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Demonstration of link protein in proteoglycan aggregates from human intervertebral disc.

ABSTRACT: Proteoglycan aggregates free of non-aggregating proteoglycan have been prepared from the annuli fibrosi and nuclei pulposi of intervertebral discs of three human lumbar spines by extraction with 4M-guanidinium chloride, associative density gradient centrifugation, and chromatography on Sepharose CL-2B. The aggregate (A1-2B.V0) was subjected to dissociative density-gradient ultracentrifugation. Three proteins of Mr 38 900, 44 200 and 50 100 found in the fraction of low buoyant density (A1-2B.V0-D4) reacted with antibodies to link protein from newborn human articular cartilage. After reduction with mercaptoethanol, two proteins of Mr 43 000 and two of Mr 20 000 and 14 000 were seen. The A1-2B.V0-D4 fraction, labelled with 125I, coeluted with both hyaluronate and a hyaluronate oligosaccharide (HA14) on a Sepharose CL-2B column. HA10 and HA14 reduced the viscosity of A1 fractions; HA4, HA6 and HA8 did not. HA14 decreased the viscosity of disc proteoglycans less than it did that of bovine cartilage proteoglycans. Thus, although a link protein was present in human intervertebral disc, it stabilized proteoglycan aggregates less well than did the link protein from bovine nasal cartilage.

SUBMITTER: Tengblad A

PROVIDER: S-EPMC1144147 | biostudies-other | 1984 Aug

REPOSITORIES: biostudies-other

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Project description:Proteoglycan aggregates (A1) were prepared from the anulus fibrosus, nucleus pulposus and cartilage-endplate tissues of postnatal (0-6-month-old)-and young-adult (20-30-year-old)-human intervertebral discs. The A1 fractions from young-adult disc contained a greater proportion of non-aggregating proteoglycans than did postnatal tissues. After dissociative CsCl-density-gradient fractionation of the A1, more than 90% of the uronic acid was found in the postnatal A1D1, whereas only 60-80% of the hexuronate was present in the A1D1 isolated from young-adult disc tissues. These results indicated that more lower-buoyant-density proteoglycans occur in the young-adult disc. Link-protein-rich fractions (A1D3) were subjected to SDS/polyacrylamide-gel electrophoresis and immunolocation analyses using monoclonal antibodies specific for epitopes on link protein or proteoglycan. Under non-reducing conditions, the major link protein present in postnatal disc tissues was link protein 1. By contrast, all three link proteins (1, 2 and 3) were detected in young-adult tissues, with the smaller link protein 3 predominating. Analyses of the A1D3 fractions under reducing conditions also indicated the presence of link-protein-degradation peptides (Mr approx. 26,000) from young-adult disc tissues, but not from postnatal tissues. Sequential Sepharose CL-6B and Sephacryl S-300 chromatography in 4 M-guanidinium chloride was employed to separate the link proteins of the A1D3 fraction from protein-rich proteoglycan. Immunolocation analyses indicated that postnatal samples contained no detectable contaminating proteoglycan fragments. However, young-adult link-protein preparations could not be separated from hyaluronic acid-binding region and other proteoglycan fragments by means of these chromatographic procedures. The studies indicate that, compared with hyaline articular cartilage, degraded link protein and proteoglycan accumulate at an early age in young-adult disc tissues. These partially degraded proteoglycan aggregate components may significantly alter the biomechanical properties of disc tissues.

Project description:IntroductionDegeneration of the intervertebral disc (IVD) is a frequent cause for back pain in humans and dogs. Link-N stabilizes proteoglycan aggregates in cartilaginous tissues and exerts growth factor-like effects. The human variant of Link-N facilitates IVD regeneration in several species in vitro by inducing Smad1 signaling, but it is not clear whether this is species specific. Dogs with IVD disease could possibly benefit from Link-N treatment, but Link-N has not been tested on canine IVD cells. If Link-N appears to be effective in canines, this would facilitate translation of Link-N into the clinic using the dog as an in vivo large animal model for human IVD degeneration.Materials and methodsThis study's objective was to determine the effect of the human and canine variant of Link-N and short (s) Link-N on canine chondrocyte-like cells (CLCs) and compare this to those on already studied species, i.e. human and bovine CLCs. Extracellular matrix (ECM) production was determined by measuring glycosaminoglycan (GAG) content and histological evaluation. Additionally, the micro-aggregates' DNA content was measured. Phosphorylated (p) Smad1 and -2 levels were determined using ELISA.ResultsHuman (s)Link-N induced GAG deposition in human and bovine CLCs, as expected. In contrast, canine (s)Link-N did not affect ECM production in human CLCs, while it mainly induced collagen type I and II deposition in bovine CLCs. In canine CLCs, both canine and human (s)Link-N induced negligible GAG deposition. Surprisingly, human and canine (s)Link-N did not induce Smad signaling in human and bovine CLCs. Human and canine (s)Link-N only mildly increased pSmad1 and Smad2 levels in canine CLCs.ConclusionsHuman and canine (s)Link-N exerted species-specific effects on CLCs from early degenerated IVDs. Both variants, however, lacked the potency as canine IVD regeneration agent. While these studies demonstrate the challenges of translational studies in large animal models, (s)Link-N still holds a regenerative potential for humans.

Project description:ObjectiveTo analyze the two major components of the intervertebral disc (IVD) in an ex vivo phantom, as well as age-related changes in patients.MethodsCollagen and chondroitin sulfate were imaged at different concentrations in agar solution. Age-related changes in disc density were retrospectively analyzed in normal-appearing discs in dual-energy computed tomography (DECT) images from a patient cohort with various spinal pathologies (n = 136). All computed tomography (CT) scans were acquired using single-source DECT at 80 and 135 kVp with automatic exposure calculation. In 136 patients, the attenuation of normal-appearing discs on collagen/chondroitin maps (cMaps) correlated with the patients' age with Pearson's r using standardized regions of interest in the anterior anulus fibrosus (AAF) and nucleus pulposus (NP).ResultsDECT collagen mapping revealed concentration-dependent Hounsfield units (HU) of IVD components. For collagen, we found Pearson's r = 0.9610 (95% CI 0.6789-0.9959), p = 0.0023 at 120 kVe, and r = 0.8824 (95% CI 0.2495-0.9871), p = 0.0199 in cMap. For chondroitin sulfate, Pearson's r was 0.9583 (95% CI 0.6603-0.9956), p = 0.0026 at 120 kVp, and r = 0.9646 (95% CI 0.7044-0.9963), p = 0.0019 in cMap. Analysis of normal-appearing IVDs revealed an inverse correlation of density with age in the AAF: Pearson's r = - 0.2294 at 135 kVp (95% CI - 0.4012 to - 0.04203; p=0.0141) and r = - 0.09341 in cMap (95% CI - 0.2777 to 0.09754; p = 0.0003). In the NP, age and density did not correlate significantly at 135 kVp (p = 0.9228) and in cMap (p = 0.3229).ConclusionsDECT-based collagen mapping allows microstructural analysis of the two main intervertebral disc components-collagen and chondroitin sulfate. IVD density declines with age, presumably due to a reduction in collagen and chondroitin sulfate content. Age-related alterations of disc microstructure appear most pronounced in the AAF.Key points• DECT-based collagen mapping allows precise analysis of the two main intervertebral disc components-collagen and chondroitin sulfate. • Intervertebral disc (IVD) density declines with age, presumably due to a reduction in collagen and chondroitin sulfate content. • Age-related alterations of disc microstructure are most pronounced in the anterior anulus fibrosus (AAF).

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Demonstration of link protein in proteoglycan aggregates from human intervertebral disc.

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