Dataset Information

Link-N: The missing link towards intervertebral disc repair is species-specific.

ABSTRACT: Degeneration 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.This 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.Human (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.Human 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.

SUBMITTER: Bach FC

PROVIDER: S-EPMC5679057 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Link-N: The missing link towards intervertebral disc repair is species-specific.

Bach Frances C FC Laagland Lisanne T LT Grant Michael P MP Creemers Laura B LB Ito Keita K Meij Björn P BP Mwale Fackson F Tryfonidou Marianna A MA

PloS one 20171108 11

<h4>Introduction</h4>Degeneration 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 can ...[more]

PMID: 29117254

Similar Datasets

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:BackgroundPreceding intervertebral disc (IVD) degeneration, the cell phenotype in the nucleus pulposus (NP) shifts from notochordal cells (NCs) to chondrocyte-like cells (CLCs). Microarray analysis showed a correlation between caveolin-1 expression and the phenotypic transition of NCs to CLCs. With a clinical directive in mind, the aim of this study was to determine the role of caveolin-1 in IVD degeneration. As a scaffolding protein, caveolin-1 influences several signaling pathways, and transforming growth factor (TGF)-β receptors have been demonstrated to colocalize with caveolin-1. Therefore, the hypothesis of this study was that caveolin-1 facilitates repair by enhancing TGF-β signaling in the IVD.MethodsProtein expression (caveolin-1, apoptosis, progenitor cell markers, extracellular matrix, and phosphorylated Smad2 [pSmad2]) was determined in IVDs of wild-type (WT) and caveolin-1-null mice and canine IVDs of different degeneration grades (immunofluorescence, immunohistochemistry, and TUNEL assay). Canine/human CLC microaggregates were treated with chondrogenic medium alone or in combination with caveolin-1 scaffolding domain (CSD) peptide and/or caveolin-1 silencing RNA. After 28 days, gene and protein expression profiles were determined.ResultsThe NP of WT mice was rich in viable NCs, whereas the NP of caveolin-1-null mice contained more collagen-rich extracellular matrix and fewer cells, together with increased progenitor cell marker expression, pSmad2 TGF-β signaling, and high apoptotic activity. During canine IVD degeneration, caveolin-1 expression and apoptotic activity increased. In vitro caveolin-1 silencing decreased the CLC microaggregate glycosaminoglycan (GAG) content, which could be rescued by CSD treatment. Furthermore, CSD increased TGF-β/pSmad2 signaling at gene and protein expression levels and enhanced the anabolic effects of TGF-β1, reflected in increased extracellular matrix deposition by the CLCs.ConclusionsCaveolin-1 plays a role in preservation of the NC phenotype. Additionally, it may be related to CLC apoptosis, given its increased expression in degenerated IVDs. Nevertheless, CSD enhanced CLC GAG deposition in vitro, and hence the increased caveolin-1 expression during IVD degeneration may also facilitate an ultimate attempt at repair. Further studies are needed to investigate how caveolin-1 modifies other signaling pathways and facilitates IVD repair.

Dataset Information

Link-N: The missing link towards intervertebral disc repair is species-specific.

Publications

Link-N: The missing link towards intervertebral disc repair is species-specific.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets