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Gene expression changes in damaged osteoarthritic cartilage identify a signature of non-chondrogenic and mechanical responses.

ABSTRACT: OBJECTIVES:Joint degeneration in osteoarthritis (OA) is characterised by damage and loss of articular cartilage. The pattern of loss is consistent with damage occurring only where the mechanical loading is high. We have investigated using RNA-sequencing (RNA-seq) and systems analyses the changes that occur in damaged OA cartilage by comparing it with intact cartilage from the same joint. METHODS:Cartilage was obtained from eight OA patients undergoing total knee replacement. RNA was extracted from cartilage on the damaged distal medial condyle (DMC) and the intact posterior lateral condyle (PLC). RNA-seq was performed to identify differentially expressed genes (DEGs) and systems analyses applied to identify dysregulated pathways. RESULTS:In the damaged OA cartilage, there was decreased expression of chondrogenic genes SOX9, SOX6, COL11A2, COL9A1/2/3, ACAN and HAPLN1; increases in non-chondrogenic genes COL1A1, COMP and FN1; an altered pattern of secreted proteinase expression; but no expression of major inflammatory cytokines. Systems analyses by PhenomeExpress revealed significant sub-networks of DEGs including mitotic cell cycle, Wnt signalling, apoptosis and matrix organisation that were influenced by a core of altered transcription factors (TFs), FOSL1, AHR, E2F1 and FOXM1. CONCLUSIONS:Gene expression changes in damaged cartilage suggested a signature non-chondrogenic response of altered matrix protein and secreted proteinase expression. There was evidence of a damage response in this late OA cartilage, which surprisingly showed features detected experimentally in the early response of cartilage to mechanical overload. PhenomeExpress analysis identified a hub of DEGs linked by a core of four differentially regulated TFs.

SUBMITTER: Dunn SL

PROVIDER: S-EPMC4989048 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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Gene expression changes in damaged osteoarthritic cartilage identify a signature of non-chondrogenic and mechanical responses.

Dunn S L SL Soul J J Anand S S Schwartz J-M JM Boot-Handford R P RP Hardingham T E TE

Osteoarthritis and cartilage 20160310 8

<h4>Objectives</h4>Joint degeneration in osteoarthritis (OA) is characterised by damage and loss of articular cartilage. The pattern of loss is consistent with damage occurring only where the mechanical loading is high. We have investigated using RNA-sequencing (RNA-seq) and systems analyses the changes that occur in damaged OA cartilage by comparing it with intact cartilage from the same joint.<h4>Methods</h4>Cartilage was obtained from eight OA patients undergoing total knee replacement. RNA w ...[more]

PMID: 26973327

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Project description:Pathologic calcification of cartilage consists of the formation of basic calcium phosphate (BCP) and/or calcium pyrophosphate dihydrate (CPPD) containing calcium crystals in mature hyaline or articular cartilage and is associated with aging, cartilage injury and likely plays a role in accelerating the pathology of osteoarthritis (OA). The pathways regulating joint calcification, in particular cartilage calcification, are not completely understood, but inflammation and the formation of reactive oxygen species (ROS) are contributory factors. The xanthine oxidase (XO) form of xanthine oxidoreductase (XOR), the key enzyme in xanthine and uric acid metabolism, is a major cellular source of superoxide. We hypothesized that XOR could be implicated in chondrocyte mineralization and cartilage calcification and degradation in OA. We showed both in murine primary chondrocyte and chondrogenic ATDC5 cells, that mineralization was inhibited by two different XOR inhibitors, febuxostat and allopurinol. In addition, XOR inhibition reduced the expression of the pro-mineralizing cytokine interleukin-6 (IL-6). We next generated XOR knock-out chondrocyte cell lines with undetectable XOR expression and XO activity. XOR knock-out chondrocyte cells showed decreased mineralization and reduced alkaline phosphatase (Alp) activity. To assess the precise form of XOR involved, primary chondrocytes of XOR mutant mice expressing either the XDH form (XDH ki) or the XO form (XO ki) were studied. We found that XO ki chondrocytes exhibited increased mineralization compared to XDH ki chondrocytes, and this was associated with enhanced Alp activity, ROS generation and IL-6 secretion. Finally, we found increased XOR expression in damaged vs. undamaged cartilage obtained from OA patients and XOR expression partially co-localized with areas showing pathologic calcification. Altogether, our results suggest that XOR, via its XO form, contribute to chondrocyte mineralization and pathological calcification in OA cartilage.

