JCP-07-0541.R2(21510)ORIGINAL ARTICLE 1

J o u r n a l o fJ o u r n a l o f

CellularPhysiologyCellularPhysiology

Internalization and Degradation

of Heparin Is Not Required forStimulus of Heparan SulfateProteoglycan Synthesis

EDVALDO S. TRINDADE,1,2 RODRIGO I. BOUCAS,1 HUGO A.O. ROCHA,3

JULIANA A. DOMINATO,1 EDGAR J. PAREDES-GAMERO,1 CELIA REGINA C. FRANCO,2

CONSTANCE OLIVER,4 MARIA C. JAMUR,4 CARL P. DIETRICH,1 AND HELENA B. NADER1*1Departamento de Bioquımica, Universidade Federal de Sao Paulo, Sao Paulo, SP, Brazil2Departamento de Biologia Celular, Universidade Federal do Parana, Curitiba, PR, Brazil3Departamento de Bioquımica, Universidade Federal do Rio Grande do Norte, Natal, RN, Brazil4Departamento de Biologia Celular e Molecular e Bioagentes Patogenicos, FMRP, USP, Ribeirao Preto, SP, Brazil

In vitro, heparin and antithrombotic drugs specifically stimulate the synthesis of an antithrombotic heparan sulfate proteoglycan (HSPG)produced by endothelial cells. The putative heparin binding site(s) that may be related to this phenomenon were investigated. In thepreceding article, using various heparin probes, it was shown that the heparin does not bind to the endothelial cell surface, but only to theextracellular matrix. The present study demonstrated that, when the cells were exposed to heparin at 378C, the heparin was internalizedand with time was localized in lysosomes. However, endocytosis of heparin was not required for the stimulation of HSPG synthesis. Therequirement for heparin degradation in the stimulus of HSPG synthesis was also investigated. When the cells were incubated withchloroquine, a lysosomotropic amine that raises the lysosomal pH thus inhibiting enzymatic degradation of internalized compounds,stimulation of HSPG synthesis was still observed. These combined results indicate that neither internalization nor degradation of heparin isrequired for stimulation of HSPG synthesis, and suggests that its binding to the extracellular matrix could be responsible for this effect.

J. Cell. Physiol. 9999: 1–7, 2008. � 2008 Wiley-Liss, Inc.

Dedicated to the memory of Professor Carl P. Dietrich.Contract grant sponsor: Fundacao de Amparo a Pesquisa do Estadode Sao Paulo (FAPESP), Brazil.Contract grant sponsor: Financiadora de Estudos e Projetos(FINEP), Brazil.Contract grant sponsor: Conselho Nacional de DesenvolvimentoCientifico e Tecnologico (CNPq), Brazil.Contract grant sponsor: Coordenacao de Aperfeicoamento dePessoal de Nıvel Superior (CAPES), Brazil.

*Correspondence to: Helena B. Nader, Departamento deBioquımica, Escola Paulista de Medicina, Universidade Federal deSao Paulo, Rua 3 de Maio, 100-CEP 04044-020, Sao Paulo, SP, Brazil.E-mail: hbnader.bioq@epm.br

Received 2 September 2007; Accepted 5 May 2008

Published online in Wiley InterScience(www.interscience.wiley.com.), 00 Month 2008.DOI: 10.1002/jcp.21510

One of the major causes of death in the United States arediseases that involve heart and blood vessels includingthrombosis. The incidence of death because of thrombosis washigher than cancer in 2005 (National Vital Statistics Reports,2008). Thus the search for compounds that may play a role inthe prevention or treatment of thrombosis has been the subjectof several investigations. Unfractionated heparins and lowmolecular weight heparins are the only sulfated polysaccharidescurrently used as anticoagulant drugs (Hirsh et al., 2001a;Messmore et al., 2004; Nader et al., 2004). Heparin wasdiscovered in 1918 as an anticoagulant compound (Howell andHolt, 1918) and was introduced in clinical use in the 1940s toprevent thrombosis in surgical patients and in the treatment ofdeep venous thrombosis (Crafoord and Jorpes, 1941; Rodenet al., 1992; Messmore et al., 2004). Heparin still remains thetherapeutic drug of choice for the management of thromboticdisorders, despite the recent design and development of newdrugs (Hirsh et al., 2001a; Nader et al., 2004; Bates and Weitz,2005).

