Nature of the interaction of chondroitin 4-sulphate and chondroitin sulphate-proteoglycan with collagen.
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ABSTRACT:
SUBMITTER: Obrink B
PROVIDER: S-EPMC1176559 | biostudies-other | 1971 Jan
REPOSITORIES: biostudies-other
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Project description:Neuron glial antigen 2 or chondroitin sulphate proteoglycan 4 (NG2/CSPG4) is expressed by immature precursors/progenitor cells and is possibly involved in malignant cell transformation. The aim of this study was to investigate its role on the progression and survival of sixty-one adult gliomas and nine glioblastoma (GB)-derived cell lines. NG2/CSPG4 protein expression was assessed by immunohistochemistry and immunofluorescence. Genetic and epigenetic alterations were detected by molecular genetic techniques. NG2/CSPG4 was frequently expressed in IDH-mutant/1p19q-codel oligodendrogliomas (59.1%) and IDH-wild type GBs (40%) and rarely expressed in IDH-mutant or IDH-wild type astrocytomas (14.3%). Besides tumor cells, NG2/CSPG4 immunoreactivity was found in the cytoplasm and/or cell membranes of reactive astrocytes and vascular pericytes/endothelial cells. In GB-derived neurospheres, it was variably detected according to the number of passages of the in vitro culture. In GB-derived adherent cells, a diffuse positivity was found in most cells. NG2/CSPG4 expression was significantly associated with EGFR gene amplification (p = 0.0005) and poor prognosis (p = 0.016) in astrocytic tumors. The immunoreactivity of NG2/CSPG4 provides information on the timing of the neoplastic transformation and could have prognostic and therapeutic relevance as a promising tumor-associated antigen for antibody-based immunotherapy in patients with malignant gliomas.
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Project description:The interplay between neural progenitors and stem cells (NPSCs), and their extracellular matrix (ECM) is a crucial regulatory mechanism that determines their behavior. Nonetheless, how the ECM dictates the state of NPSCs remains elusive. The hindbrain is valuable to examine this relationship, as cells in the ventricular surface of hindbrain boundaries (HBs), which arise between any two neighboring rhombomeres, express the NPSC marker Sox2, while being surrounded with the membrane-bound ECM molecule chondroitin sulphate proteoglycan (CSPG), in chick and mouse embryos. CSPG expression was used to isolate HB Sox2+ cells for RNA-sequencing, revealing their distinguished molecular properties as typical NPSCs, which express known and newly identified genes relating to stem cells, cancer, the matrisome and cell cycle. In contrast, the CSPG- non-HB cells, displayed clear neural-differentiation transcriptome. To address whether CSPG is significant for hindbrain development, its expression was manipulated in vivo and in vitro. CSPG manipulations shifted the stem versus differentiation state of HB cells, evident by their behavior and altered gene expression. These results provide further understanding of the uniqueness of hindbrain boundaries as repetitive pools of NPSCs in-between the rapidly growing rhombomeres, which rely on their microenvironment to maintain their undifferentiated state during development.
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Project description:Olfactory ensheathing cell (OEC) transplantation is a promising strategy for treating spinal cord injury (SCI), as has been demonstrated in experimental SCI models and naturally occurring SCI in dogs. However, the presence of chondroitin sulphate proteoglycans within the extracellular matrix of the glial scar can inhibit efficient axonal repair and limit the therapeutic potential of OECs. Here we have used lentiviral vectors to genetically modify canine OECs to continuously deliver mammalian chondroitinase ABC at the lesion site in order to degrade the inhibitory chondroitin sulphate proteoglycans in a rodent model of spinal cord injury. We demonstrate that these chondroitinase producing canine OECs survived at 4 weeks following transplantation into the spinal cord lesion and effectively digested chondroitin sulphate proteoglycans at the site of injury. There was evidence of sprouting within the corticospinal tract rostral to the lesion and an increase in the number of corticospinal axons caudal to the lesion, suggestive of axonal regeneration. Our results indicate that delivery of the chondroitinase enzyme can be achieved with the genetically modified OECs to increase axon growth following SCI. The combination of these two promising approaches is a potential strategy for promoting neural regeneration following SCI in veterinary practice and human patients.
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Project description:The excretion and degradation was studied of (35)S-labelled 4-chondroitin sulphate injected into guinea pigs in the form of proteoglycan isolated from cartilage and in the form of free chondroitin 4-sulphate prepared from the same proteoglycan by proteolysis. When the proteoglycan was injected there was a delay of about 15-20min before significant amounts or radioactivity were excreted, whereas after injection of chondroitin 4-sulphate a considerable amount of radioactivity was excreted within 10min and a much higher proportion of the radioactive dose was excreted in 1h or 24h compared with the proteoglycan. In both cases, however, a major part of the radioactivity was not excreted even in 24h. Sterile conditions were used to collect the radioactive material directly from the bladder. When chondroitin 4-sulphate was injected, the molecular sizes of injected and excreted materials were similar, as assessed by gel chromatography on Sephadex G-200, whereas when proteoglycan was injected the molecular size of the excreted labelled material was similar to that of the chondroitin 4-sulphate chains in the original proteoglycan. In neither case did the size of the excreted labelled material change with time over 1h, and low-molecular-weight labelled material was virtually absent. In contrast, when urine was collected for 24h without preservative the labelled material in it was extensively degraded after either the proteoglycan or chondroitin 4-sulphate had been given. Chondroitin 4-sulphate became similarly degraded when incubated with non-sterile urine, but not when the urine was passed through a bacterial filter, suggesting that degradation was caused by contaminating micro-organisms in the experiments in which urine was collected for 24 h. It is concluded that chondroitin 4-sulphate chains of about 18000 molecular weight can be excreted readily as such, whereas intact proteoglycans must be degraded to free glycosaminoglycans first, although both are taken up by the tissues more rapidly than they are excreted.
