Effect ofin vitro differentiation on proteoglycan structure in cultured human monocytes

Parellel toin vitro differentiation of human monocytes into macrophage-like cells, the cells change their synthesis of glycosaminoglycans from chondroitin 4-sulfate to highly sulfated chondroitin sulfate, containing 4,6-disulfatedN-acetylgalactosamine units [Kolsetet al. (1983) Biochem J 210:661–67]. After exposure of monocyte cultures to [³⁵S]sulfate for 24h either from the onset of cultivation, prior to differentiation, or from day 4, after differentiation,³⁵S-macromolecules from medium and cell-layer were isolated and characterized. The cell-layer of day 5 cultures contained both proteoglycans and free polysaccharide chains, while the³⁵S-macromolecules present in the cell-layer of day 1 cultures and in medium of both monocytes and macrophage-like cells were almost exclusively of proteoglycan nature. Proteoglycans produced by macrophage-like cells were of larger size than the monocyte proteoglycans, most likely due to an increased polysaccharide chain length. These proteoglycans, in contrast to the monocyte-derived species, also showed affinity for fibronectin at physiological ionic strength.     

Proteoglycan biosynthesis in relation to differentiation of cord blood monocytes in vitro

Human monocytes were obtained from umbilical cord blood and cultured in vitro. By morphological criteria, the neonatal monocytes developed into macrophage-like cells in the course of 3-5 days in culture. The cells were exposed to [35S]sulphate for 24 h, either from day 0-1 or day 9-10 in vitro. The 35S-labelled macromolecules recovered were mainly associated with the medium fraction (approximately 75%) in both day 1 and day 10 cultures. These secretory macromolecules were demonstrated by the use of chondroitinase ABC-digestions to contain predominantly chondroitin sulphate proteoglycan (CSPG). [35S]galactosaminoglycan chains from day 10 cultures were more highly sulphated than the corresponding day 1 species due to the appearance of (glucuronosyl-4,6-diS-N-acetylgalactosamine) disulphated disaccharide units. The galactosaminoglycan chains in neonatal CSPG were found to increase in Mr during cultivation in vitro; from mean Mr of 20,400 to 30,200 (n = 5) in day 1 and day 10 medium proteoglycans, respectively. The corresponding Mr values for adult monocyte [35S]galactosaminoglycan chains were 21,300 and 22,800. On the basis of the concomitant changes in cellular morphology and glycosaminoglycan structure, it is concluded that neonatal monocytes, like monocytes from adults, differentiate into macrophage-like cells in vitro.     

Effects of 4-methyl umbelliferyl-beta-D-xylopyranoside on chondrogenesis and proteoglycan synthesis in chick limb bud mesenchymal cell cultures

When chick limb bud mesenchyme cells from stage 23 to 24 embryos are plated at high density, they rapidly divide and a large proportion initiate chondrogenic expression during the first 2 to 3 days in culture. Between Days 4 and 8, the emergent chondrocytes mature and elaborate a cartilaginous matrix. The proteoglycans synthesized by the newly emergent Day 3 to 4 chondrocytes differ from those synthesized by either the prechondrogenic mesenchyme cells or the mature Day 8 chondrocytes. Cultures were grown from initial plating (Day 0) or from Day 2 in the continuous presence of 1 mM 4-methyl umbelliferyl-beta-D-xyloside, which acts intracellularly as a competitive acceptor with the endogenous core protein of proteoglycans for chondroitin sulfate synthesis. The proteoglycans synthesized by Day 8 cultures which had been maintained on xyloside or to which xyloside was added only 1 h prior to labeling were essentially identical. They were able to form aggregates, and they contained the same number of keratan sulfate chains, but only about 40% as many chondroitin sulfate chains, as normal. Additionally, both the chondroitin sulfate and keratan sulfate chains were 25% shorter than in the normal proteoglycans. The proteoglycans synthesized by cells in a culture maintained on xyloside until Day 8, and then switched to medium with no xyloside 1 h prior to labeling, were characteristic of those synthesized by normal mature Day 8 chondrocytes. These data suggest that stage 23 to 24 mesenchyme cells undergo normal chondrogenic maturation in culture in the presence of xylosides even though (a) most of the polysaccharides are synthesized onto the exogenously supplied xyloside substrate and released into the medium, (b) the proteoglycans that are synthesized are greatly reduced in polysaccharide content, and (c) the extracellular matrix as a consequence is greatly depleted in chondroitin sulfate content and, therefore, is abnormal in general morphology.     

