Effect of chondroitinase on dermatan sulfate-facilitated arginine amidase released from rabbit ear artery.

We examined the effects of chondroitinases on the release of dermatan sulfate (DS)-induced arginine amidase (AA) from rabbit ear artery. DS-induced AA release was significantly decreased by treatment with chondroitinase ABC (ABCase) in the rabbit ear artery. On the other hand, Chondroitinase ACII (ACIIase) enhanced spontaneous and DS-induced AA release. Heat-inactivated ABCase and ACIIase did not affect spontaneous and DS-induced AA release. Furthermore, ABCase, but not ACIIase and heat-inactivated chondroitinases, degraded DS. These results indicate that the facilitatory effect of DS-induced AA release from the rabbit ear artery is affected by the molecular size of DS.

A cell surface chondroitin sulfate proteoglycan, immunologically related to CD44, is involved in type I collagen-mediated melanoma cell motility and invasion.

The metastatic spread of tumor cells occurs through a complex series of events, one of which involves the adhesion of tumor cells to extracellular matrix (ECM) components. Multiple interactions between cell surface receptors of an adherent tumor cell and the surrounding ECM contribute to cell motility and invasion. The current studies evaluate the role of a cell surface chondroitin sulfate proteoglycan (CSPG) in the adhesion, motility, and invasive behavior of a highly metastatic mouse melanoma cell line (K1735 M4) on type I collagen matrices. By blocking mouse melanoma cell production of CSPG with p-nitrophenyl beta-D-xylopyranoside (beta-D-xyloside), a compound that uncouples chondroitin sulfate from CSPG core protein synthesis, we observed a corresponding decrease in melanoma cell motility on type I collagen and invasive behavior into type I collagen gels. Melanoma cell motility on type I collagen could also be inhibited by removing cell surface chondroitin sulfate with chondroitinase. In contrast, type I collagen-mediated melanoma cell adhesion and spreading were not affected by either beta-D-xyloside or chondroitinase treatments. These results suggest that mouse melanoma CSPG is not a primary cell adhesion receptor, but may play a role in melanoma cell motility and invasion at the level of cellular translocation. Furthermore, purified mouse melanoma cell surface CSPG was shown, by affinity chromatography and in solid phase binding assays, to bind to type I collagen and this interaction was shown to be mediated, at least in part, by chondroitin sulfate. Additionally we have determined that mouse melanoma CSPG is composed of a 110-kD core protein that is recognized by anti-CD44 antibodies on Western blots. Collectively, our data suggests that interactions between a cell surface CD44-related CSPG and type I collagen in the ECM may play an important role in mouse melanoma cell motility and invasion, and that the chondroitin sulfate portion of the proteoglycan seems to be a critical component in mediating this effect.

Leukocyte interleukins induce cultured endothelial cells to produce a highly organized, glycosaminoglycan-rich pericellular matrix.

We report here that interleukins have a dramatic effect on extracellular matrix production by cultured endothelial cells. Human umbilical vein endothelial cells incubated with growth media conditioned by lectin-activated human peripheral blood mononuclear leukocytes undergo marked changes in cell shape and elaborate a highly organized extracellular material that is not detectable in untreated cultures. This material has the following characteristics: (a) it is not recognizable by electron microscopy unless the cationic dye, Alcian blue, is added to the fixative; (b) it is visualized as a network of branching and anastomosing fibrils of various thickness that can be resolved into bundles of fine filaments; (c) it is associated with the cell surface, extends between contiguous cells, and coats the culture substrate; (d) it is removed by digestion with glycosaminoglycan-degrading enzymes, such as crude heparinase and chondroitinase ABC. These results demonstrate that soluble factors released by activated peripheral blood mononuclear leukocytes (interleukins) stimulate cultured human umbilical vein endothelial cells to produce a highly structured pericellular matrix containing glycosaminoglycans (probably chondroitin sulfate and/or hyaluronic acid) as a major constituent. We speculate that this phenomenon corresponds to an early step of angiogenesis as observed in vivo as a consequence of interleukin release.

