.4

Derivatized dextrans stimulate in vitro EC growth

Two CMDBS termed JCk and JC29, which exhibitsigniücant anticoagulant activity compared with thatof native dextran (Table 1) were assessed. We addedthe polysaccharides to quiescent EC in 2% serum.Control cells which did not receive polysaccharideswere grown in medium containing 2% serum. Themitogenic activity was estimated by cell counting.

The dose-response data collected after 5 days ofexposure are shown in Fig. 2. A maximum was

Figure 2. Dos e-res pons e of polys accharides on EC growth.

EAhy.926 cells s upplemented with 10% FCS were s eeded on24-well culture plates . Then, the medium was changed and

s upplemented with 2% FCS. Control cells were grown in mediumwith 2% FCS without polys accharide. Derivatized dextrans JC29

JCk and CMDB were added at 0.1, 1, 10 or 100lgml(…), (=) (>)in medium with 2% FCS. The cell number was determined with aCoulter counter after 5 days . Mean values ^SD (n\ 4) of growths imulation are indicated for a repres entative experiment.

observed for the mitogenic activities of derivatizeddextran-treated cultures at about 1lg ml~1. Thelevel of stimulation was more evident with JCk thanwith JC29. Under these conditions, a slight inhibi-tory eþect with heparin is observed,21,35,36 whereasnative dextran, dextran sulphate and carboxymethyldextran display no eþect.21 However, carboxymethyldextran substituted with benzylamine (CMDB) alsoexhibits a stimulatory eþect on EC growth (Fig 2).

Even if the precise structure–function relationshipseems complex, we can draw two main conclusionswith the help of previous data:21 (i) B and S groupson the polymer interact with EC, leading to prolifer-ative eþects ; (ii) the mitogenic eþect is not related tothe anticoagulant property of polysaccharides. Forexample, non-anticoagulant CMDB presents a pro-

Table 1. Chemical characterizations and anticoagulant activity of derivatized dextrans

Analys es a Compos ition (ds )b Msec

APTT

(g molÉ1)c (IU mgÉ1)d

COONa N S CM B S

(meqgÉ1) (g 100gÉ1) (g 100gÉ1)

Dextran (D) È È È È È È 35 000 0

CMD 4.76 È È 1.24 È È 95 000 0

CMDB 3.81 1.42 È 1.10 0.29 0 109 000 0

CMDBS JCk 3.51 1.25 3.40 1.09 0.06 0.22 40 000 1.7

CMDBS JC29 2.63 2.30 4.23 0.96 0.22 0.39 57 000 5.0

CMDSu 3.63 È 5.78 1.0 È 0.37 65 000 1.5

reduced CMDSu 0.40 È 5.92 0.08 È 0.37 55 000 0.4

a SD, COONa, 0.02meqgÉ1, N, 0.05 g (100 g)É1 ; S, 0.05 g (100 g)É1.

b SD, CM, 0.05 ; B, 0.05 ; S, 0.05.

c SD, 3000 gmolÉ1.

d SD, 0.3 IUmgÉ1.

316 Polym Int 48 :313–319 (1999)

Sulphated polysaccharides derived from dextran

Figure 3. SMC growth inhibition by derivatized dextrans and

heparin. After 5 days in culture media containing 10% s erum andthe indicated concentrations of polys accharides , the cell numbers

were determined. The growth inhibition was calculated as

des cribed in Materials and Methods . Native dextran, D (+) ;carboxymethyl dextran, CMD CMDBS JCk heparin(L) ; (=) ; (K).Mean values ^SD (n\ 4) are indicated in a repres entativeexperiment.

liferative capacity, while anticoagulant heparin(170IU mg~1) has no stimulatory eþect. In addition,sample JC29 with a speciüc anticoagulant activity of5 IU mg~1 has a lower stimulatory eþect than JCk(1.7 IU mg~1). We therefore conclude that the anti-coagulant and antiproliferative properties are quiteseparate on the derivatized dextrans.

Derivatized dextrans inhibit in vitro SMC growth

We have isolated vascular SMC from rat aorta by theexplant method after partial digestion with collage-nase.22,37 SMC growth in the presence of 10%serum and CMDBS or heparin was studied aftergrowth arrest for 48h in 0.4% serum. After 5 days inthe presence of polysaccharides, cell numbers weredetermined and the percentage inhibition was calcu-lated. In the dose-response experiments (Fig 3),SMC growth inhibition was observed with thesample JCk. The growth inhibitory eþects are notdue to a cytotoxic eþect as assayed by trypan blueassay, with a viability of the SMCs always above95%.

The IC50, deüned as the concentration requiredto yield 50% inhibition, is in the 10lg ml~1 range.The value compares well with that of heparin (2–5lg ml~1), but native dextran and CMD had noeþect on SMC growth even with high doses (Fig 3).

DISCUSSION

Natural products used in an increasing number ofbiological and medical applications are oftenextracted from animal and human tissues or ýuidssuch as blood. There are numerous problems associ-ated with the preparation and use of these com-pounds. These problems include reproducibilityfrom batch to batch, contaminant-free preparations

(ie HIV, BSE), and the availability of products inquantities large enough for industrial scale prep-aration. In addition, the poor stability of natural pro-ducts and their high costs are also critical pointsagainst their extensive use. To avoid these draw-backs, investigations have been focused on syntheticproducts for biomedical applications. In theory,these synthetic products should exhibit similar orbetter biological activities than their natural ana-logues and also fewer side-eþects. With the dataavailable on chemical groups or sequences withinnatural products that confer biological properties,the research and development of new products canbe oriented towards the synthesis of analogues.Because functional groups are usually disposed oncomplex macromolecules, synthetic products couldbe polymers with suitable distributions of thesefunctional groups.

