Sulfated polysaccharides inhibit the catabolism and loss of both large and small proteoglycans in explant cultures of tendon

This study investigated the effects of two highly sulfated polysaccharides, calcium pentosan polysulfate and heparin, on the loss of newly synthesized proteoglycans from the matrix of explant cultures of bovine tendon. The tensional region of deep flexor tendon was incubated with [35S]sulfate for 6 h and then placed in culture for up to 15 days. The amount of radiolabel associated with proteoglycans lost to the medium and retained in the matrix was determined for each day in culture. It was shown that both sulfated polysaccharides at concentrations of 1000 microg x mL(-1) inhibited the loss of 35S-labeled large and small proteoglycans from the matrix and concomitant with this was a retention of chemical levels of proteoglycans in the explant cultures. In other explant cultures that were maintained in culture in the presence of both agents for more than 5 days after incubation with [35S]sulfate, inhibition of the intracellular catabolic pathway was evident, indicating that these highly sulfated polysaccharides also interfered with the intracellular uptake of small proteoglycans by tendon cells.     

Inhibition of aggrecan turnover in short-term explant cultures of bovine tendon.

The large aggregating proteoglycan, aggrecan, better known for its physiological role in articular cartilage where it serves to facilitate resistance of compressive forces during joint articulation, is also present within the distinct functional regions of tendon (i.e., compressed/fibrocartilaginous and tensional). Previous studies demonstrate that an increased turnover of aggrecan occurs in tendon, which is mediated principally by the 'aggrecanases' and, as such, these proteinases may play an important role in the normal functioning of the tissue. In the present study, utilising bovine tendon explant culture systems, we demonstrated that aggrecanase-mediated tendon aggrecan turnover may be modulated by generic metalloproteinase inhibitors (i.e., the aggrecanase inhibitor, actinonin and the broad-spectrum MMP inhibitor, marimistat). As expected, no MMP-generated aggrecan catabolites were detected in the culture system, suggesting that tendon aggrecanases may be inhibited by marimistat. Furthermore, immunohistochemical analyses revealed that aggrecan metabolites are present in the endotenon, surrounding the collagen fibre bundles, suggesting that aggrecan may provide functions of water imbibement and resistance of reversible and repeated compressive loads manifest between the collagen fibres; these functions, in turn, may be associated with increased aggrecan turnover in this tissue. Thus, inhibition of tendon aggrecanases and consequently aggrecan turnover in this tissue, may be related to some of the deleterious effects observed in the tendons of patients undergoing drug therapy with broad-spectrum MMP inhibitors for cancer and arthritis.     

Catabolism of aggrecan proteoglycan aggregate components in short-term explant cultures of tendon.

Hyaluronan (HA) and link protein are essential components of the aggrecan proteoglycan aggregate, whereby HA binds multiple aggrecan monomers, an interaction which is stabilised by link protein. In this study, we have examined the turnover of the aggregate components, HA, link protein, and the N-terminal G1 domain of aggrecan, in explant cultures of tissue from compressed and tensional regions of young and mature bovine tendons. Western blot analyses revealed the release of highly processed link protein and G1-containing metabolites, in the absence of catabolic agents, indicating an increased turnover of these components in tendon. In addition, significant levels of HA were released from the tissue matrix into the media compartment. Furthermore, RT-PCR analysis showed that the mRNA expression of link protein and enzymes/proteins associated with HA metabolism may be modulated in the distinct functional regions of tendon with development. Perturbation of normal aggrecan aggregate metabolism may lead to tissue dysfunction.     

Turnover of proteoglycans in articular-cartilage cultures. Characterization of proteoglycans released into the medium.

