Catabolic Response In Chondrocytes Research Articles

Exosomes derived from diabetic serum accelerate the progression of osteoarthritis

Diabetes mellitus (DM) has been demonstrated to accelerate the progression of osteoarthritis (OA) by largely unknown mechanisms. Studies have shown that DM dysfunctional adipocyte-derived exosomes play a crucial role in the pathogenesis of remote organ functions. The present study aimed to clarify whether and how diabetic adipocyte-derived exosomes mediate the pathological regulation of OA. We found that intraarticular injection of DM serum exosomes in the non-diabetic mice significantly exacerbated OA injury as evidenced by a rough and fractured cartilage surface as well as increased chondrocyte apoptosis, decreased mitochondrial membrane potential (△Ψ) and increased expression of cleaved caspase-3. Mechanistic investigation identified that miR-130b-3p was significantly increased in circulating exosomes derived from DM mice and exosomes derived from HG-treated normal adipocytes, and we demonstrated that transfection of miR-130b-3p mimics significantly exacerbated the mitochondrial function of chondrocytes. Our data also indicated that miR-130b-3p impaired the △Ψ, increased cleaved caspase-3 levels, and decreased the expression of 5′-adenosine monophosphate-activated protein kinase α1 (AMPKα1), Silent mating-type information regulation 2 homolog 1 (SIRT1), and peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) in chondrocytes. Pharmacologic activation of AMPKα1 using AICAR reversed the △Ψ and catabolic responses in chondrocytes transfected with miR-130b-3p mimics. Moreover, AICAR decreased the effects of miR-130b-3p mimics on chondrocytes transfected with SIRT1-siRNA or PGC-1α-siRNA. The current study demonstrated that adipocyte-derived exosomal miR-130b-3p under DM conditions suppresses mitochondrial function in chondrocytes through targeting the AMPKα1/SIRT1/PGC1-α pathway, thus exacerbating OA injury.

Mitochondrial dysfunction triggers a catabolic response in chondrocytes via ROS-mediated activation of the JNK/AP1 pathway.

Mitochondrial function is impaired in osteoarthritis (OA) but its impact on cartilage catabolism is not fully understood. Here, we investigated the molecular mechanism of mitochondrial dysfunction-induced activation of the catabolic response in chondrocytes. Using cartilage slices from normal and OA cartilage, we showed that mitochondrial membrane potential was lower in OA cartilage, and that this was associated with increased production of mitochondrial superoxide and catabolic genes [interleukin 6 (IL-6), COX-2 (also known as PTGS2), MMP-3, -9, -13 and ADAMTS5]. Pharmacological induction of mitochondrial dysfunction in chondrocytes and cartilage explants using carbonyl cyanide 3-chlorophenylhydrazone increased mitochondrial superoxide production and the expression of IL-6, COX-2, MMP-3, -9, -13 and ADAMTS5, and cartilage matrix degradation. Mitochondrial dysfunction-induced expression of catabolic genes was dependent on the JNK (herein referring to the JNK family)/activator protein 1 (AP1) pathway but not the NFκB pathway. Scavenging of mitochondrial superoxide with MitoTEMPO, or pharmacological inhibition of JNK or cFos and cJun, blocked the mitochondrial dysfunction-induced expression of the catabolic genes in chondrocytes. We demonstrate here that mitochondrial dysfunction contributes to OA pathogenesis via JNK/AP1-mediated expression of catabolic genes. Our data shows that AP1 could be used as a therapeutic target for OA management.This article has an associated First Person interview with the first author of the paper.

First person – Mohammad Yunus Ansari

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Mohammad Yunus Ansari is first author on ‘Mitochondrial dysfunction triggers a catabolic response in chondrocytes via ROS-mediated activation of the JNK/AP1 pathway’, published in JCS. Mohammad Yunus is a Research Assistant Professor in the lab of Tariq M Haqqi, at the Department of Anatomy and Neurobiology, Northeast Ohio Medical University, USA, investigating the mechanism of regulation of mitochondrial function in articular cartilage and its role in cartilage homeostasis in health and disease.

The chondrocyte channelome: A narrative review.