Oversulfated chondroitin sulfate proteoglycan in cultured human peritoneal macrophages

Human peritoneal macrophages were cultured in vitro and labeled with [35S]-sulfate. Both on day 1 and day 6 in culture the cells were found to synthesize exclusively chondroitin sulfate proteoglycan, the main part (70%) being associated with the medium after a 20 hour pulse. The glycosaminoglycan chains were found to be oversulfated both after 1 and 6 days in culture, due to the presence of disulfated disaccharide units.     

Characterization of β-D-xyloside-initiated glycosaminoglycan synthesized by human skin fibroblasts in the presence of tunicamycin

Human skin fibroblasts were incubated with a fluorogenic xyloside, 4-methylumbelliferyl--D-xyloside (Xyl-MU), in the presence or absence of tunicamycin. The xyloside-initiated glycosaminoglycans (GAG-MUs) were isolated from the culture medium, and their structures characterized. When the cells were incubated with Xyl-MU in the presence of 0.2 g ml–1 tunicamycin, the synthesis of GAG-MU was increased about three fold, compared with the control value in the absence of tunicamycin (cells exposed to Xyl-MU alone). The structures of GAG-MUs synthesized in the presence or absence of tunicamycin were compared by HPLC analysis using gel-filtration and ion-exchange columns, enzymatic digestion, and unsaturated disaccharide composition analysis. The data indicated that cells incubated with tunicamycin produced more undersulfated and shorter GAG-MUs than cells without tynicamycin. These results suggest that tunicamycin inhibits the elongation and sulfation of glycosaminoglycan (GAG) chains and that, as a result, GAG-MUs with shorter chains and undersulfated residues, but possessing a large number of GAG chains, are synthesized in the presence of tunicamycin.     

Role of proteoglycans in renal development

The role of proteoglycans (PGs) in morphogenesis was investigated. Fetal kidneys were obtained from 13-day-old mouse embryos and maintained for 7 days in culture. The biosynthesis of PGs was perturbed by addition of p-nitrophenyl-beta-D-xylopyranoside in the culture medium. The kidneys were processed for morphological and biochemical studies. The morphological studies included staining of tissues with anti-basement membrane antibodies and ruthenium red. [35S]sulfate was used as the precursor product for biosynthetic and autoradiographic studies. The kidneys treated with xyloside had loose mesenchyme, inhibition of ureteric bud branching, diminution in the population of developing nephron elements, decreased immunofluorescence with anti-proteoglycan antibodies and staining with ruthenium red, and a reduced [35S]sulfate incorporation into poorly organized extracellular matrices. The biochemical studies included characterization of PGs/glycosaminoglycans (GAGs) by Sepharose CL-4B, -6B, and DEAE-Sephacel chromatographies and cellulose acetate electrophoresis. Under the influence of xyloside, the total radioactivities decreased 2 to 4-fold in tissues and increased 18 to 42-fold in media fractions. A reduction in the size of macromolecular form of PGs, i.e., from MW approximately 2.5 X 10(6) to approximately 2.5 X 10(4), was noted. The PGs/GAGs synthesized were mainly made up of heparan sulfate and small amounts of chondroitin sulfate. They eluted at a lower salt concentration as compared to the controls. A similar diminution in the size of media PGs, i.e., from MW approximately 1.8 X 10(5) to approximately 2.8 X 10(4), was observed. Additional studies with [3H]xyloside indicated that the chains initiated on xyloside residues were similar in size and composition to GAG-chains. These findings indicate that a perturbance in the biosynthesis of PGs/GAGs leads to abnormalities in renal organogenesis.     

Biosynthesis of heparin. Modulation of polysaccharide chain length in a cell-free system.