By using an e.l.i.s.a. method it was demonstrated that the majority of proteoglycans released into the medium of both control and retinoic acid-treated explant cultures of bovine articular cartilage did not contain a hyaluronate-binding region. This supports our previous findings [Campbell & Handley (1987) Arch. Biochem. Biophys. 258, 143-155] that proteoglycans released into the medium of both cultures were of smaller hydrodynamic size, more polydisperse and unable to form aggregates with hyaluronate. Analysis of 35S-labelled core proteins associated with proteoglycans released into the medium of both cultures by using SDS/polyacrylamide-gel electrophoresis and fluorography indicated the presence of a series of core-protein bands (Mr approx. 300,000, 230,000, 215,000, 200,000, 180,000, 140,000, 135,000, 105,000, 85,000 and 60,000) compared with three core proteins derived from the proteoglycans remaining in the matrix (Mr 300,000, 230,000 and 215,000). Further analysis of the core proteins released into the medium indicated that the larger core proteins associated with medium proteoglycans contain both chondroitin sulphate and keratan sulphate glycosaminoglycans whereas the smaller core proteins contain only chondroitin sulphate chains. These experiments provide definitive evidence that the loss of proteoglycans from the matrix involves proteolytic cleavage at various sites along the proteoglycan core protein.     

Natural bovine osteogenin and recombinant human bone morphogenetic protein-2B are equipotent in the maintenance of proteoglycans in bovine articular cartilage explant cultures.

Osteogenin and related bone morphogenetic proteins are members of the transforming growth factor-beta superfamily, and were isolated by their ability to induce cartilage and bone formation in vivo. The influence of osteogenin, purified from bovine bone, and of recombinant human bone morphogenetic protein-2B (BMP-2B) has been examined in bovine articular cartilage explants. Both differentiation factors stimulated in a dose-dependent manner the synthesis of proteoglycans and decreased their rate of degradation. At a dose of 30 ng/ml, proteoglycan synthesis was increased to levels observed with either 20 ng/ml insulin-like growth factor I, 10 ng/ml transforming growth factor-beta, or 20% fetal bovine serum. This increase of biosynthetic rates above basal medium levels was observed in young, adolescent, and adult tissues. Analysis of the size of the newly synthesized proteoglycans, the glycosaminoglycan chain size, and the glycosaminoglycan type of explants treated with osteogenin or BMP-2B were very comparable to each other, and to proteoglycans isolated from cartilage treated with either insulin-like growth factor I or fetal bovine serum. These results demonstrate that osteogenin and BMP-2B alone are capable of stimulating and maintaining the chondrocyte phenotype in vitro.     

Collagen--proteoglycan interactions. Localization of proteoglycans in tendon by electron microscopy.

Proteoglycan in foetal- and adult-rat tail tendon and adult-rabbit achilles tendon was stained for electron microscopy with a cationic phthalocyanin-like dye, based on cinchomeronic acid, in a 'critical electrolyte concentration' method [Scott (1973) Biochem. Soc. Trans. 1, 787-806). Provided that the tissue was fixed with glutaraldehyde or formaldehyde, regular orthogonal perifibrillar arrays of filamentous material (proteoglycan) were observed, but no intra-fibrillar proteoglycan was seen. Specific proteoglycan-collagen interactions are inferred, and a model is proposed. Without fixation, the filamentous arrays disaggregated in the MgCl2 solutions (0.3 M) used during staining. End-to-end proteoglycan aggregation is implied. Tendon and cartilage are compared. Problems of electron-histochemical localization of extended space-filling polyanions by the use of cationic electron-dense precipitants are discussed, particularly polyanion-domain collapse, specificity of staining and fixation. A two-stage staining procedure that markedly enhances contrast is described, based on the multivalent nature of the dye, and the consequent anion-exchange properties of the dye-polyanion complex.     

Biosynthesis of proteoglycans and their assembly into aggregates in cultures of chondrocytes from the Swarm rat chondrosarcoma.

Cultured chondrocytes from the Swarm rat chondrosarcoma incorporate [35S]sulfate into proteoglycans typical of hyaline cartilage. The movement of newly synthesized proteoglycans from inside the cells into the extracellular matrix and, finally, into the culture medium was examined by measuring the distribution of 35S-labeled proteoglycans in the medium, a 4 M guanidine HCl extract of the cell layer, and in the remaining residue for a number of chase times following a 5-min pulse with [35S]sulfate. When hyaluronate oligosaccharides containing greater than or equal to 10 monosaccharides were included in the chase media, a proportion of newly synthesized proteoglycans were displaced from the matrix (4 M extract) into the culture medium. This displacement was greatest when oligomers were in the chase media between 10 and 20 min after the pulse, approximately the time when the molecules are being secreted from the cells. The proportion of link-stabilized aggregate in the medium was examined by Sepharose 2B chromatography after adding an excess of unlabeled monomer which displaces labeled monomer from complexes with hyaluronate which are not link-stabilized. The proportion of link-stabilized aggregate increased from 12% to about 70% between 12 and 120 min of chase. The presence of 40 micron hyaluronate oligosaccharides of 16 monosaccharides in the chase media retarded but did not prevent aggregate formation. Oligomers of about 50 monosaccharides, which are large enough to bind both a monomer proteoglycan and a link protein, almost completely prevented the formation of the large link-stabilized aggregates. The results suggest: (a) newly synthesized proteoglycans are not bound into link-stabilized aggregates at the time of secretion; (b) hyaluronic acid oligomers which are long enough to interact only with the hyaluronic acid-binding site of proteoglycans will retard but not prevent link-stabilized aggregation; and (c) hyaluronic acid oligomers long enough to accommodate additionally a link protein form a link-stabilized ternary complex and prevent aggregation with larger hyaluronic acid molecules.     

Agarose/polyacrylamide minislab gel electrophoresis of intact cartilage proteoglycans and their proteolytic degradation products.

We have developed a procedure for the use of minislab gels to electrophoretically separate proteoglycans (PGs), large macromolecules with molecular masses greater than 2.5 million Da. Our procedure is a modification of the method of C.A. McDevitt and H. Muir (Anal. Biochem. 44, 612-622, 1971) for agarose/polyacrylamide, composite tube gels. These 1% agarose/1.2% acrylamide minigels are run at 35 mA for 75 min; bands are visualized by toluidine blue staining. The subtle size differences between the large aggregating PGs isolated from rat chondrosarcoma, bovine nasal septal cartilage, and adult bovine articular cartilage (which consists of two subpopulations) can be distinguished by their migration on these large pore gels. Chondroitin sulfate chains, added to all wells as a marker of constant mobility, ran immediately behind the dye front. The distance of migration into the gel of PGs incubated overnight with cathepsin B, carboxypeptidase A, papain, plasmin, elastase, or cathepsin G varied with the size of the cleavage products. We propose the use of this procedure for a convenient assessment of cartilage PGs and a rapid, reproducible assay for proteoglycanase activity.     

Hyaluronan and its catabolic products in tissue injury and repair.

Hyaluronan is an unbiquitous glycosaminoglycan present in most tissues. Under homeostatic conditions hyaluronan exists as a high molecular mass polymer that has important roles in tissue structural integrity. Under conditions of stress such as following tissue injury, hyaluronan becomes depolymerized and lower molecular mass polymers are generated. The biological properties of these hyaluronan fragments appear to be distinct from the larger precursor molecules. This review examines the biological role of hyaluronan fragments in tissue injury and repair.     