The possible sites of action for antithrombotic compoundsare the blood itself, composed of plasma proteins, lipids andcells (Hirsh et al., 2001b; Nader et al., 2004), and the vessel wall(Nader et al., 2001, 2004; Munoz and Linhardt, 2004). In the1980s, Colburn and Buonassisi (1982) reported that anendothelial cell line in culture showed blood compatibility, asthe surface of these cells had antithrombotic activity. Thus,when the surface of endothelial cells changes, there is a chancein thrombus formation. Among the compounds present on thecell surface and in the extracellular matrix of endothelial cellsare heparan sulfate proteoglycans (HSPG). This heparan sulfatehas been shown to possess antithrombotic activity in differentmodels (Colburn and Buonassisi, 1982; Marcum andRosenberg, 1989; Shimada et al., 1991; Shibata et al., 1994;

� 2 0 0 8 W I L E Y - L I S S , I N C .

HajMohammadi et al., 2003; Weitz, 2003; Rocha et al., 2005; Liuand Pedersen, 2007).

As described in the previous article, when endothelial cellsare exposed to heparin or other antithrombotic compounds(i.e., dextran sulfate (Nader et al., 1991), low molecular weightheparins, an oligosaccharide prepared from heparin byheparitinase II digestion; chemically sulfatedglycosaminoglycans and polysaccharides such as Suleparoid(SyntexQ1), Aprosulate (Luitpold-WerkQ2); chemicallymodified glycosaminoglycans GAGPS and MPS(Luitpold-Werk) (Pinhal et al., 1994a), cyclicoctaphenol–octasulfonic acid (GL-522-Y-1) (Pinhal et al.,1994b, 2001) and sulfated galactofucan (Rocha et al., 2005))

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there is a twofold increase in the synthesis of theantithrombotic HS secreted by the cells (Nader et al., 1989,1991, 2001, 2004; Pinhal et al., 1994a,b, 1995, 2001; Rocha et al.,2005). The mechanism of action by which heparin couldstimulate synthesis of HSPG in endothelial cells wasinvestigated. In the preceding article it was reported thatheparin binds only to the extracellular matrix (ECM). Thepresent work demonstrates that while heparin is internalizedby endothelial cells, neither heparin internalization nordegradation are required for stimulation of HSPG synthesis.

Materials and MethodsCell cultures

An endothelial cell line derived from rabbit thoracic aorta was usedin this study. This cell line has many endothelial characteristicsincluding blood compatibility, expression of von Willebrand factor,tissue factor pathway inhibitor, and estrogen receptor, productionof anticoagulant heparan sulfate, and fibronectin as well asdevelopment of capillary-like structures in vitro (Buonassisi andVenter, 1976; Colburn and Buonassisi, 1978, 1982, 1988;Buonassisi and Colburn, 1983; Colburn et al., 1987, 1991; Naderet al., 1987, 1991; Rocha et al., 2005). Cells were grown in Ham’sNutrient Mixture F-12 (Invitrogen, Carlsbad, CA) supplementedwith 10% fetal calf serum (FCS) (Cultilab, Campinas, SP, Brazil),streptomycin (100mg/ml) and penicillin (100 IU/ml) (Sigma–AldrichChemical Co., St. Louis, MO) at 378C in a humidified atmospherewith 2.5% CO2.

Antibodies and chemicals

Goat polyclonal anti-human lysosome-associated membraneprotein 1 IgG (anti-LAMP1) was purchased from Santa CruzBiotechnology (Santa Cruz, CA) (5mg/ml). The secondary antibodyconjugated to Texas Red and streptavidin conjugated tofluorescein (DATF), Texas Red or horseradish peroxidase (HRP)were purchased from Jackson ImmunoResearch (West Grove,PA). DAPI (40,6-diamidino-2-phenylindole, dihydrochloride), AlexaFluor 488-hydrazide and LysoTracker Red DND99 were obtainedfrom Molecular Probes (Invitrogen, Molecular Probes, Camarillo,CA). Heparin from bovine lung was obtained from Kim Master(Passo Fundo, RS, Brazil). The heparan sulfate from bovinepancreas was a gift from Dr. P. Bianchini (Opocrin ResearchLaboratories, Modena, Italy). The maxatase (alkaline protease fromSporobacillus) was obtained from Biobras (Belo Horizonte, MG,Brazil). Chloroquine, DAB (3,30-diamino benzamidinetetrahydrochloride), EDAC (1-(30-dimethylaminopropil)-3-ethylcarbodiimide), ethylenediamine (1,2-diaminoethane) andsaponin were purchased from Sigma–Aldrich. Biotin-hydrazide wasobtained from Pierce Biotechnology (Rockford, IL), agaroselow-Mr from Biorad (Richmond, CA) and carrier free[35S]-inorganic sodium sulfate from Instituto de PesquisasNucleares-IPEN (Sao Paulo, SP, Brazil). Paraformaldehyde,glutaraldehyde, cacodylic acid, osmium tetroxide (OsO4), Embed812 and Fluoromont G were purchased from Electron MicroscopySciences (Hatfield, PA).