The formation of heparin-precursor polysaccharide (N-acetylheparosan) was studied with a mouse mastocytoma microsomal fraction. Incubation of this fraction with UDP-[3H]GlcA and UDP-GlcNAc yielded labelled macromolecules that could be depolymerized, apparently to single polysaccharide chains, by alkali treatment, and thus were assumed to be proteoglycans. Label from UDP-[3H]GlcA (approx. 3 microM) is transiently incorporated into microsomal polysaccharide even in the absence of added UDP-GlcNAc, probably owing to the presence of endogenous sugar nucleotide. When the concentration of exogenous UDP-GlcNAc was increased to 25 microM the rate of incorporation of 3H increased and proteoglycans carrying polysaccharide chains with an Mr of approx. 110,000 were produced. Increasing the UDP-GlcNAc concentration to 5 mM led to an approx. 4-fold decrease in the rate of 3H incorporation and a decrease in the Mr of the resulting polysaccharide chains to approx. 6000 (predominant component). When both UDP-GlcA and UDP-GlcNAc were present at high concentrations (5 mM) the rate of polymerization and the polysaccharide chain size were again increased. The results suggest that the inhibition of polymerization observed at grossly different concentrations of the two sugar nucleotides, UDP-GlcA and UDP-GlcNAc, may be due either to interference with the transport of one of these precursors across the Golgi membrane or to competitive inhibition of one of the glycosyltransferases. The maximal rate of chain elongation obtained, under the conditions employed, was about 40 disaccharide units/min. The final length of the polysaccharide chains was directly related to the rate of the polymerization reaction.     

Sulfated proteoglycan synthesis by confluent cultures of rabbit costal chondrocytes grown in the presence of fibroblast growth factor

We examined the effect of fibroblast growth factor (FGF) on proteoglycan synthesis by rabbit costal chondrocyte cultures maintained on plastic tissue culture dishes. Low density rabbit costal chondrocyte cultures grown in the absence of FGF gave rise at confluency to a heterogeneous cell population composed of fibroblastic cells and poorly differentiated chondrocytes. When similar cultures were grown in the presence of FGF, the confluent cultures organized into a homogenous cartilage-like tissue composed of rounded cells surrounded by a refractile matrix. The cell ultrastructure and that of the pericellular matrix were similar to those seen in vivo. The expression of the cartilage phenotype in confluent chondrocyte cultures grown from the sparse stage in the presence vs. absence of FGF was reflected by a fivefold increase in the rate of incorporation of [35S]sulfate into proteoglycans. These FGF effects were only observed when FGF was present during the cell logarithmic growth phase, but not when it was added after chondrocyte cultures became confluent. High molecular weight, chondroitin sulfate proteoglycans synthesized by confluent chondrocyte cultures grown in the presence of FGF were slightly larger in size than that produced by confluent cultures grown in the absence of FGF. The major sulfated glycosaminoglycans associated with low molecular weight proteoglycan in FGF-exposed cultures were chondroitin sulfate, while in cultures not exposed to FGF they were chondroitin sulfate and dermatan sulfate. Regardless of whether or not cells were grown in the presence or absence of FGF, the 6S/4S disaccharide ratio of chondroitin sulfate chains associated with high and low molecular weight proteoglycans synthesized by confluent cultures was the same. These results provide evidence that when low density chondrocyte cultures maintained on plastic tissue culture dishes are grown in the presence of FGF, it results in a stimulation of the expression and stabilization of the chondrocyte phenotype once cultures become confluent.     

Structure and antithrombin-binding properties of heparin isolated from the clams Anomalocardia brasiliana and Tivela mactroides

Heparin with high anticoagulant activity was isolated from the two marine clam species Anomalocardia brasiliana and Tivela mactroides. A large portion of the polysaccharide chains of both preparations bound with high affinity to immobilized antithrombin. Titrations monitored by tryptophan fluorescence showed that clam polysaccharide chains with Mr approximately 22,500 contained up to three binding sites for antithrombin and that the binding constants for the interaction of these chains with antithrombin were higher than those reported for mammalian heparin of comparable size. Structural analysis of clam heparin fractions and subfractions of clam heparin with differing affinity for immobilized antithrombin revealed the presence of large amounts (up to 25-30% of the total disaccharide units) of the 3-O-sulfated saccharide sequences (-GlcNSO3)-GlcA-GlcNSO3(3-OSO3)- and (-GlcNSO3)-GlcA-GlcNSO3(3,6-di-OSO3)-, previously identified as unique markers for the antithrombin-binding region of heparin. The content of these saccharide sequences was found to increase with increasing affinity of the parent polysaccharide for antithrombin. Structural analysis of the clam heparins also demonstrated the occurrence of a novel saccharide sequence, tentatively identified as (-GlcNSO3)-IdA-GlcNSO3(3,6-di-OSO3)-, that has not previously been found in heparin or related polysaccharides. The contents of this latter sequence, at most 3-4% of the total disaccharide units, showed no correlation with the affinity for antithrombin.     

Somatic antigens of Pseudomonas aeruginosa. The structure of O-specific polysaccharide chains of the lipopolysaccharides from P. aeruginosa O5 (Lányi) and immunotype 6 (Fisher)

Lipopolysaccharides were isolated from dry bacterial cells of Pseudomonas aeruginosa O5a,b,c, O5a,b,d, O5a,d (Lányi classification) and immunotype 6 (Fisher classification) by the Westphal procedure. Their polysaccharide chains were built up of trisaccharide repeating units containing D-xylose, 2-acetamido-2,6-dideoxy-D-galactose and a new sialic acid-like sugar, the di-N-acyl derivative of 5,7-diamino-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic (pseudaminic) acid. Formyl, acetyl and (R)-3-hydroxybutyryl groups were identified as the N-acyl substituents of the last monosaccharide; O5a,b,c and O5a,b,d lipopolysaccharides also contained O-acetyl groups. The glycosidic linkage of pseudaminic acid was extremely labile towards acids, and mild acid degradation of the lipopolysaccharides produced, instead of the O-specific polysaccharides, their trisaccharide fragments with pseudaminic acid at the reducing terminus. Similar degradation of immunotype 6 lipopolysaccharides, followed by oxidation with sodium metaperiodate, resulted in a disaccharide fragment due to destruction of xylose. In contrast the glycosidic linkage of pseudaminic acid proved to be more stable towards treatment with hydrogen fluoride than those of xylose and N-acetylfucosamine. As a result, solvolysis of immunotype 6 lipopolysaccharide with hydrogen fluoride in methanol gave methyl glycosides of a disaccharide and a trisaccharide with pseudaminic acid at the non-reducing terminus. Mild acid hydrolysis of these oligosides afforded free 5-N-acetyl-7-N-formylpseudaminic acid, which was identified by the 1H ande 13C nuclear magnetic resonance data, as well as by the mass spectrum of the corresponding fully methylated aldonic acid. As a result of the identification of all oligosaccharides obtained and comparative analysis of the 13C nuclear magnetic resonance spectra of the oligosaccharides and lipopolysaccharides the following structures were established for the repeating units of the polysaccharide chains of the lipopolysaccharides: (Formula: see text) where D-Xyl = D-xylose, D-FucNAc = 2-acetamido-2,6-dideoxy-D-galactose, Pse5N7NFm = 5-amino-3,5,7,9-tetradeoxy-7-formamido-L-glycero-L-manno-nonulosonic+ ++ acid (7-N-formylpseudaminic acid). All the polysaccharides have an identical carbohydrate skeleton and differ from each other by the acyl substituent at N-5 of pseudaminic acid [acetyl or (R)-3-hydroxybutyryl group] or by the presence or absence of the O-acetyl group at position 4 of N-acetylfucosamine. The data obtained account properly for the O specificity of the studied P. aeruginosa strains.     

Stimulation of synthesis of free chondroitin sulfate chains by beta-D-xylosides in cultured cells.

Beta-Xylosides stimulate 2- to 6-fold the synthesis of glycosaminoglycans by three types of nonconnective tissue cells (RG-C6, NB41A, and rat hepatoma cells, and normal and simian virus 40 (SV40)-transformed normal human skin fibroblasts. The effect, which is specific for the anomeric linkage and the glycone, is observed in the presence and absence of puromycin. Beta-Xylosides may substitute for xylosylated core protein as initiators of synthesis of chondroitin sulfate chains. No stimulation of synthesis of heparan sulfate was observed. With the use of a fluorogenic xyloside, 4-methylumbelliferyl-beta-D-xyloside, it was demonstrated that the free chondroitin sulfate chains secreted into the medium bear the xyloside at the reducing end, and have an average molecular weight of 16,500.     