Adherence of Plasmodium falciparum to chondroitin sulfate A in the human placenta.

Women are particularly susceptible to malaria during first and second pregnancies, even though they may have developed immunity over years of residence in endemic areas. Plasmodium falciparum-infected red blood cells (IRBCs) were obtained from human placentas. These IRBCs bound to purified chondroitin sulfate A (CSA) but not to other extracellular matrix proteins or to other known IRBC receptors. IRBCs from nonpregnant donors did not bind to CSA. Placental IRBCs adhered to sections of fresh-frozen human placenta with an anatomic distribution similar to that of naturally infected placentas, and this adhesion was competitively inhibited by purified CSA. Thus, adhesion to CSA appears to select for a subpopulation of parasites that causes maternal malaria.

Chondroitin sulfate as a regulator of neuronal patterning in the retina.

Highly sulfated proteoglycans are correlated with axon boundaries in the developing central nervous system which suggests that these molecules affect neural pattern formation. In the developing mammalian retina, gradual regression of chondroitin sulfate may help control the onset of ganglion cell differentiation and initial direction of their axons. Changes induced by the removal of chondroitin sulfate from intact retinas in culture confirm the function of chondroitin sulfate in retinal histogenesis.

Antitumor effect of chondroitin AC lyase (PsPL8A) from Pedobacter saltans on melanoma and fibrosarcoma cell lines by in vitro analysis.

BACKGROUND: PGs are involved in cellular communication and cancer biology. The role of CS in melanoma and fibrosarcoma cell lines was explored by using chondroitin AC lyase (PsPL8A). METHODS: The proliferation of mouse fibroblast L929, human melanoma (SK-Mel 28) and fibrosarcoma (HT-1080) cell lines after treatment with chondroitin AC lyase (PsPL8A) was studied by MTT assay. The mode of cell death was studied by Annexin-V FITC using flow cytometry and fluorescence microscopy. The alteration in mitochondrial cell potential was studied by JC-1 dye using fluorescence microscopy and flow cytometry. RESULTS: Treatment of L929 cells with PsPL8A imparts no cytotoxicity and showed no alteration in proliferation with nearly 95-98% cell viability. An overall 58% and 59% inhibition of SK-Mel 28 and HT-1080 cell proliferation was observed with 1.3 µM of PsPL8A after 24 h of incubation. The PsPL8A (1.3 µM) treated SK-Mel 28 and HT-1080 cells showed significant green fluorescence with annexin-V FITC under fluorescence microscopy and 56.6% and 35.5% apoptosis, respectively by flow cytometry analysis. The results of fluorescence microscopy and flow cytometry of SK-Mel 28 and HT-1080 upon treatment with PsPL8A (1.3 µM) for 24 h, gave green fluorescence due to dissipation of mitochondrial potential with JC-1 dye. CONCLUSIONS: Chondroitin AC lyase (PsPL8A) displayed anti-tumor potential against human melanoma SK-Mel 28 and fibrosarcoma HT-1080 cell lines, while the mouse fibroblast L929 cells were unaffected.

Inhibitors and promoters of thalamic neuron adhesion and outgrowth in embryonic neocortex: functional association with chondroitin sulfate.

When embryonic thalamic neurons are plated onto living slices of mouse forebrain, cell attachment and neurite outgrowth on different layers of the developing cerebral cortex vary dramatically, in ways that correlate with the timing and pattern of thalamocortical innervation. These layer-specific differences can be eliminated from embryonic day 16 slices by enzymatic removal of chondroitin sulfate (CS). The cortical plate (a zone avoided by thalamic axons in vivo) possesses inhibitory activity (anti-adhesive, neurite repelling) and the intermediate zone and subplate (in which thalamic axons normally grow) possess stimulatory activity (adhesive, neurite promoting), both of which are chondroitinase sensitive. These opposing activities appear not to reflect the presence of different CS proteoglycans (CSPGs) in different zones, but rather the presence of differentially localized CS-binding molecules, which can be competed away by soluble CS. This model reconciles conflicting reports on the actions of CSPGs in neural development, and suggests a role for CSPGs in the organization of matrix-bound cues in the brain.