Heparin probes

Heparin (10 mg, �20 mmol of uronic acid) and biotin-hydrazide(20mmol) were dissolved in 0.1 M HCl (20 ml) and the pH adjustedto 4.8. After addition of 20 mmol of EDAC the pH of the reactionwas kept at 4.8 by dropwise addition of 0.01 M HCl for 5 min withstirring. The reaction was stopped by the addition of sodiumacetate to a final concentration of 0.5 M, pH 4.8, with stirring for anadditional 60 min. The solution was then dialyzed against water andlyophilized. Biotin-hydrazide and non-biotinylated heparin wereused as controls. With this method, biotin is conjugated to thecarboxyl groups of the uronic acid residues yielding the biotinylatedheparin (BiotHep). This probe contains 16% of the uronic acid

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residues covalently linked to biotin. Alexa Fluor 488 was alsoconjugated to heparin by a similar procedure. Heparin (1 mg) andAlexa Fluor 488-hydrazide (1 mg) were dissolved in 0.1 M HCl(20 ml) and the pH adjusted to 4.8. After, addition of 2 mmoles ofEDAC the reaction followed the procedure described above. Bothcompounds retain several chemical and enzymatic characteristicsof the mother compound, such as similar behavior in gelelectrophoresis and degradation by heparinase.

Synthesis of [35S]-sulfated glycosaminoglycans

Post confluent endothelial cells, grown in 35 mm culture dishes,were exposed or not (control) to heparin (100 mg/ml) in F-12medium containing 150 mCi/ml of [35S]-sulfate in presence orabsence of chloroquine (100 mM) (24 h in F-12 medium, at 378C).For some assays, the cells were washed and pre-treated withheparin (1 h in F-12 medium, at 4 or 378C) followed by washing andaddition of fresh medium containing 150mCi/ml [35S]-sulfate in theabsence of heparin and then cultured for an additional 2 h at 378C.In both assays the conditioned medium was removed and the cellswere washed with 0.1 M phosphate buffered saline (PBS) (3�) andscraped from the dishes using 3.5 M urea in 0.05 M Tris–HCl,pH 7.4. Protein-free glycosaminoglycan chains were prepared fromthe cells and conditioned medium after incubation with maxatase(0.1 mg/ml in 0.05 M Tris–HCl pH 8.0 containing 0.15 M NaCl, 4 h,608C, final volume of 100 ml) and identified and quantified usingagarose gel electrophoresis and enzymatic degradation withglycosaminoglycan lyases (chondroitinases AC and ABC,heparinase and heparitinases I and II) as previously described(Nader et al., 1989; Pinhal et al., 2001). Cell protein was determinedas described by Spector (1978).

Cytochemistry

Post-confluent cultures grown on 12-mm-diameter glasscoverslips in 24-well plates (Corning Costar Corp., Cambridge,MA) were used in all assays.

Internalization. Cells were incubated in the presence or absence(control) of BiotHep (100 mg/ml, 24 h in F-12 medium, at 378C).Afterwards the cells were rinsed several times and fixed with 2%paraformaldehyde (30 min at 228C). The BiotHep bound outside thecells was detected after incubation with streptavidin conjugated toTexas Red (5 mg/ml, 30 min, 228C). After thorough rinsing the cellswere permeabilized with 0.01% saponin in PBS and the internalizedBiotHep was detected with streptavidin-DATF (5 mg/ml, 30 min,228C). After washing, the cells were incubated with DAPI (3 mM,2 min), washed, mounted in fluoromount-G and examined using a laserscanning confocal microscope (Zeiss LSM-510 NLO, Carl Zeiss,Jena, DE).