Tumor attenuation by 2(6-hydroxynaphthyl)-beta-D-xylopyranoside requires priming of heparan sulfate and nuclear targeting of the products

We have previously reported that the heparan sulfate-priming glycoside 2-(6-hydroxynaphthyl)-beta-D-xylopyranoside selectively inhibits growth of transformed or tumor-derived cells. To investigate the specificity of this xyloside various analogs were synthesized and tested in vitro. Selective growth inhibition was dependent on the presence of a free 6-hydroxyl in the aglycon. Because cells deficient in heparan sulfate synthesis were insensitive to the xyloside, we conclude that priming of heparan sulfate synthesis was required for growth inhibition. In growth-inhibited cells, heparan sulfate chains primed by the active xyloside were degraded to products that contained anhydromannose and appeared in the nuclei. Hence the degradation products were generated by nitric oxide-dependent cleavage. Accordingly, nitric oxide depletion reduced nuclear localization of the degradation products and counteracted the growth-inhibitory effect of the xyloside. We propose that 2-(6-hydroxynaphthyl)-beta-D-xylopyranoside entered cells and primed synthesis of heparan sulfate chains that were subsequently degraded by nitric oxide into products that accumulated in the nucleus. In vivo experiments demonstrated that the xyloside administered subcutaneously, perorally, or intraperitoneally was adsorbed and made available to tumor cells located subcutaneously. Treatment with the xyloside reduced the average tumor load by 70-97% in SCID mice. The present xyloside may serve as a lead compound for the development of novel antitumor strategies.     

Modulation of the expression of chondroitin sulfate proteoglycan in stimulated human monocytes

Proteoglycan biosynthesis was studied in human monocytes and monocyte-derived macrophages (MDM) after exposure to typical activators of the monocyte/macrophage system: interferon-gamma (IFN-gamma), lipopolysaccharide (LPS), and phorbol 12-myristate 13-acetate (PMA). By morphological examination, both monocytes and MDM were stimulated by these activators. Treatment with IFN-gamma resulted in a slight decrease in the expression of [35S]chondroitin sulfate proteoglycan (CSPG) in both monocytes and MDM, whereas LPS treatment increased the [35S]CSPG expression 1.8 and 2.2 times, respectively. PMA, in contrast, decreased the CSPG expression 0.4 times in monocytes, whereas MDM were stimulated to increase the biosynthesis 1.9 times. An increase in the sulfate density of the chondroitin sulfate chains was evident following differentiation of monocytes into MDM due to the expression of disulfated disaccharide units of the chondroitin sulfate E type (CS-E). However, monocytes exposed to PMA did also express disaccharides of the chondroitin sulfate E type. Furthermore, the expression of CS-E in MDM was increased 2 times following PMA treatment. An inactive phorbol ester, phorbol 12,13-diacetate, did not affect the expression of CS-E in either monocytes or MDM when compared with control cultures, suggesting that protein kinase C-dependent signal pathways may be involved in the regulation of sulfation of CSPG. Exposure to LPS or IFN-gamma did not lead to any changes in the sulfation of the chondroitin sulfate chains.     

Structure of a fucose-branched chondroitin sulfate from sea cucumber. Evidence for the presence of 3-O-sulfo-beta-D-glucuronosyl residues

The structure of a unique focose-branched chondroitin sulfate isolated from the body wall of a sea cucumber was examined in detail. This glycosaminoglycan contains side chain disaccharide units of sulfated fucopyranosyl units linked to approximately one-half of the glucuronic acid moieties through the O-3 position of the acid. The intact polysaccharide is totally resistant to chondroitinase degradation, whereas, after defucosylation, it is partially degraded by the enzyme. However, only after an additional step of desulfation, the chondroitin from sea cucumber is almost totally degraded by chondroitinase AC or ABC. This result, together with the methylation and NMR studies of the native and chemically modified polysaccharide, suggest that besides the fucose branches, the sea cucumber chondroitin sulfate contains sulfate esters at position O-3 of the beta-D-glucuronic acid units. Furthermore, the proteoglycan from the sea cucumber chondroitin sulfate is recognized by anti-Leu-7 monoclonal antibody, which specifically recognizes 3-sulfoglucuronic acid residues. In analogy with the fucose branched units, the 3-O-sulfo-beta-D-glucuronosyl residues are resistant to chondroitinase degradation. Regarding the position of the glycosidic linkage and site of sulfation in the fucose branches, our results suggest high heterogeneity. Tentatively, it is possible to suggest the preponderance of disaccharide units formed by 3,4-di-O-sulfo-alpha-L-fucopyranosyl units glycosidically linked through position 1----2 to 4-O-sulfo-alpha-L-fucopyranose. Finally, the presence of unusual 4/6-disulfated disaccharide units, together with the common 6-sulfated and non-sulfated units, was detected in the chondroitin sulfate core of this polysaccharide.     