CD44-related chondroitin sulfate proteoglycan, a cell surface receptor implicated with tumor cell invasion, mediates endothelial cell migration on fibrinogen and invasion into a fibrin matrix.

Microvascular endothelial cell invasion into the fibrin provisional matrix is an integral component of angiogenesis during wound repair. Cell surface receptors which interact with extracellular matrix proteins participate in cell migration and invasion. Malignant cells use CD44-related chondroitin sulfate proteoglycan (CSPG) as a matrix receptor to mediate migration and invasion. In this study, we examine whether cell surface CSPG can mediate similar events in nonmalignant wound microvascular endothelial cells or whether use of CSPG for migration and invasion is a property largely restricted to malignant cells. After inhibiting CSPG synthesis with p-nitrophenyl beta-d xylopyranoside (beta-d xyloside), wound microvascular endothelial cells were capable of attaching and spreading on the surface of a fibrin gel; however, their ability to invade the fibrin matrix was virtually eliminated. To begin to examine the mechanism by which endothelial cells use CSPG to invade fibrin matrices, cell adhesion and migration on fibrinogen was examined. Endothelial cell adhesion and migration on fibrinogen were inhibited by both beta-d xyloside and after cleavage of chondroitin sulfate from the core protein by chondroitinase ABC. We have determined that wound microvascular endothelial cells express the majority of their proteoglycan as CSPG and that the CSPG core protein is immunologically related to CD44. PCR studies show that these cells express both the "standard" (CD44H) isoform and an isoform containing the variably spliced exon V3. In addition, anti-CD44 antibody blocks endothelial cell migration on fibrinogen. Affinity chromatography studies reveal that partially purified microvascular endothelial cell CSPG binds fibrinogen. These findings suggest that CD44-related CSPG, a molecule implicated in the invasive behavior of tumor cells, is capable of binding fibrinogen/fibrin, thereby mediating endothelial cell migration and invasion into the fibrin provisional matrix during wound repair.

Subendothelial retention of lipoprotein (a). Evidence that reduced heparan sulfate promotes lipoprotein binding to subendothelial matrix.

Vessel wall subendothelial extracellular matrix, a dense mesh formed of collagens, fibronectin, laminin, and proteoglycans, has important roles in lipid and lipoprotein retention and cell adhesion. In atherosclerosis, vessel wall heparan sulfate proteoglycans (HSPG) are decreased and we therefore tested whether selective loss of HSPG affects lipoprotein retention. A matrix synthesized by aortic endothelial cells and a commercially available matrix (Matrigel; , Rutherford, NJ) were used. Treatment of matrix with heparinase/heparitinase (1 U/ml each) increased LDL binding by approximately 1.5-fold. Binding of lipoprotein (a) [Lp(a)] to both subendothelial matrix and Matrigel(R) increased 2-10-fold when the HSPG were removed by heparinase treatment. Incubation of endothelial cells with oxidized LDL (OxLDL) or lysolecithin resulted in decreased matrix proteoglycans and increased Lp(a) retention by matrix. The effect of OxLDL or lysolecithin on endothelial PG was abolished in the presence of HDL. The decrease in matrix HSPG was associated with production of a heparanase-like activity by OxLDL-stimulated endothelial cells. To test whether removal of HSPG exposes fibronectin, a candidate Lp(a) binding protein in the matrix, antifibronectin antibodies were used. The increased Lp(a) binding after HSPG removal was inhibited 60% by antifibronectin antibodies. Similarly, the increased Lp(a) binding to matrix from OxLDL-treated endothelial cells was inhibited by antifibronectin antibodies. We hypothesize that atherogenic lipoproteins stimulate endothelial cell production of heparanase. This enzyme reduces HSPG which in turn promotes Lp(a) retention.