Project description:The repair capability of traumatized articular cartilage is highly limited so that joint injuries often lead to osteoarthritis. Migratory chondrogenic progenitor cells (CPC) might represent a target cell population for in situ regeneration. This study aims to clarify, whether 1) CPC are present in regions of macroscopically intact cartilage from human osteoarthritic joints, 2) CPC migration is stimulated by single growth factors and the cocktail of factors released from traumatized cartilage and 3) CPC migration is influenced by cytokines present in traumatized joints.We characterized the cells growing out from macroscopically intact human osteoarthritic cartilage using a panel of positive and negative surface markers and analyzed their differentiation capacity. The migratory response to platelet-derived growth factor (PDGF)-BB, insulin-like growth factor 1 (IGF-1), supernatants obtained from in vitro traumatized cartilage and interleukin-1 beta (IL-1?) as well as tumor necrosis factor alpha (TNF-?) were tested with a modified Boyden chamber assay. The influence of IL-1? and TNF-? was additionally examined by scratch assays and outgrowth experiments.A comparison of 25 quadruplicate marker combinations in CPC and bone-marrow derived mesenchymal stromal cells showed a similar expression profile. CPC cultures had the potential for adipogenic, osteogenic and chondrogenic differentiation. PDGF-BB and IGF-1, such as the supernatant from traumatized cartilage, induced a significant site-directed migratory response. IL-1? and TNF-? significantly reduced basal cell migration and abrogated the stimulative effect of the growth factors and the trauma supernatant. Both cytokines also inhibited cell migration in the scratch assay and primary outgrowth of CPC from cartilage tissue. In contrast, the cytokine IL-6, which is present in trauma supernatant, did not affect growth factor induced migration of CPC.These results indicate that traumatized cartilage releases chemoattractive factors for CPC but IL-1? and TNF-? inhibit their migratory activity which might contribute to the low regenerative potential of cartilage in vivo.

Project description:ObjectivePain is the most common symptom of osteoarthritis (OA), yet where it originates in the joint and how it is driven are unknown. The aim of this study was to identify pain-sensitizing molecules that are regulated in the joint when mice subjected to surgical joint destabilization develop OA-related pain behavior, the tissues in which these molecules are being regulated, and the factors that control their regulation.MethodsTen-week-old mice underwent sham surgery, partial meniscectomy, or surgical destabilization of the medial meniscus (DMM). Pain-related behavior as determined by a variety of methods (testing of responses to von Frey filaments, cold plate testing for cold sensitivity, analgesiometry, incapacitance testing, and forced flexion testing) was assessed weekly. Once pain-related behavior was established, RNA was extracted from either whole joints or microdissected tissue samples (articular cartilage, meniscus, and bone). Reverse transcription-polymerase chain reaction analysis was performed to analyze the expression of 54 genes known to regulate pain sensitization. Cartilage injury assays were performed using avulsed immature hips from wild-type or genetically modified mice or by explanting articular cartilage from porcine joints preinjected with pharmacologic inhibitors. Levels of nerve growth factor (NGF) protein were measured by enzyme-linked immunosorbent assay.ResultsMice developed pain-related behavior 8 weeks after undergoing partial meniscectomy or 12 weeks after undergoing DMM. NGF, bradykinin receptors B1 and B2, tachykinin, and tachykinin receptor 1 were significantly regulated in the joints of mice displaying pain-related behavior. Little regulation of inflammatory cytokines, leukocyte activation markers, or chemokines was observed. When tissue samples from articular cartilage, meniscus, and bone were analyzed separately, NGF was consistently regulated in the articular cartilage. The other pain sensitizers were also largely regulated in the articular cartilage, although there were some differences between the 2 models. NGF and tachykinin were strongly regulated by simple mechanical injury of cartilage in vitro in a transforming growth factor ?-activated kinase 1-, fibroblast growth factor 2-, and Src kinase-dependent manner.ConclusionDamaged joint tissues produce proalgesic molecules, including NGF, in murine OA.

Project description:BACKGROUND:While bone marrow-derived mesenchymal stem cells (BMSC) are established sources for stem cell-based cartilage repair therapy, articular cartilage-derived mesenchymal stem cells from osteoarthritis patients (OA-MSC) are new and potentially attractive candidates. We compared OA-MSC and BMSC in chondrogenic potentials, gene expression, and regulation by miR-365, a mechanical-responsive microRNA in cartilage (Guan et al., FASEB J 25: 4457-4466, 2011). METHODS:To overcome the limited number of OA-MSC, a newly established human OA-MSC cell line (Jayasuriya et al., Sci Rep 8: 7044, 2018) was utilized for analysis and comparison to BMSC. Chondrogenesis was induced by the chondrogenic medium in monolayer cell culture. After chondrogenic induction, chondrogenesis and mineralization were assessed by Alcian blue and Alizarin red staining respectively. MiRNA and mRNA levels were quantified by real-time PCR while protein levels were determined by western blot analysis at different time points. Immunohistochemistry was performed with cartilage-specific miR-365 over-expression transgenic mice. RESULTS:Upon chondrogenic induction, OA-MSC underwent rapid chondrogenesis in comparison to BMSC as shown by Alcian blue staining and activation of ACAN and COL2A1 gene expression. Chondrogenic induction also activated mineralization and the expression of hypertrophic and osteogenic genes in OA-MSC while only hypertrophic genes were activated in BMSC. MiR-365 expression was activated by chondrogenic induction in both OA-MSC and BMSC. Transfection of miR-365 in OA-MSC induced chondrogenic, hypertrophic, and osteogenic genes expression while miR-365 inhibition suppressed the expression of these genes. Over-expression of miR-365 upregulated markers of OA-MSC and hypertrophy and increased OA scores in adult mouse articular cartilage. CONCLUSIONS:Induction of chondrogenesis can activate mineralization, hypertrophic, and osteogenic genes in OA-MSC. MiR-365 appears to be a master regulator of these differentiation processes in OA-MSC during OA pathogenesis. These findings have important implications for cartilage repair therapy using cartilage derived stem cells from OA patients.

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Gene expression changes in damaged osteoarthritic cartilage identify a signature of non-chondrogenic and mechanical responses.

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Gene expression changes in damaged osteoarthritic cartilage identify a signature of non-chondrogenic and mechanical responses.

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