Kinetics of Determination in the Differentiation of Isolated Mesophyll Cells of Zinnia elegans to Tracheary Elements

Mechanically isolated mesophyll cells of Zinnia elegans L. cv Envy differentiate to tracheary elements when cultured in inductive medium containing 0.5 micromolar α-naphthaleneacetic acid and 0.5 micromolar benzyladenine. The cells do not differentiate when cultured in medium in which the concentration of auxin and/or cytokinin has been reduced to 0.005 micromolar. Cells require an initial 24-hour exposure to inductive cytokinin and 56-hour exposure to inductive auxin for differentiation at 72 hours of culture. Freshly isolated Zinnia cells can be maintained in medium having low concentrations of both auxin and cytokinin for only 1 day without significant loss of potential to differentiate upon transfer to inductive medium. Initial culture for up to 2 days in medium having high auxin and low cytokinin, or low auxin and high cytokinin, allows full differentiation on the third day after transfer to inductive medium and potentiates the early differentiation of some cells.     

The isolation and cultivation of rabbit bone marrow mononuclear phagocytes

Rabbit bone marrow cells were cultured in a liquid medium in the presence of high concentrations of serum but in the absence of other added exogenous stimulating substances. Conditions were established that allow the selective proliferation of the precursor cells of the mononuclear phagocytes and their differentiation into macrophages. These cells were identified both on the basis of their morphological appearance as well as on several other properties (presence of Fc receptors at their surface, rapid phagocytosis of latex or India ink, continuous secretion of lysozyme). Their proliferation is preceded by the rapid degeneration of all the other cell types, including the granulocytes that are no longer found after 4 days of culture. Macrophage precursors (monoblasts and promonocytes) proliferate and persist in suspension until the 8th–10th day of culture when they become adherent. Essentially pure populations of macrophages could be maintained in culture for at least 4 weeks.     

Photodynamic inhibition of enzymatic detachment of human cancer cells from a substratum

The metabolism of poly(ADP-ribose) is known to play important roles in the nuclear function of the mammalian cells. In this study, changes in the activities and gene expressions of poly(ADP-ribose) glycohydrolases (PARG) in HL-60 cells treated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or a PARG inhibitor, tannic acid, were investigated. Nuclear PARG activities of HL-60 cells treated with TPA were reduced to 30-40% of the activity in untreated cells at 24 h, while PARG activities in the cytoplasm remained unchanged. The transient decrease in the nuclear PARG activity by TPA treatment was accompanied by differentiation as measured by the nitroblue tetrazolium (NBT) reducing activity and adhesion to the culture dishes. In the presence of H7, an inhibitor of protein kinase C (PKC), both the decrease in nuclear PARG activity and the induction of differentiation by TPA treatment were suppressed. On the other hand, treatment with tannic acid caused the nuclear PARG activity to decrease continuously while the NBT reducing activity increased, but no morphological differentiation to macrophage-like cells was apparent. In order to analyze PARG gene expression, we isolated the human PARG cDNA by the RT-PCR technique. RT-PCR analysis revealed that TPA treatment leads to a reduction in the PARG gene expression prior to the phenotypic expression of macrophage-like cell differentiation, which was diminished by the presence of H7. Also, PARG gene expression was reduced by tannic acid treatment. These results provide the first evidence that a transient decrease in nuclear PARG activity is important for the onset of differentiation of HL-60 cells to macrophage-like cells.     

Effect of p-nitrophenyl-beta-D-xyloside on proteoglycan synthesis and extracellular matrix formation by bovine corneal endothelial cell cultures

