Extraction and characterization of proteoglycan from human meniscus.
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ABSTRACT:
SUBMITTER: McNicol D
PROVIDER: S-EPMC1161448 | biostudies-other | 1980 Mar
REPOSITORIES: biostudies-other
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Project description:Proteoglycans were extracted from the adult human meniscus under dissociative conditions and purified by CsCl-density-gradient centrifugation. The preparations of highest density contained proteoglycan that possessed the ability to interact with hyaluronic acid, was of large subunit size and was composed of chondroitin sulphate, keratan sulphate and sialic acid-containing oligosaccharides. This 'cartilage-like' proteoglycan also exhibited subunit and aggregate structures analogous to those of hyaline-cartilage proteoglycans when examined by electron microscopy. However, the composition of this proteoglycan was more comparable with proteoglycans from immature cartilage than from age-matched cartilage. The preparations from lower density, which were enriched in dermatan sulphate, contained smaller proteoglycan that was not able to interact with hyaluronic acid. This non-aggregating proteoglycan may be structurally distinct from the 'cartilage-like' proteoglycan, which does not contain dermatan sulphate.
| S-EPMC1163056 | biostudies-other
Project description:BACKGROUND:Lubricin, or proteoglycan 4 (PRG4), is a glycoprotein responsible for joint boundary lubrication. PRG4 has been shown previously to be down-regulated after traumatic joint injury such as a meniscal tear. Preliminary evidence suggests that intra-articular injection of PRG4 after injury will reduce cartilage damage in rat models of surgically induced posttraumatic osteoarthritis. OBJECTIVE:To determine the efficacy of intra-articular injection of full-length recombinant human lubricin (rhPRG4) for reducing cartilage damage after medial meniscal destabilization (DMM) in a preclinical large animal model. STUDY DESIGN:Controlled laboratory study. METHODS:Unilateral DMM was performed in 29 Yucatan minipigs. One week after DMM, animals received 3 weekly intra-articular injections (3 mL per injection): (1) rhPRG4 (1.3 mg/mL; n = 10); (2) rhPRG4+hyaluronan (1.3 mg/mL rhPRG4 and 3 mg/mL hyaluronan [~950 kDA]; n = 10); and (3) phosphate-buffered saline (PBS; n = 9). Hindlimbs were harvested 26 weeks after surgery. Cartilage integrity was evaluated by use of macroscopic (India ink) and microscopic (safranin O-fast green and hematoxylin and eosin) scoring systems. Secondary outcomes evaluated via enzyme-linked immunosorbent assay (ELISA) included PRG4 levels in synovial fluid, carboxy-terminal telepeptide of type II collagen (CTX-II) concentrations in urine and serum, and interleukin 1? (IL-1?) levels in synovial fluid and serum. RESULTS:The rhPRG4 group had significantly less macroscopic cartilage damage in the medial tibial plateau compared with the PBS group ( P = .002). No difference was found between the rhPRG4+hyaluronan and PBS groups ( P = .23). However, no differences in microscopic damage scores were observed between the 3 groups ( P = .70). PRG4 production was elevated in the rhPRG4 group synovial fluid compared with the PBS group ( P = .033). The rhPRG4 group presented significantly lower urinary CTX-II levels, but not serum levels, when compared with the PBS ( P = .013) and rhPRG4+hyaluronan ( P = .011) groups. In serum and synovial fluid, both rhPRG4 ( P = .006; P = .017) and rhPRG4+hyaluronan groups ( P = .009; P = .03) presented decreased IL-1? levels. CONCLUSION:All groups exhibited significant cartilage degeneration after DMM surgery. However, animals treated with rhPRG4 had the least amount of cartilage damage and less inflammation, providing evidence that intra-articular injections of rhPRG4 may slow the progression of posttraumatic osteoarthritis. CLINICAL RELEVANCE:Patients with meniscal trauma are at high risk for posttraumatic osteoarthritis. This study demonstrates that an intra-articular injection regimen of rhPRG4 may attenuate cartilage damage after meniscal injury.