Lysosomal markers. To verify if the internalized heparin waslocalized in lysosomes, assays similar to those described above wereperformed using lysosomal markers. Anti-LAMP1 recognizes a proteinassociated with the lysosome membrane. Cells were incubated withBiotHep, fixed as described above, and after permeabilization withsaponin, BiotHep was detected using streptavidin-DATF. The cellswere then incubated with anti-LAMP1 (5 mg/ml in PBS with 1% BSA for1 h at 228C) followed by incubation with Texas Red-labeled secondaryantibody (5 mg/ml, 30 min, 228C). In other experiments LysoTrackerRed was used as a lysosomal marker. In these experiments, the heparinprobe was conjugated to Alexa Fluor 488 (AlexaHep). The cells wereexposed to AlexaHep (100 mg/ml, 22 h in F-12 medium, at 378C) andthen LysoTracker Red (500 nM) was added and the cells wereincubated for an additional 2 h, at 378C). Afterwards the cells werewashed several times with PBS and observed in a confocal microscope.

Degradation of biotinylated heparin. Post-confluent cells wereexposed or not (control) to BiotHep (100 mg/ml, 24 h in F-12 medium,at 378C) in the presence or absence of 100 mM chloroquine. After theincubation, the medium was replaced with fresh medium supplementedwith 10% FBS with or without chloroquine. The coverslips wereremoved at 24 h intervals up to 144 h. The cells were then fixed with 2%paraformaldehyde. BiotHep was detected both outside and inside thecells as described above.

Fig. 2. Transmission electron microscopy reveals that theinternalized heparin is in vesicles morphologically similar tolysosomes. The endothelial cells were incubated with BiotHep whichwas localized with streptavidin-peroxidase. A,B are sections ofdifferent cells. A,B: The internalized heparin is present in vesicles,which are morphologically similar to lysosomes (arrows).C: No reaction product was seen when the cells were exposed tonon-biotinylated heparin and incubated with streptavidin-peroxidase.n, nucleus. Bars, A U 0.62 mm, B U 0.39 mm, C U 0.31 mm.

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Time lapse. Post-confluent cultures were washed and pre-treatedwith AlexaHep (10 mg/ml in F-12 medium for 1 h at 4 or 378C).Afterwards the medium was removed and the cell washed 3 times withF-12 medium, placed in a fresh medium without AlexaHep at 378C andobserved using a fluorescence inverted microscope (TE300, NikonInstrument Inc., MelvilleQ3). The images were captured in intervals of10 min, using a video camera.

Transmission electron microscopy

For transmission electron microscopy, post-confluent cells grownin 35 mm-tissue culture dishes were incubated in the presence orabsence (control) of BiotHep (24 h in F-12 medium, at 378C),rinsed several times with PBS and fixed by microwave (Jamur et al.,1995) in 2% paraformaldehyde, 0.05% glutaraldehyde, 0.025%CaCl2 in 0.1 M cacodylate buffer, pH 7.4. After washing severaltimes with 0.1 M cacodylate buffer, the BiotHep bound to the ECMwas blocked with streptavidin (1:100 in PBS for 30 min). The cellswere then washed five times with 0.1 M cacodylate and incubatedwith streptavidin conjugated to HRP (1:40 in PBS with 1% BSA and0.01% saponin, 1 h, 228C) and washed again several times with thesame buffer. The streptavidin-HRP was detected by incubating thecells with DAB–H2O2 (1 mg/ml in 0.1 M cacodylate buffercontaining 0.02% H2O2, 30 min, 228C). After several washes withcacodylate buffer, the cells were postfixed in 2% OsO4 for 1 h at228C, rinsed in distilled water, dehydrated through a graded seriesof ethanol, removed from the dishes and pelleted in propyleneoxide, rinsed in acetone and embedded in Embed 812 (ElectronMicroscopy Sciences). Thin sections were cut with a diamond knifeand stained for 10 min each in Reynold’s lead citrate (Reynolds,1963) and uranyl acetate. Sections were examined in a Phillips208 electron microscope (FEI Company, Eindhoven, TheNetherlands).

ResultsEndothelial cells internalize heparin

In order to determine if the cells could internalize heparin, postconfluent cell cultures were incubated with BiotHep for 24 h at378C. The BiotHep outside the cells is bound to the ECM(Fig. 1). When the cells are permeabilized, BiotHep is detectedinside the cells in vesicles (Fig. 1, arrows).