A novel use of TAT-EGFP to validate techniques to alter osteosarcoma cell surface glycosaminoglycan expression.

Several methods to alter cell surface glycosaminoglycan (GAG) expression have previously been described, including treatments with chlorate to reduce the addition of charged sulfate groups, xyloside compounds to displace GAGs from their core proteins, and GAG lyases, such as heparinase and chondroitinase, to release GAG fragments from the cell layer. While these methods are useful in identifying cellular mechanisms which are dependent on GAGs, they must be stringently validated to assess results in the appropriate context. To determine the most useful technique for the evaluation of GAG function in osteogenesis, MG-63 osteosarcoma cells were systematically treated with these agents and evaluated for changes in cell surface GAGs using a TAT-EGFP fusion protein. TAT, a protein transduction domain from the HIV-1 virus, requires cell surface GAGs to traverse cell membranes. The EGFP component provides a method to assess protein entry into cells in both qualitative and quantitative tests. Here, TAT-EGFP transduction analysis confirmed radiochemical and physiological data that chlorate effectively disrupts GAG expression. TAT-EGFP entry into cells was also inhibited by the exogenous application of commercial heparin and GAGs extracted from MG-63 cells as well as by the pre-treatment of cells with chondroitinase ABC. However, neither heparinase III treatment nor the addition of exogenous chondroitin-6-sulfate affected TAT-EGFP entry into cells. In addition, xyloside-beta-D-naphthol and xyloside-beta-D-cis/trans-decahydro-2-naphthol treatment could not induce significant phenotypic change in these cells, and the unaffected TAT-EGFP transduction confirmed that this was due to an inability to efficiently prime GAG synthesis. The use of TAT-EGFP is thus a useful technique to specifically evaluate cell surface GAG expression in a simple, quantifiable manner, and avoids the complications involved with conventional radiochemical assays or analytical chromatography.

Role of glycosaminoglycans in calcium metabolism of human platelets.

Changes in intracellular Ca2+, [Ca2+]i, were measured in control and chondroitin ABC lyase-pretreated platelets. [Ca2+]i was measured with the fluorescent calcium probe Quin2. Chondroitin ABC lyase removed chondroitin 4-sulfate from the platelet surface without inducing shape change or release of serotonin. Compared to similarly prepared controls, enzyme treated platelets showed an increase of [Ca2+]i in response to stimulation by various agonists at high (1 mM) extracellular Ca2+ concentration. At low Ca2+ in the medium (1 mM EGTA), such platelets responded to agonists with a decreased rise in [Ca2+]i compared to the controls. These studies indicate that selective removal of glycosaminoglycans may sensitize platelets to the action of platelet aggregating agents. In addition, glycosaminoglycans may have a calcium storage function.

Platelet surface glycosaminoglycans are an effective shield for distinct platelet receptors.

The presence of glycosaminoglycans on the platelet surface was demonstrated by electronmicroscopy and biochemical analysis. Chondroitin ABC lyase was able to remove a substantial portion of the Ruthenium red-stained outer coat of platelets. Analysis of the reaction product released by the enzyme revealed chondroitin 4-sulfate. To determine the biological function of this glycosaminoglycan coat, binding studies with a variety of potential platelet ligands were performed. In decreasing order of effectiveness, chondroitin ABC lyase was able to increase the binding sites of von Willebrand factor, fibrinogen, antibody to platelet-specific antigen P1A1, Fc fragments of IgG, and monomeric IgG. No change in binding was observed with F(ab)2 fragments of IgG, wheat germ agglutinin and pokeweed mitogen. These studies indicate that glycosaminoglycans shield some platelet receptor sites from their respective ligands. Upon release of the heteropolysaccharide from the platelet surface more of these sites become accessible to the ligand. It may be significant that especially glycoproteins involved in platelet adhesion and aggregation are involved in this process.

The proteoglycans of the canine intervertebral disc.