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Project description:A biologically active, high-strength tissue adhesive is needed for numerous medical applications in tissue engineering and regenerative medicine. Integration of biomaterials or implants with surrounding native tissue is crucial for both immediate functionality and long-term performance of the tissue. Here, we use the biopolymer chondroitin sulphate (CS), one of the major components of cartilage extracellular matrix, to develop a novel bioadhesive that is readily applied and acts quickly. CS was chemically functionalized with methacrylate and aldehyde groups on the polysaccharide backbone to chemically bridge biomaterials and tissue proteins via a twofold covalent link. Three-dimensional hydrogels (with and without cells) bonded to articular cartilage defects. In in vitro and in vivo functional studies this approach led to mechanical stability of the hydrogel and tissue repair in cartilage defects.
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Project description:Proteoglycans were isolated from cartilage by extraction with 4M-guanidinium chloride followed by direct centrifugation in 4M-guanidinium chloride/CsCl at a low starting density, 1.34 g/ml. N-Ethylmaleimide was included in the extraction solvent as a precaution against contamination of proteoglycans with unrelated proteins mediated by disulphide exchange. A novel, discrete, low-buoyant-density proteoglycan (1.40--1.35 g/ml) was demonstrated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Its proteoglycan nature was revealed by the shift in the molecular size observed on gel electrophoresis after treatment with chondroitinase ABC. The core protein was monodisperse. The proteoglycan was further purified by gel chromatography with and without addition of hyaluronate. The proteoglycan constitutes less than 2% (by weight) of the total extracted proteoglycans and is not capable of interacting with hyaluronate. The same proteoglycan was purified in larger quantities by sequential associative and dissociative CsCl-density-gradient centrifugation, zonal rate sedimentation in a sucrose gradient and gel chromatography on Sepharose CL-4B. The pure proteoglycan had a molecular weight of 76 300 determined by sedimentation-equilibrium centrifugation and an apparent partial specific volume of 0.59 ml/g. It contained about 25% protein (of dry weight) and had remarkably high contents of leucine and cysteine as compared with other proteoglycans. The proteoglycan contained two to three large chondroitin sulphate chains and some oligosaccharides.
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Project description:Afferent lymph vessels entering the popliteal lymph nodes of sheep were infused with [3H]acetyl-labelled hyaluronan [HA; Mr of (0.85-1.2) x 10(5)] for up to 4 h at a rate of 17.4-23.1 micrograms/h. As much as 22.8 micrograms (99%) of infused [3H]HA was taken up by the node per h and degraded. During this interval it was observed that infused HA polymers of higher Mr were absorbed by the node to a greater degree than those of lower Mr. When proteoglycan monomer (PG; Mr 5 x 10(5); 400 micrograms of hexuronic acid/h) was infused concurrently with [3H]HA, the absolute amount of radioactivity appearing in efferent lymph (i.e. labelled material not absorbed by the node) increased, whereas the amount of labelled metabolites of low Mr was reduced considerably. During this period the Mr distribution of labelled HA in efferent outflow reverted to that of the infused material within 30-60 min. Our findings suggest that PG subunits and their chondroitin sulphate chains compete with HA for uptake into the peripheral lymph node of sheep. This indicates that PG, chondroitin sulphate and HA share the same pathway of elimination in this tissue, and is consistent with the view that the lymph node is involved in the metabolic turnover of normal intracellular matrix.
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Project description:Crystal growth in native collagen gels has been used to determine the role of extracellular matrix macromolecules in biological calcification phenomena. In this system, type I collagen gels containing sodium phosphate and buffered at pH 7.4 are overlayed with a solution containing CaCl2. Crystals form in the collagen gel adjacent to the gel-solution interface. Conditions were determined which permit the growth of crystals of hydroxyapatite [Ca10(PO4)6(OH)2]. At a Ca/P molar ratio of 2:1, the minimum concentrations of calcium and phosphate necessary for precipitation of hydroxyapatite are 10 mM and 5 mM, respectively. Under these conditions, precipitation is initiated at 18-24h, and is maximal between 24h and 6 days. Addition of high concentrations of chondroitin 4-sulphate inhibits the formation of hydroxyapatite in collagen gels; initiation of precipitation is delayed, and the final (equilibrium) amount of precipitation is decreased. Inhibition of hydroxyapatite formation requires concentrations of chondroitin sulphate higher than those required to inhibit calcium pyrophosphate crystal formation.
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