Heparin is internalized in lysosomes

The fate of the internalized heparin inside the cells wasinvestigated by electron microscopy. The cells were incubatedwith BiotHep, permeabilized and incubated withstreptavidin-HRP. The cells were then incubated with

Fig. 1. Heparin is internalized by endothelial cells. Confluent cells were idetected with streptavidin conjugated to Texas red (Red). Note that theInternalizedBiotHepwasdetectedwithstreptavidinconjugatedtoDATF(G(arrows).Overlay:Theheparin isboundextracellularly(red)toECMandintwith DAPI (Blue). Bar, 10 mm.

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H2O2–DAB to reveal peroxidase activity. The internalizedheparin is localized inside the cells in vesicles thatmorphologically resemble lysosomes (Fig. 2A,B). Control cells(Fig. 2C), incubated with non-biotinylated heparin, do notcontain any reaction product, indicating that the electron densematerial seen inside the vesicles in Figure 2A,B is BiotHep. The

ncubated with BiotHep for 24 h at 37-C. Non-permeabilized: BiotHepBiotHep shows a fiber-like pattern outside the cells. Permeabilized:reen).Thegreenfluorescenceshowsheparin invesicles insidethecells

racellulary(green) is locatedinvesicles(arrows).Thenucleiarestained

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nature of these vesicles was further investigated usinglysosomal markers. Initially the cells were incubated withBiotHep overnight and after fixation and permeabilization wereincubated with anti-LAMP1 which recognizes a proteinassociated with the lysosomal membrane. LAMP1 and Heparincolocalize in the same vesicles (Fig. 3A) indicating that thevesicles containing the BiotHep could be lysosomes. In order toconfirm that the BiotHep was in lysosomes, the experiment wasrepeated using LysoTracker Red to mark the lysosomes. Boththe LysoTracker and Heparin were found in the same vesicles(Fig. 3B). These results confirm that the vesicles containing theBiotHep are lysosomes.

Heparin degradation is not required for stimulationof HSPG synthesis

The fate of the internalized heparin inside the lysosomes wasfollowed for up to 72 h of incubation. Figure 4A shows that theBiotHep slowly disappears from the cells, most likely bylysosomal degradation of the heparin. In order to test thispossibility, the cells were incubated in with chloroquine, alysosomotropic amine that inhibits enzymatic degradation byraising the lysosomal pH. In the presence of chloroquine theclearance of heparin from the cells is retarded (Fig. 4B). Theseresults further indicate that heparin is sequestered anddegraded in lysosomes. Therefore it was important to verify ifthe stimulation of synthesis of HSPG by heparin was related toits degradation by lysosomal enzymes. However, thestimulation of HSPG synthesis elicited by incubation withheparin is not affected by chloroquine (Fig. 5). Thus, thedegradation of heparin is not necessary in order to stimulateHSPG synthesis.

Internalization of heparin is not required for thestimulation of HSPG synthesis

In order to verify if internalization of heparin is necessary tostimulate the synthesis of HSPG, experiments were performedin which endocytosis of heparin was inhibited. The cells wereincubated for 1 h with heparin at 37 or 48C. After washing and

Fig. 3. The internalized heparin colocalizes with lysosomes. The cells wedetected with streptavidin conjugated to DATF (green; arrows) and the ly(green; arrows) is observed in vesicles. The lysosomes (red; arrows) are laobserve the co-localization (yellow; arrows) between internalized heparin

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the addition of fresh medium without heparin, the synthesis ofheparan sulfate was investigated. No differences in the amountsof HS synthesized were found when the cells were incubatedwith heparin at either 37 or 48C (Fig. 6A), thus indicating thatinternalization is not a necessary step for the increase in thesynthesis HSPG. Time lapse experiments were also performedto follow the fate of the heparin. The cells were incubated withAlexaHep at 37 or 48C for 1 h, the medium removed, and freshmedium without heparin was added. The fate of heparin wasthen followed at 378C for up to 2 h using a video camera(Fig. 6B). It is clear that the cells that were initially exposed toheparin at 378C have this glycosaminoglycan inside the cells invesicles. On the other hand, the cells that were exposed toheparin at 48C, and later transferred to 378C, do not containheparin inside the cells. Comparing the results shown inFigure 6A,B it can be concluded that the internalization ofheparin is not required to stimulate in the synthesis of HS inendothelial cells.