| S-EPMC5453820 | biostudies-literature
Project description:BackgroundRheumatoid arthritis (RA) is an autoimmune disease of the synovial joints. The autoimmune character of RA is underscored by prominent production of autoantibodies such as those against IgG (rheumatoid factor), and a broad array of joint tissue-specific and other endogenous citrullinated proteins. Anti-citrullinated protein antibodies (ACPA) can be detected in the sera and synovial fluids of RA patients and ACPA seropositivity is one of the diagnostic criteria of RA. Studies have demonstrated that RA T cells respond to citrullinated peptides (epitopes) of proteoglycan (PG) aggrecan, which is one of the most abundant macromolecules of articular cartilage. However, it is not known if the PG molecule is citrullinated in vivo in human cartilage, and if so, whether citrulline-containing neoepitopes of PG (CitPG) can contribute to autoimmunity in RA.MethodsCitPG was detected in human cartilage extracts using ACPA+ RA sera in dot blot and Western blot. Citrullination status of in vitro citrullinated recombinant G1 domain of human PG (rhG1) was confirmed by antibody-based and chemical methods, and potential sites of citrullination in rhG1 were explored by molecular modeling. CitPG-specific serum autoantibodies were quantified by enzyme-linked immunosorbent assays, and CitPG was localized in osteoarthritic (OA) and RA cartilage using immunohistochemistry.FindingsSera from ACPA+ RA patients reacted with PG purified from normal human cartilage specimens. PG fragments (mainly those containing the G1 domain) from OA or RA cartilage extracts were recognized by ACPA+ sera but not by serum from ACPA- individuals. ACPA+ sera also reacted with in vitro citrullinated rhG1 and G3 domain-containing fragment(s) of PG. Molecular modeling suggested multiple sites of potential citrullination within the G1 domain. The immunohistochemical localization of CitPG was different in OA and RA cartilage.ConclusionsCitPG is a new member of citrullinated proteins identified in human joints. CitPG could be found in both normal and diseased cartilage specimens. Antibodies against CitPG may trigger or augment arthritis by forming immune complexes with this autoantigen in the joints of ACPA+ RA patients.
| S-EPMC4778950 | biostudies-literature
Project description:Freshly prepared platelets were shown to contain glycosaminoglycans equivalent to 530 micrograms of hexuronate/10(11) platelets. When the platelets were extracted with 4 M-guanidinium chloride containing proteinase inhibitors, and the extract was dialysed extensively against 7 M-urea solution, almost all of proteoglycan was recovered in the urea-soluble fraction. The proteoglycan was purified from the urea-soluble fraction with a yield of 47% by DEAE-Sephacel chromatography, CsCl-density-gradient centrifugation, Bio-Gel A-15m gel filtration and then rechromatography on DEAE-Sephacel. The purified proteoglycan contained 30% glucuronic acid, 32% N-acetylgalactosamine, 14% sulphate and 15% protein. Serine, glutamic acid, glycine, aspartic acid and leucine accounted for 64% of the total amino acids. The Mr of the proteoglycan was assessed to be approx. 136000 by sedimentation-equilibrium methods. The galactosaminoglycan released by alkaline-borohydride treatment of the proteoglycan was converted stoichiometrically into 4-sulphated unsaturated disaccharide by digestion with chondroitinase AC-II, indicating that the galactosaminoglycan was fully sulphated chondroitin 4-sulphate. The apparent Mr of the chondroitin sulphate was assessed to be 28000 by gel filtration on Bio-Gel A-0.5m (KD 0.18). On two-dimensional electrophoresis on a cellulose acetate membrane, the chondroitin sulphate gave a single compact spot co-migrating with a reference chondroitin sulphate, indicating that the chondroitin sulphate chains were homogeneous in both length and charge density. On the basis of these results, the proteoglycan in human platelets was concluded to be a macromolecule of Mr 136000 containing four chondroitin 4-sulphate chains each with the apparent Mr of 28000.