Growth and myelination of explant cultures in defined medium

Summary The purpose of this study was to compare the development of organotypic cultures in defined medium versus nutrient containing serum and embryo extract (EE). Explant cultures of cerebellum with or without locus ceruleus were grown in the Maximow system and monitored in the living state and with histological stains. Thinner explants, fibronectin and a more frequent feeding schedule were required to overcome the growth differences encountered using a defined medium. The final medium formulation was arrived at by evaluation of living cultures and consisted of a basal medium (Dulbecco's minimal essential medium), a number of hormones and other supplements, and a final glucose concentration of 750 mg %. Using a Golgi stain and histofluorescence, it was shown that the three major types of neurons—Purkinje, deep nuclear, and locus ceruleus—developed similarly in the defined medium and in serum-EE cultures. Myelination occurred in virtually all cerebellar cultures in defined medium and the onset was earlier than in serum-EE cultures. These results indicate that differentiation of oligodendroglia and maturation of neurons occur in a defined medium. Elimination of thyroid hormone delayed the maturation of the cultures, both neurons and myelin, by 3–4 days. This project was supported by a grant from Supply and Services (Canada) and from the Department of Health and Welfare (Canada). The findings and opinions are the sole responsibility of the authors. EDITOR'S STATEMENT This article describes adaptations of serum-free cell culture methods previously developed by other laboratories to the organ culture of central nervous system tissues. Although it is difficult to develop reliable procedures for quantitative analyses in cultures of this type, organ cultures provide unique advantages in the study of development, regeneration and response to damage, organismal and cellular senescence and genetic abnormalities of the nervous system. Observations reported here regarding effects of thyroid hormone on cellular maturation in this culture system may be valuable in future studies in these areas.     

Characterization of proteoglycans from adult bovine tendon

Proteoglycans were extracted in good yield from the proximal, fibrous portion of adult bovine tendon with 4 m guanidine HCl. They comprise less than 1% of the dry weight of the tissue. Using CsCl density gradient centrifugation, gel chromatography, and ion exchange chromatography, two populations of proteoglycans were separated and purified from other tissue proteins. One was a large, chondroitin sulfate proteoglycan with high buoyant density in CsCl. This component appeared to be composed of two or three subpopulations as detected by agarose/polyacrylamide electrophoresis, although they could not be effectively separated from one another for individual characterization. As a group, the large proteoglycans eluted from Sepharose CL-2B with Kav from 0.1-0.5 and their core protein had Mr greater than 200,000 with high contents of glutamic acid, serine, and glycine. The glycosaminoglycan chains had a weight average Mr of 17,000 and more than 98% of the uronic acid was glucuronic acid. This group comprised only 12% of the total proteoglycan of the tissue. The other 88% of the proteoglycans appeared to represent one group of small molecules that eluted from Sepharose CL-2B at Kav = 0.70. They demonstrated buoyant densities in a CsCl gradient ranging from greater than or equal to 1.51 to 1.30 g/ml. Their core protein had an apparent Mr = 48,000 following removal of the glycosaminoglycan chains by digestion with chondroitinase ABC. This core protein had a particularly high content of aspartic acid/asparagine and leucine. The glycosaminoglycan chains had a weight average Mr of 37,000 and were dermatan sulfate containing 73% iduronic acid. Those molecules found at highest buoyant density appeared to have additional glycosaminoglycan chains that were shorter. Proteoglycans were also extracted from the pressure-bearing distal region of this tendon, where contents of proteoglycan per wet weight of tissue were 3-fold higher and as much as 50% of this was as large as the large proteoglycans from the proximal tissue. Preparations of large proteoglycans from both tendon regions contained molecules capable of interacting with hyaluronic acid.     

Rodlet cells in epidermal explant cultures of Lepomis Macrochirus

Sunfish rodlet cells were examined in vitro using a novel tissue explant system. Outgrowth of epidermal cell layers from explanted fish scales enabled both live cell videomicroscopy and immunocytochemical analysis of rodlet cells within the cell layer. Cells stained with fluorescent phallotoxin and antibody to tubulin showed that F‐actin is a component of the fibrous capsule that envelopes the cell and a microtubule network extends from the basal to apical ends of the cell interior. The fibrous capsule is also enriched for phosphotyrosine suggesting a potential signal‐transducing capability is present in this structure. Videomicroscopy analysis of live explant cultures demonstrated that rodlet cells are immobile and that interior structures are highly dynamic. Rodlet sacs can undergo extension and retraction, while intracellular particles can move rapidly within these cells. Fish scale tissue explants provide a useful system for analyzing the molecular composition and dynamic behavior of rodlet cells.     