Discussion

Virtually all cells, from simple invertebrates to humans, have thecapacity to produce heparan sulfate (Dietrich, 1984; Esko andLindahl, 2001; Kreuger et al., 2006; Sampaio et al., 2006).Heparan sulfate proteoglycans are ubiquitous components of alltissue-organized animal life forms (Dietrich et al., 1998;Bernfield et al., 1999; Nader et al., 2004) and are present at thecell surface and associated with the ECM of mammalian cells inculture (Dietrich and De Oca, 1970; Bernfield et al., 1999;Medeiros et al., 2000; Weitz, 2003; Whitelock and Iozzo, 2005;Gallagher, 2006; Taylor and Gallo, 2006; Imberty et al., 2007).Endothelial cells express an HSPG which have anantithrombotic action (Damus et al., 1973; Colburn andBuonassisi, 1982; Marcum and Rosenberg, 1989;HajMohammadi et al., 2003; Weitz, 2003; Liu and Pedersen,2007). By using an antibody against heparan sulfate, it wasconfirmed that this effect was at least in part heparansulfate-dependent (Colburn and Buonassisi, 1982). Since an

re incubated with heparin and lysosomal markers. A: BiotHep wassosomes were marked with anti-LAMP1 (red; arrows); (B) AlexaHep

beled with LysoTracker Red. A,B: In the image overlay it is possible toand lysosomes. Bar, 20 mm.

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Fig. 4. The internalized heparin is degraded by endothelial cells. The cells were incubated with BiotHep for 24 h. Afterwards the medium wasremoved, the cells washed and incubated in a fresh medium without heparin. The coverslips were removed at 24 h intervals and processed asdescribed in Figure1. A:Control assay in theabsence ofchloroquine. Theamountofheparin inside thecells (green) decreaseswith time, indicatingdegradationof theheparin. Notethat theheparinoutsidethecells (red) isnotvisibleafter24h.B: Inthepresenceofchloroquine thedegradationofinternalized heparin (green) was blocked and the heparin bound outside the cells (red) slowly decreases with time. Arrows: intracellular heparin(green). The nuclei were stained with DAPI (blue). Bar, 20 mm.

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increased synthesis of heparan sulfate chains is observed whencells are exposed to heparin and other antithrombotic agents,the hypothesis has been raised that the in vivo antithromboticactivity of these compounds is related, at least in part, to theincreased production of this unique heparan sulfate byendothelial cells. In favor of this hypothesis we have recentlyshown that the conditioned medium from endothelial cellsexhibits anti-FXa activity. This effect is increased when the cellsare exposed to sulfated galactofucan and the anti-FXa activity

Fig. 5. The degradation of heparin is not necessary in order tostimulate HSPG synthesis. Post confluent cells were metabolicallylabeled with [35S]-sulfate (37-C, 24 h, 2.5% CO2) in the absence(Control) or presence of 100 mg/ml of non-biotinylated (Hep) and inthe presence or absence of chloroquine (100 mM). The sulfatedglycosaminoglycans from the medium were extracted and analyzedby agarose gel electrophoresis using 0.05 M 1,3-diaminopropaneacetate buffer pH 9.0. HS, heparan sulfate; CS, chondroitin sulfate.These results are the mean W SEM of four different experiments donein triplicate. (M) P < 0.05, t-test.

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disappeared after heparitinase treatment, indicating that thisactivity may be related to the heparan sulfate produced byendothelial cells (Rocha et al., 2005). Possibly heparin, and otherantithrombotic agents, prevents thrombosis because of itscombined effect as an anticoagulant and an inducer of thesynthesis of heparan sulfate by the endothelial cells.

In an accompanying article in this issue we have shown thatusing different probes of heparin and different conditions, theheparin does not bind to the cell surface, but only to the ECM.On the other hand, several studies demonstrated that heparin isinternalized by the cells. Some authors connect the short halftime of heparin (Hirsh et al., 2001b) with the fast uptake by livercells (Watanabe et al., 1983, 1985, 1993; Nakamura et al., 2002).Montes de Oca and Dietrich (1969) were the first to report theinternalization of heparin. These authors, using mammaliancells, observed that after 48 h of incubation, heparin was foundinside these cells, showing a perinuclear distribution. Otherstudies observed this phenomenon in vascular endothelium(Hiebert and Jaques, 1976a; Mahadoo et al., 1978, 1980).Endothelial cells in culture were also able to internalize heparin(Hiebert and Jaques, 1976a,b; Barzu et al., 1985, 1987;Vannucchi et al., 1986, 1988; Herbert and Maffrand, 1989;Hiebert, 1989; Hiebert and McDuffie, 1989, 1990; Dawes andPepper, 1992).