To investigate the structure–bioactivity relation-ships for synthetic polymers, we have undertakenstudies directed according to the hypothesis that arandom distribution of particular chemical groups onpolymer backbones can confer speciüc interactions.6Random substitution of macromolecular chains withsuitable ýexibility would permit the creation of spe-ciüc ‘keys’ which could match protein domains. Inthis work, we have studied the ability of a family ofsynthetic polysaccharides to exhibit anticoagulantactivity and/or capacity to stimulate EC proliferationand/or to inhibit SMC growth. This family is model-led on heparin, a natural glycosaminoglycan mainlyused as an anticoagulant in clinical practice.

The anticoagulant activity of CMDBS was pre-viously described as closely depending on three mainparameters : the CM content higher than 0.43 themolecular weight greater than 40000g mol~1without signiücant increase above this value,19 andünally the amount of sulphur-containing groups(sulphate and sulphonate groups) promoting anenhancement of clotting time.3,17 Although B groupsare not directly implied in the anticoagulant activityof CMDBS, we postulate that the spatial arrange-ment of sulphate groups along a polymer backbonecould be oriented by these groups. Newly inducedconformations would promote better interactionswith blood proteins.4 The mechanisms underlyingthe anticoagulant activities of heparin and CMDBSare diþerent. The former is a catalysis of the throm-bin inhibition by AT through interaction between awell-deüned oligosaccharidic structure belonging toheparin and the protease.16 The latter is mainlyrelated to preferential inhibition of thrombin andcatalysis of thrombin inhibition by HCII.17 Due toits high anticoagulant capacity, prolonged use ofheparin is not recommended in vascular therapy,especially when patients are subjected to haemor-rhages. CMDBS endowed with a low but signiücantanticoagulant activity as compared to that of heparinrepresents a potential alternative.

Proliferation of EC in response to injury is impor-

Polym Int 48 :313–319 (1999) 317

F Chaubet et al

tant in wound healing. In this report, we show thatderivatized dextrans stimulate the in vitro growth ofhuman endothelial cells. The results indicate thatchemical substitution of dextran has a direct role inthe EC stimulatory process. We present evidencethat these synthetic polysaccharides retain a prolifer-ative activity quite diþerent from that of heparin.According to their substitution rates, the derivatizeddextrans diþer signiücantly in their ability to stimu-late EC growth. From studies reported here, wedemonstrated that CMDB and CMDBS are able tostimulate EC growth in the 1–10lg ml~1 range. Wehave also shown that sulphonated and sulphatedgroups alone on the dextran chain are not able toinduce any proliferative eþect.21 Therefore, deriva-tized dextrans which contain hydrophobic benzyl-amide groups have a stimulatory eþect on ECgrowth.

The results also imply that the proliferative natureof the derivatized dextrans is not related to theiranticoagulant properties. The CMDB sample doesnot bear S groups, and consequently has no anti-coagulant capacity. Moreover, heparin which pos-sesses high anticoagulant capacity has noproliferative property. These studies indicate that onderivatized dextran, the structural determinantsresponsible for anticoagulant activity are distinctfrom those which are required for proliferative activ-ity. Finally, our data have shown that charge density(carboxyl and/or sulphate/sulphonate) cannot induceproliferative activity.21 On the contrary, they revealthat a speciüc sequence or speciüc groups distrib-uted along the macromolecular chains are requiredfor the proliferative activity of CMDBS.

The proliferation of vascular SMCs plays a crucialrole in the pathogenesis of atherosclerosis and in per-sistent pulmonary hypertension of the newborn.38The hyperplastic response of SMCs to vessel injuryduring vascular surgery is yet another area of seriousconcern. A large number of vascular surgical pro-cedures such as vein grafts, bypasses, angioplasties,arteriovenous shunts, endarterectomies and hearttransplants fail because of SMCs proliferation.38,39A highly antiproliferative molecule would be ideal toachieve adequate therapeutic control of the diseasesand surgically-induced pathological states. To avoidhaemorrhagic complications, the antiproliferativedrug should not be an anticoagulant. Unfortunately,heparin and heparin sulphate which are demon-strated inhibitors of in vivo and in vitro SMCs pro-liferation, are more or less naturally anticoagulant.The development of non-anticoagulant anti-proliferative heparin analogues may have clinical sig-niücance. Our approach was to prepare newsynthetic polysaccharides with SMCs anti-proliferative activity. We have shown that dextrangrafted with carboxymethyl benzylamide sul-phonated groups (CMDBS) inhibits SMCs prolifer-ation. In comparison, native dextran or dextransubstituted with carboxyl units (ie anionic charged

CMD) or dextran substituted with carboxyl benzyl-amide units (CMDB) have no eþect on SMCsgrowth. Further experiments will contribute to abetter understanding of the precise mechanisms ofaction of derivatized dextrans on vascular cells.

CONCLUSIONS

The synthetic dextran derivatives are prepared vialimited and easy to perform modiücations of a lowcost natural polysaccharide. The nature and distribu-tion of chemical groups and the composition can bealtered, leading to polymers which mimic the actionof heparin in diþerent biological systems.3,19,40,41 Inaddition to anticoagulant activities, derivatized dex-trans have the capacity to inhibit SMC growth andstimulate EC proliferation. With the examples ofbioactive derivates described in this study, wepropose that CMDBS could be considered as newinteresting macromolecular drugs for vasculartherapy.

ACKNOWLEDGEMENTS

This work was supported by the Centre National dela Recherche Scientiüque (CNRS), France. Dr Step-hane La Barre (LRM, Villetaneuse) is gratefullyacknowledged for his readership and SandrinePrigent-Richard (LRM, Villetaneuse) for help withthe SMCs experiments.

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