| S-EPMC1152987 | biostudies-other
Project description:Recent research suggests an important role of the meniscus in the development of knee osteoarthritis. We, therefore, aimed to analyze the proteome of the normal human meniscus body, and specifically to gain new knowledge on global protein expression in the different radial zones. Medial menisci were retrieved from the right knees of 10 human cadaveric donors, from which we cut a 2 mm radial slice from the mid-portion of the meniscal body. This slice was further divided into three zones: inner, middle, and peripheral. Proteins were extracted and prepared for mass spectrometric analysis using data-independent acquisition. We performed subsequent data searches using Spectronaut Pulsar and used fixed-effect linear regression models for statistical analysis. We identified 638 proteins and after statistical analysis, we observed the greatest number of differentially expressed proteins between the inner and peripheral zones (163 proteins) and the peripheral and middle zones (136 proteins), with myocilin being the protein with the largest fold-change in both comparisons. Chondroadherin was one of eight proteins that differed between the inner and middle zones. Functional enrichment analyses showed that the peripheral one-third of the medial meniscus body differed substantially from the two more centrally located zones, which were more similar to each other. This is probably related to the higher content of cells and vascularization in the peripheral zone, whereas the middle and inner zones of the meniscal body appear to be more similar to hyaline cartilage, with high levels of extracellular matrix proteins such as aggrecan and collagen type II.
| S-EPMC7610686 | biostudies-literature
Project description:There is a need to clarify the role of the meniscus in knee OA which may be aided by the identification of biomarkers that reflect the molecular events involved in the tissue degradation. This is of particular importance in the early stages of the degenerative processes, a crucial phase when it might still be possible to interfere with disease progression before tearing and critical loss of meniscal function occurs. One way to achieve this goal is exploratory proteomics by mass spectrometry (MS), which has the potential to analyse the proteome of complex samples. Thus, our aim was to gain new knowledge about the molecular processes within the human meniscus by comparing the proteomic profiles of both the lateral and medial meniscus from healthy controls, subjects with mild signs of joint degeneration, and patients with end-stage knee OA, using a non-targeted MS approach operating in data-independent acquisition (DIA) mode. For the research purpose six groups were established: medial and lateral menisci from control donors (n=12), medial and lateral menisci from donors with mild signs of joint degeneration (n=13) and medial and lateral menisci from end-stage knee OA patients (n=12).
2024-04-03 | PXD042122 | Pride
Project description:Human platelet proteoglycan (P.PG) was prepared from a 4 M-guanidinium chloride platelet extract in the presence of proteinase inhibitors. The purification procedure included CsCl-density-gradient centrifugation, DEAE-Sepharose CL-6B ion-exchange chromatography and f.p.l.c. on a Mono Q HR 5/5 column. P.PG was recovered as a polydisperse molecule, but the protein core appeared to be at least 90% homogeneous. This observation could be due to partial proteolysis of the core protein during extraction. The N-terminal sequence of the human P.PG core protein was determined up to residue 66 and was shown to be highly homologous to the propeptide of an embryonic rat yolk-sac tumour proteoglycan (PG19); the significance of this homology is discussed.
| S-EPMC1135341 | biostudies-other
Project description:Meniscus tears are associated with a heightened risk of osteoarthritis. This study aimed to advance our understanding of the metabolic state of injured human meniscus at the time of arthroscopic partial meniscectomy through transcriptome-wide analysis of gene expression in relation to the patient's age and degree of cartilage chondrosis.The degree of chondrosis of knee cartilage was recorded at the time of meniscectomy in symptomatic patients without radiographic osteoarthritis. RNA preparations from resected menisci (n = 12) were subjected to transcriptome-wide microarray and QuantiGene Plex analyses. Variations in the relative changes in gene expression with age and chondrosis were analyzed, and integrated biologic processes were investigated computationally.We identified a set of genes in torn menisci that were differentially expressed with age and chondrosis. There were 866 genes that were differentially regulated (?1.5-fold difference and P < 0.05) with age and 49 with chondrosis. In older patients, genes associated with cartilage and skeletal development and extracellular matrix synthesis were repressed, while those involved in immune response, inflammation, cell cycle, and cellular proliferation were stimulated. With chondrosis, genes representing cell catabolism (cAMP catabolic process) and tissue and endothelial cell development were repressed, and those involved in T cell differentiation and apoptosis were elevated.Differences in age-related gene expression suggest that in older adults, meniscal cells might dedifferentiate and initiate a proliferative phenotype. Conversely, meniscal cells in younger patients appear to respond to injury, but they maintain the differentiated phenotype. Definitive molecular signatures identified in damaged meniscus could be segregated largely with age and, to a lesser extent, with chondrosis.