In the experiments reported in this and in the precedingarticle, we used bovine lung heparin, that was biotinylated orconjugated to Alexa 488 in our laboratory as well as heparinconjugated to FITC purchased from Molecular Probes (H7482),that is from a different source than ours. All of the compoundsused in our experiments bind to the extracellular matrix as wellas being internalized when incubated at 378C. Thus, in thepresent study, we investigated if the site of action of heparininvolved in the stimulus of HSPG synthesis is located extra- orintracellularly. Using transmission electron microscopy, theinternalized heparin was localized inside vesiclesmorphologically similar to lysosomes (Fig. 2). The localization ofheparin in lysosomes was confirmed using the lysosomalmarkers LAMP1 and LysoTracker Red (Fig. 3A,B). Clearanceassays shows that the heparin can be detected inside the cell for

Fig. 6. Heparin internalization is not required for stimulation ofHSPG synthesis. Post confluent cells were pre-treated with 100mg/mlof non-modified heparin for 1 h at 37 or 4-C. Afterwards the cells werewashed and metabolically labeled with [35S]-sulfate (37-C, 2 h,2.5% CO2) in the absence of heparin. The sulfated glycosaminoglycansfrom the medium were extracted and analyzed by agarose gelelectrophoresis using 0.05 M 1,3-diaminopropane acetate buffer pH9.0. A: Inhibition of heparin endocytosis by incubation in at 4-C did notalter the stimulation of HSPG synthesis caused by heparin. HS,heparan sulfate; CS, chondroitin sulfate. These results aremean W SEM of three different experiments done in triplicate.(M) P < 0.05, t-test. B: The cells were incubated with AlexaHep at 37 or4-C for 1 h. Afterwards the medium was removed, the cells werewashed and fresh medium without heparin was added. The cultureswere maintained for an additional 2 h, as described. The AlexaHepwas monitored using a fluorescence microscope equipped with adigital camera. Images were collected for up to 2 h. Images are shownfor zero (0), 60 and 120 min after washing the cells free of heparin.Note that at 37-C heparin can be detected inside the cells (arrows) aswell as bound to the ECM, whereas at 4-C it can observed bound onlyto the ECM (arrowheads). Bar, 30 mm.

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144 h (data not shown), although its concentration graduallydecreases (Fig. 4) suggesting a slow degradation of the heparin.Similar results were obtained by Hiebert and McDuffie (1990)who showed heparin remained inside porcine aorticendothelial cells for up to 6 days. Several authors using [3H]- or[125I]-heparin have shown its internalization and subsequentdegradation (Vannucchi et al., 1986, 1988; Barzu et al., 1987;Herbert and Maffrand, 1989; Dawes and Pepper, 1992). Pinhalet al. (1995) showed that a pentasulfated tetrasaccharide is theminimum heparin fragment able to increase HSPG synthesis.In order to investigate if these degradation fragments could

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related to the stimulation of HSPG synthesis, cells wereincubated with heparin in the presence chloroquine (Ohkumaand Poole, 1978; Yanagishita and Hascall, 1984; Iozzo, 1987).Our results showed that even after blockage of heparindegradation with chloroquine, the cells were still capable ofenhanced HSPG synthesis (Fig. 5) thus demonstrating thatdegradation is not necessary for this effect. Furthermore whenthe cells were incubated with heparin at 48C, in order to inhibitinternalization, there was no difference in the level ofstimulation of HSPG synthesis between these cells and cellsincubated at 378C (Fig. 6A). Thus showing that internalization ofheparin was not required for this effect.

Taken together, these data suggest that the signal for thisstimulus in the increase in heparan sulfate synthesis byendothelial cells that is elicited by heparin is mediated byheparin binding to ECM components.

Acknowledgments

The authors would like to thank Maria Tereza Picinot Maglia,Maria Dolores Ferreira and Jose Augusto Maulin for technicalsupport with the electron microscopy (Department of Cell andMolecular Biology and Pathogenic Bioagents, Faculdade deMedicina de Ribeirao Preto, USP).

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