Paclobutrazol stimulates bud regeneration in Solanum tuberosum L. primary explant cultures

The growth retardant paclobutrazol (PBZ) inhibited stem internode growth of in vitro cloned potato plants. The extent of growth inhibition caused by10-9 M PBZ in Murashige and Skoog medium was genotype-specific, varying between 10 - 60% of the stem growth of untreated controls in ten cultivars examined. An increase in percentage of de novo bud regenerating stem internode segments (SIS) as well as in the total number of buds per explant was observed in SIS taken from PBZ pretreated plants. PBZ applied directly into the regenerative media had no stimulatory effect on there generation process. We assume that the enhancing effect of PBZ on regeneration may be attributed to its interaction with cytokinin metabolism. This revised version was published online in July 2006 with corrections to the Cover Date.     

Preparation and square wave electroporation of retinal explant cultures

This protocol details organotypic cultures of developing mouse, monkey and human retinas, which can be maintained for up to 2 weeks. Intact retinas are placed on polycarbonate filters floating on explant culture medium and fed every day with previously prepared retinal conditioned medium. Developing mouse retinas from E12.5 to P12 have been successfully cultured using this protocol as well as retinas from the equivalent stages of human and monkey development. Although this protocol does not require any special equipment, it provides a relatively high throughput. Retinal explant cultures lend themselves to complex pharmacological and genetic manipulations that are currently not feasible in vivo. A detailed procedure for square wave electroporation of retinal explants is also included to provide a high-throughput means to alter gene expression in the developing retina. This protocol for the preparation of retinal conditioned explant medium requires 4 d. Other steps of this protocol can be completed in 2 h.     

Four classes of cell-associated proteoglycans in suspension cultures of articular-cartilage chondrocytes.

The characteristics of cell-associated proteoglycans were studied and compared with those from the medium in suspension cultures of calf articular-cartilage chondrocytes. By including hyaluronic acid or proteoglycan in the medium during [35S]sulphate labelling the proportion of cell-surface-associated proteoglycans could be decreased from 34% to about 15% of all incorporated label. A pulse-chase experiment indicated that this decrease was probably due to blocking of the reassociation with the cells of proteoglycans exported to the medium. Three peaks of [35S]sulphate-labelled proteoglycans from cell extracts and two from the medium were isolated by gel chromatography on Sephacryl S-500. These were characterized by agarose/polyacrylamide-gel electrophoresis, by SDS/polyacrylamide-gel electrophoresis of core proteins, by glycosaminoglycan composition and chain size as well as by distribution of glycosaminoglycans in proteolytic fragments. The results showed that associated with the cells were (a) large proteoglycans, typical for cartilage, apparently bound to hyaluronic acid at the cell surface, (b) an intermediate-size proteoglycan with chondroitin sulphate side chains (this proteoglycan, which had a large core protein, was only found associated with the cells and is apparently not related to the large proteoglycans), (c) a small proteoglycan with dermatan sulphate side chains with a low degree of epimerization, and (d) a somewhat smaller proteoglycan containing heparan sulphate side chains. The medium contained a large aggregating proteoglycan of similar nature to the large cell-associated proteoglycan and small proteoglycans with dermatan sulphate side chains with a higher degree of epimerization than those of the cells, i.e. containing some 20% iduronic acid.     

Basal-cell subpopulations and cell-cycle kinetics in human epidermal explant cultures

Cultured human epidermal cells were studied by cell sorting and autoradiography after different 3H-thymidine (3H-dThd)-labelling procedures and after labelling with DNA precursors that are incorporated via salvage or de novo pathways. It was shown that 3H-dThd incorporation was the best measure of the rate of DNA replication. Dose-response experiments with pulse and continuous labelling revealed that all S- and G2-phase cells were cycling, whereas some 20% of the cells stayed in G1-phase for long periods of time. Most, if not all of these cells were probably non-proliferating differentiated keratinocytes. At least two subpopulations of S-phase cells could be discriminated on the basis of the rate of incorporation of DNA precursors. The difference in precursor incorporation did not seem to be caused by differences in nucleotide metabolism but rather to reflect true differences in the rate of DNA replication. Continuous labelling experiments showed that these subpopulations also were apparent in the G1- and G2-phases. Studies of the grain-count distribution revealed that cells that appeared to move rapidly through the S-phase moved slowly through the G2-phase, and vice versa. Cells stained with acridine orange were subjected to a two-parameter analysis in the cell sorter by simultaneous measurement of the DNA and RNA fluorescence. Autoradiography of sorted cells revealed that, on average, cells with low RNA contents incorporated 3H-dThd at a higher rate than cells with high RNA contents.     