| S-EPMC3873730 | biostudies-literature
Project description:Meniscus fibrochondrocytes (MFCs) experience simultaneous hypoxia and mechanical loading in the knee joint. Experimental conditions based on these aspects of the native MFC environment may have promising applications in human meniscus tissue engineering. We hypothesized that in vitro "mechano-hypoxia conditioning" with mechanical loading such as dynamic compression (DC) and cyclic hydrostatic pressure (CHP) would enhance development of human meniscus fibrocartilage extracellular matrix in vitro. MFCs from inner human meniscus surgical discards were pre-cultured on porous type I collagen scaffolds with TGF-β3 supplementation to form baseline tissues with newly formed matrix that were used in a series of experiments. First, baseline tissues were treated with DC or CHP under hypoxia (HYP, 3% O2) for 5 days. DC was the more effective load regime in inducing gene expression changes, and combined HYP/DC enhanced gene expression of fibrocartilage precursors. The individual treatments of DC and HYP regulated thousands of genes, such as chondrogenic markers SOX5/6, in an overwhelmingly additive rather than synergistic manner. Similar baseline tissues were then treated with a short course of DC (5 vs 60 min, 10-20% vs 30-40% strain) with different pre-culture duration (3 vs 6 weeks). The longer course of loading (60 min) had diminishing returns in regulating mechano-sensitive and inflammatory genes such as c-FOS and PTGS2, suggesting that as few as 5 min of DC was adequate. There was a dose-effect in gene regulation by higher DC strains, whereas outcomes were inconsistent for different MFC donors in pre-culture durations. A final set of baseline tissues was then cultured for 3 weeks with mechano-hypoxia conditioning to assess mechanical and protein-level outcomes. There were 1.8-5.1-fold gains in the dynamic modulus relative to baseline in HYP/DC, but matrix outcomes were equal or inferior to static controls. Long-term mechano-hypoxia conditioning was effective in suppressing hypertrophic markers (e.g., COL10A1 10-fold suppression vs static/normoxia). Taken together, these results indicate that appropriately applied mechano-hypoxia conditioning can support meniscus fibrocartilage development in vitro and may be useful as a strategy for developing non-hypertrophic articular cartilage using mesenchymal stem cells.
| S-EPMC8446439 | biostudies-literature
Project description:Proteoglycans were extracted from human uterine cervix with 4 M-guanidinium chloride in the presence of proteinase inhibitors. They were purified by density-gradient centrifugation in 4 M-guanidinium chloride/CsCl (starting density 1.32 g/ml) followed by DEAE-cellulose and Sepharose chromatography. Only one polydisperse proteoglycan was found. s020,w was 2.1S and the weight-average molecular weight was 73 000 (sedimentation-equilibrium centrifugation) to 110 500 (light-scattering). The core protein was monodisperse, with an apparent molecular weight of 47 000. The proteoglycan contained about 30% protein and probably two or three glycosaminoglycan side chains per molecule. High contents of aspartate, glutamate and leucine were found. The glycan moiety of the proteoglycan was exclusively dermatan sulphate, with a co-polymeric structure with approximately equal quantities of iduronic acid- and glucuronic acid-containing disaccharides.
| S-EPMC1154117 | biostudies-other