The high-buoyant-density proteoglycans of the nucleus pulposus and annulus fibrosus of the beagle intervertebral disc have been isolated by CsCl density gradient ultracentrifugation. The sulphated proteoglycans were labelled in vivo with 35SO4, 24 h and 60 days prior to killing. The hydrodynamic size and aggregation of the 24 h, 60 day and resident (from hexuronic acid and hexosamine analysis) proteoglycan subunit populations were determined by Sepharose CL-2B chromatography in the presence or absence of excess hyaluronic acid. The hydrodynamic size of the keratan sulphate-proteoglycan core protein complexes were also determined by Sepharose CL-2B chromatography after chondroitinase ABC digestion of proteoglycans. When initially synthesised (24 h) or after 60 days, the percentage aggregation and hydrodynamic size of the proteoglycans derived from the annulus fibrosus were larger than those present in the nucleus pulposus. Hexosamine, hexuronic and protein determination of the high-buoyant-density fractions showed that the proteoglycans of the nucleus pulposus were richer in chondroitin sulphate than those in the annulus. However there was no difference in Mr of the chondroitin sulphate and keratan sulphate attached to the proteoglycans of the two disc regions, nor were differences detected by HPLC between the proportions of chondroitin 4-sulphate and chondroitin 6-sulphate present in these high-density fractions. In contrast, the low-buoyant-density (1.54 greater than p greater than 1.45) proteoglycan fractions and tissue residues remaining after 4 M GuHCl extraction were found to contain dermatan sulphate, suggesting the presence of a third proteoglycan species possibly associated with the collagen of the fibrocartilagenous matrix.

Secretion of mucoid material by lymphokine-activated killer cells: study by light and electron microscopy.

When lymph node cells from nude mice were grown on embryonic fibroblast monolayers together with rat interleukin-2, only one type of colonies developed. These colonies were composed of cytotoxic cells termed "granular/lymphokine-activated killer/mucus-secreting cells" (LAK-GM). An extensive differentiation course, in which all the cellular components were involved, ended with a population of short-lived, mature, nondividing large cells that apparently synthesized and deposited a flowing mucoid material (FMM) that stained distinctly blue with periodic acid-Schiff/alcian blue (PAS-Ab) at pH 1 and distinctly red by the naphthol AS-D-chloracetate method for specific esterase. So far, the best monolayers to trigger the FMM synthesis were those prepared from 16- to 18-day-old whole embryos. These cells were compared with LAK cells that developed on monolayers (such as embryonic skin or adult kidney) that did not trigger FMM synthesis. They were also compared with other cell types that differentiated in colonies on the fibroblast monolayers: histiocytes (fixed macrophages), mixed granulocytes/monocytes, mucosal mast cells; and with populations of mature rat T-killer cells developed on same mouse monolayers. Features distinctive to the secreting LAK-GM cells were presence of masses of membrane-limited vesicles that were strictly confined to the surface of the cells in FMM-containing colonies. All transitional forms of budding activity could be seen on the cell surface facing the masses. Within the same cells, many granules displayed varying degrees of degradation, the granular material being transformed into flocculent material that formed small pools facing each degraded surface. Other characteristics of the LAK-GM lineage were the accumulation of glycogen prior to the appearance of the FMM, the presence of several structures of a ribosome-lamella complex in the LAK-GM in colonies that did not accumulate FMM, and filopodia commonly emerging from the pole proximal to the nucleus. Of various fixation methods tried, only after treatment with absolute alcohol and subsequent drying was the FMM stained with PAS-Ab. By subsequent wetting, the capacity to be stained was irreversibly lost. After incubation of the living cultures with the enzymes hyaluronidase or chondroitinases AC or ABC, the FMM disappeared. These observations suggest a triggering mechanism by the embryonic mesenchymal fibroblastoid cells for synthesis and secretion of mucous material that is a proteoglycan of the chondroitin sulfate group.

Characterization of a human melanosome-associated antigen recognized by monoclonal antibody, HMSA-2.