Soluble recombinant neprilysin induces aggrecanase-mediated cleavage of aggrecan in cartilage explant cultures.

Neprilysin (neutral endopeptidase, enkephalinase, CALLA, CD10, NEP) is a regulatory Zn metallopeptidase expressed in the brush border membranes of the kidney and has been found in porcine chondrocytes and rat articular cartilage as well as other cell types and tissues. Although its function in cartilage is not currently known, previous observations of high levels of NEP enzymatic activity in the synovial fluid of arthritic patients and on the chondrocyte membranes of human osteoarthritic cartilage have led to the hypothesis that NEP is involved in the inflammation or degradation pathways in articular cartilage. Our study localized endogenous NEP to the membranes of mature bovine articular chondrocytes in a tissue explant model and demonstrated that the addition of soluble recombinant NEP (sNEP) to the culture medium of bovine cartilage explants leads to the degradation of aggrecan through the action of aggrecanase. A 6-day exposure to sNEP was necessary to initiate the degradation, suggesting that the chondrocytes were responding in a delayed manner to an altered composition of regulatory peptides. This NEP-induced degradation was completely inhibited by the NEP inhibitors thiorphan and phosphoramidon. These results suggest that NEP is present as a transmembrane enzyme on articular chondrocytes where it can cleave regulatory peptides and lead to the induction of aggrecanase.     

The polyamines and their catabolic products are significant players in the turnover of nitrogenous molecules in plants

Polyamines (PAs) are nitrogenous molecules which play a well-established role in most cellular processes during growth and development under physiological or biotic/abiotic stress conditions. The molecular mode(s) of PA action have only recently started to be unveiled, and comprehensive models for their molecular interactions have been proposed. Their multiple roles are exerted, at least partially, through signalling by hydrogen peroxide (H(2)O(2)), which is generated by the oxidation/back-conversion of PAs by copper amine oxidases and PA oxidases. Accumulating evidence suggests that in plants the cellular titres of PAs are affected by other nitrogenous compounds. Here, we discuss the state of the art on the possible nitrogen flow in PAs, their interconnection with nitrogen metabolism, as well as the signalling roles of PA-derived H(2)O(2) during some developmental processes and stress responses.     

Catabolism of newly synthesized decorin by explant cultures of bovine ligament.

The catabolism of newly synthesized decorin by explant cultures of bovine collateral ligament was investigated. The tissue was placed in explant culture for 6 days then incubated with radiolabeled sulfate for 6 h and replaced in culture for 5 days to allow for the loss of the radiolabeled large proteoglycan. The metabolic fate of the remaining radiolabeled decorin present in the matrix of the tissue over the next 9-day period was determined. It was shown that this pool of decorin was lost from ligament explant cultures either directly into the culture medium or taken up and degraded within the cells of the tissue. The intracellular degradation of the radiolabeled pool of decorin by ligament explant cultures was shown to result in the generation of [35S]sulfate. This process required metabolically active cells and involved the lysosomal system since sulfate generation was inhibited when cultures were maintained at 4 degrees C or in the presence of either 10 mM ammonium chloride or 0. 05 mM chloroquine. The inhibition of intracellular processing of decorin resulted in an increase in the rate of loss of this proteoglycan into the medium of the cultures. The inhibition of intracellular degradation of decorin was reversible on incubation of the explant cultures at 37 degrees C or removal of ammonium chloride from the culture medium. After removal of the ammonium chloride from the culture medium the rate of intracellular catabolism was greater than that observed in cultures maintained in medium alone, which suggested that there was an intracellular accumulation of native and/or partially degraded material within the cells.