The effect of p-nitrophenyl-beta-D-xyloside on proteoglycan synthesis and extracellular matrix (ECM) formation by cultured bovine corneal endothelial (BCE) cells was investigated. BCE cells actively proliferating on plastic dishes produced in the absence of xyloside an ECM containing various proteoglycans. Heparan sulfate was the main 35S-labeled glycosaminoglycan component (83%). Dermatan sulfate (14%) and chondroitin sulfate (3%) were also present. Exposure of actively proliferating BCE cells to xyloside totally inhibited synthesis of proteoglycans containing dermatan sulfate or chondroitin sulfate and caused an 86% inhibition of heparan sulfate proteoglycan synthesis. The heparan sulfate proteoglycans that were extracted from the ECM produced by BCE cells exposed to xyloside had a smaller size and a reduced charge density compared to their counterparts extracted from the ECM of cultures not exposed to xyloside. In contrast to the inhibitory effect of the xyloside on proteoglycan synthesis, exposure of actively proliferating BCE cells to xyloside stimulated synthesis of free chondroitin sulfate and heparan sulfate chains. All of the xyloside-initiated glycosaminoglycan chains were secreted into the culture medium. The proteoglycan-depleted matrices produced by BCE cells exposed to xyloside were used to study the effect of these matrices on proteoglycan synthesis by BCE cells. BCE cells growing on proteoglycan-depleted ECM showed a considerable increase in the rate of proteoglycan synthesis compared to BCE cells growing on normal ECM. Moreover, the pattern of glycosaminoglycan synthesis by BCE cells growing on proteoglycan-depleted ECM was changed to one which resembled that of BCE cells actively proliferating on plastic dishes. It is postulated that BCE cells are able to recognize when an ECM is depleted of proteoglycan and to respond to it by increasing their rate of proteoglycan synthesis and incorporation into the ECM.     

Sodium spirulan as a potent inhibitor of arterial smooth muscle cell proliferation in vitro

Sodium spirulan (Na-SP) is a sulfated polysaccharide with M(r) approximately 220,000 isolated from the blue-green alga Spirulina platensis. The polysaccharide consists of two types of disaccharide repeating units, O-hexuronosyl-rhamnose (aldobiuronic acid) and O-rhamnosyl-3-O-methylrhamnose (acofriose) with sulfate groups, other minor saccharides and sodium ion. Since vascular smooth muscle cell proliferation is a crucial event in the progression of atherosclerosis, we investigated the effect of Na-SP on the proliferation of bovine arterial smooth muscle cells in culture. It was found that Na-SP markedly inhibits the proliferation without nonspecific cell damage. Either replacement of sodium ion with calcium ion or depolymerization of the Na-SP molecule to M(r) approximately 14,700 maintained the inhibitory activity, however, removal of sodium ion or desulfation markedly reduced the activity. Heparin and heparan sulfate also inhibited vascular smooth muscle cell growth but their effect was weaker than that of Na-SP; dextran sulfate, chondroitin sulfate, dermatan sulfate and hyaluronan failed to inhibit the cell growth. The present data suggest that Na-SP is a potent inhibitor of arterial smooth muscle cell proliferation, and the inhibitory effect requires a certain minimum sequence of polysaccharide structure whose molecular conformation is maintained by sodium ion bound to sulfate group.     

Biosynthesis of heparin. Effects of n-butyrate on cultured mast cells

Murine mastocytoma cells were incubated in vitro with inorganic [35S]sulfate, in the absence or presence of 2.5 mM n-butyrate, and labeled heparin was isolated. The polysaccharide produced in the presence of butyrate showed a lower charge density on anion exchange chromatography than did the control material and a 3-fold increased proportion (54 versus 17% for the control) of components with high affinity for antithrombin. Structural analysis of heparin labeled with [3H] glucosamine in the presence of butyrate showed that approximately 35% of the glucosamine units were N-acetylated, as compared to approximately 10% in the control material; the nonacetylated glucosamine residues were N-sulfated. The presence of butyrate thus leads to an inhibition of the N-deacetylation/N-sulfation process in heparin biosynthesis, along with an augmented formation of molecules with high affinity for antithrombin. Preincubation of the mastocytoma cells with butyrate was required for manifestation of either effect; when the preincubation period was reduced from 24 to 10 h the effects of butyrate were no longer observed. Assays for microsomal N-acetylheparosan deacetylase activity failed to show any significant inhibition of the enzyme at butyrate concentrations well above those found to affect heparin biosynthesis in intact mastocytoma cells. Moreover, a polysaccharide formed on incubating mastocytoma microsomal fraction with UDP-[3H]glucuronic acid, UDP-N-acetylglucosamine, and 3'-phosphoadenylylsulfate in the presence of 5 mM butyrate showed the same N-acetyl/N-sulfate ratio as did the corresponding control polysaccharide, produced in the absence of butyrate. These findings suggest that the effect of butyrate on heparin biosynthesis depends on the integrity of the cell.