This study elucidates the nature of antigens recognized by monoclonal antibody (MoAb) HMSA-2, which was developed against human melanosome-associated antigen (HMSA) of malignant melanoma (Maeda and Jimbow, Cancer 59:415-423, 1987). Through flow cytometry analyses, indirect immunoprecipitation of antigen biosynthetically labeled with 35S-methionine, enzyme-linked immunosorbent assays and immunoelectron microscopy, we found that a) the antigens recognized by MoAb HMSA-2 were melanosomal matrix glycoproteins; b) these antigens were expressed mainly in the cytoplasm, although they could also be detected on the cell surface; c) the cytoplasmic expression of MoAb HMSA-2 was cell-cycle dependent; d) large amounts of these antigens were released into culture supernatants; e) MoAb HMSA-2 immunoprecipitated two major glycoproteins with molecular weights of 94 and 53 kDa from culture supernatants, and f) both components have complex N-linked oligosaccharide chains with sialic acid, suggesting that these melanosomal proteins are derived from the trans-cisternae of the Golgi. These human melanosome-associated antigens may prove useful not only for studying the immunobiology of melanogenesis, but also for the immunodiagnosis of melanocytic disorders.

Heparin inhibition of insulin-like growth factor-binding protein-3 binding to human fibroblasts and rat glioma cells: role of heparan sulfate proteoglycans.

The mitogenic action of insulin-like growth factors (IGFs) on target cells is determined by interaction with signaling IGF-I receptors and modulated by interactions with IGF-binding proteins (IGFBPs). IGFBP-3, an abundant IGFBP that binds IGF-I and IGF-II with high affinity, can form soluble inhibitory complexes with the IGFs that prevent them from binding to IGF-I receptors. Alternatively, IGFBP-3 can bind to the cell surface and possibly potentiate IGF action or act independently of the IGFs. Previous studies showed that heparin inhibited IGFBP-3 binding to the cell surface and increased its accumulation in the medium, suggesting that it might act as a competitive inhibitor of IGFBP-3 binding to structurally similar heparan sulfate proteoglycans on the cell surface. We evaluated this hypothesis by binding 125I-labeled recombinant glycosylated human IGFBP-3 to human fetal skin fibroblasts (GM-10) and to C6 rat glioma cells at 12 C. Heparin inhibited [125I]IGFBP-3 binding more effectively than chondroitin sulfate and dextran sulfate. Complete digestion of cell surface heparan sulfate and chondroitin sulfate glycosaminoglycans using heparitinase and chondroitinase ABC, however, did not significantly decrease IGFBP-3 binding. Quantitative removal was demonstrated by analysis of parallel cultures of cells whose glycosaminoglycans had been biosynthetically labeled using Na2 35SO4. These results suggested that IGFBP-3 did not bind to heparan sulfate glycosaminoglycans on the cell surface, and that the inhibition of IGFBP-3 binding by heparin most likely resulted from its direct interaction with the heparin-binding domains of IGFBP-3. When [125I]IGFBP-3 was incubated with GM-10 fibroblasts or C6 glioma cells at 37 C for 4 h, only 10% of the bound ligand remained associated with the cell surface; approximately 90% of the cell-associated radio-activity was internalized and could be recovered in lysates of acid-washed cells. Incubation with IGF-I or heparin decreased the total cell-associated radioactivity, but did not affect internalization. These results suggest that direct interaction of heparin or IGF-I with IGFBP-3 inhibits its ability to bind to the surface of GM-10 fibroblasts and C6 glioma cells.

Glycosaminoglycan and collagen fibrillar interactions in the mouse corneal stroma.

When sections of mouse corneal stroma were treated with 20 mM adenosine 5'-triphosphate (ATP) in phosphate buffered saline, pH 4.0, at 37 degrees C and observed by electron microscopy, numerous periodic fibrils with about 100-nm periodicity appeared which were the aggregated form of type VI collagen (type VI collagen fibrils). They occurred in close association with D-periodic fibrillar collagens (striated collagen fibrils). However, when the tissue was digested with chondroitinase ABC or testicular hyaluronidase prior to the ATP treatment, type IV collagen fibrils were segregated from striated collagen fibrils, even though the type VI collagen fibrils themselves aggregated to form the 100 nm-periodic structures. Keratanase or Streptomyces hyaluronidase had no such effect. One possible suggestion is that the ATP-aggregated type VI collagen fibrils are connected with striated collagen fibrils through chondroitin/dermatan sulfate glycosaminoglycans.

Ultrastructural visualization of carbohydrates in oxytalan fibers in monkey periodontal ligaments.

Fullmer's oxytalan fibers appear to be special connective tissue fibers belonging to elastic system fibers. We have ultrastructurally examined carbohydrates in oxytalan fibers in monkey periodontal ligaments after glutaraldehyde fixation and ethylenediaminetetraacetic acid (EDTA) decalcification using: Thiéry's periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method for thin-section staining of vicinal glycol-containing complex carbohydrates, and the concanavalin A-ferritin (Con A-ferritin) and Con A-horseradish peroxidase (Con-A-HRP) en bloc staining methods specific for alpha-D-mannosyl and alpha-D-glucosyl groups. PA-TCH-SP stained collagen fibrils weakly to moderately and stained oxytalan fibers moderately. Con A-ferritin and Con A-HRP stained collagen fibrils weakly or moderately and stained oxytalan fibers intensely within the superficial region of specimen blocks. The penetration of staining reagents was improved by prior saponin treatment and/or chondroitinase ABC digestion. Thus, these studies demonstrate that PA-TCH-SP and Con A staining of carbohydrates is very useful in identifying oxytalan fibers at the ultrastructural level and that more carbohydrate components are present in oxytalan fibers than in collagen fibrils.

Polyethyleneimine as a contrast agent for ultrastructural localization and characterization of proteoglycans in the matrix of cartilage and bone.

We examined the presence of proteoglycans in the extracellular matrix of cartilage and bone in fetal mouse radii at the ultrastructural level, using the cationic dye polyethyleneimine (PEI). After staining with this dye, the proteoglycans appeared as granules in the uncalcified bone matrix and as extended winding structures in the cartilage matrix. PEI-positive material was removed after treatment of the tissue with chondroitinase ABC. Inhibition of the proteoglycan synthesis by beta-D-xyloside resulted in smaller PEI-positive windings in the cartilage matrix. These observations suggest that the winding, PEI-positive structures represent proteoglycan aggregates. No loss of PEI-positive material in the calcified cartilage matrix was seen, suggesting that proteoglycans do not need to be removed to make the matrix calcifiable.

A role for pericellular proteoglycan in the binding of thrombin or antithrombin III by the blood vessel endothelium? The effects of proteoglycan-degrading enzymes and glycosaminoglycan-binding proteins on 125I-thrombin binding by the rabbit thoracic aorta in vitro.

Rabbit thoracic aorta segments were treated with either proteoglycan-degrading enzymes or with glycosaminoglycan-binding proteins to examine the nature of the endothelial and subendothelial binding sites of 125I-thrombin. Treatment (5-30 min) with enzymes (heparitinase, chondroitinases AC or ABC) caused a decrease in 125I-thrombin binding by the endothelium (30-70%) and by the subendothelial (intima-media) layer (20-50%); a low-specificity protease destroyed endothelial binding almost entirely and reduced binding to the subendothelium by approximately 60% over a similar period. Of the glycosaminoglycan-binding proteins, pretreatment of the aorta wall with protamine caused a 30% decrease in thrombin binding to the endothelium whereas lipoprotein lipase (present during 125I-thrombin uptake) decreased binding by up to 40%. Pretreatment with antithrombin III did not significantly affect binding of either 125I-thrombin or 125I-FPR-inactivated thrombin. In contrast to thrombin, 125I-antithrombin III was not readily uptaken by the aorta segments. These observations indicate that, whereas the minimal binding by 125I-antithrombin III probably does not involve endothelial proteoglycan, a strong case can be made for endothelial and subendothelial proteoglycan binding sites for thrombin.