Chondrocytes are the main cells in the extracellular matrix (ECM) of articular cartilage and possess a highly differentiated phenotype that is the hallmark of the unique physiological functions of this specialised load-bearing connective tissue. The plasma membrane of articular chondrocytes contains a rich and diverse complement of membrane proteins, known as the membranome, which defines the cell surface phenotype of the cells. The membranome is a key target of pharmacological agents and is important for chondrocyte function. It includes channels, transporters, enzymes, receptors, and anchors for intracellular, cytoskeletal and ECM proteins and other macromolecular complexes. The chondrocyte channelome is a sub-compartment of the membranome and includes a complete set of ion channels and porins expressed in these cells. Many of these are multi-functional proteins with "moonlighting" roles, serving as channels, receptors and signalling components of larger molecular assemblies. The aim of this review is to summarise our current knowledge of the fundamental aspects of the chondrocyte channelome, discuss its relevance to cartilage biology and highlight its possible role in the pathogenesis of osteoarthritis (OA). Excessive and inappropriate mechanical loads, an inflammatory micro-environment, alternative splicing of channel components or accumulation of basic calcium phosphate crystals can result in an altered chondrocyte channelome impairing its function. Alterations in Ca2+ signalling may lead to defective synthesis of ECM macromolecules and aggravated catabolic responses in chondrocytes, which is an important and relatively unexplored aspect of the complex and poorly understood mechanism of OA development.

Biophysical Stimuli: A Review of Electrical and Mechanical Stimulation in Hyaline Cartilage.

Hyaline cartilage degenerative pathologies induce morphologic and biomechanical changes resulting in cartilage tissue damage. In pursuit of therapeutic options, electrical and mechanical stimulation have been proposed for improving tissue engineering approaches for cartilage repair. The purpose of this review was to highlight the effect of electrical stimulation and mechanical stimuli in chondrocyte behavior. Different information sources and the MEDLINE database were systematically revised to summarize the different contributions for the past 40 years. It has been shown that electric stimulation may increase cell proliferation and stimulate the synthesis of molecules associated with the extracellular matrix of the articular cartilage, such as collagen type II, aggrecan and glycosaminoglycans, while mechanical loads trigger anabolic and catabolic responses in chondrocytes. The biophysical stimuli can increase cell proliferation and stimulate molecules associated with hyaline cartilage extracellular matrix maintenance.

Sodium Thiosulfate Prevents Chondrocyte Mineralization and Reduces the Severity of Murine Osteoarthritis.

ObjectivesCalcium-containing crystals participate in the pathogenesis of OA. Sodium thiosulfate (STS) has been shown to be an effective treatment in calcification disorders such as calciphylaxis and vascular calcification. This study investigated the effects and mechanisms of action of STS in a murine model of OA and in chondrocyte calcification.MethodsHydroxyapatite (HA) crystals-stimulated murine chondrocytes and macrophages were treated with STS. Mineralization and cellular production of IL-6, MCP-1 and reactive oxygen species (ROS) were assayed. STS's effects on genes involved in calcification, inflammation and cartilage matrix degradation were studied by RT-PCR. STS was administered in the menisectomy model of murine OA, and the effect on periarticular calcific deposits and cartilage degeneration was investigated by micro-CT-scan and histology.ResultsIn vitro, STS prevented in a dose-dependent manner calcium crystal deposition in chondrocytes and inhibited Annexin V gene expression. In addition, there was a reduction in crystal-induced IL-6 and MCP-1 production. STS also had an antioxidant effect, diminished HA-induced ROS generation and abrogated HA-induced catabolic responses in chondrocytes. In vivo, administration of STS reduced the histological severity of OA, by limiting the size of new periarticular calcific deposits and reducing the severity of cartilage damage.ConclusionsSTS reduces the severity of periarticular calcification and cartilage damage in an animal model of OA via its effects on chondrocyte mineralization and its attenuation of crystal-induced inflammation as well as catabolic enzymes and ROS generation. Our study suggests that STS may be a disease-modifying drug in crystal-associated OA.

PEP-1-GRX-1 Modulates Matrix Metalloproteinase-13 and Nitric Oxide Expression of Human Articular Chondrocytes

Background: The protein transduction domain (PTD) enables therapeutic proteins to directly penetrate the membranes of cells and tissues, and has been increasingly utilized. Glutaredoxin-1 (GRX-1) is an endogenous antioxidant enzyme involved in the cellular redox homeostasis system. In this study, we investigated whether PEP-1-GRX-1, a fusion protein of GRX-1 and PEP-1 peptide, a PTD, could suppress catabolic responses in primary human articular chondrocytes and a mouse carrageenan-induced paw edema model. Methods: Human articular chondrocytes were isolated enzymatically from articular cartilage and cultured in a monolayer. The transduction efficiency of PEP-1-GRX-1 into articular chondrocytes was measured by western blot and immunohistochemistry. The effects of PEP-1-GRX-1 on matrix metalloproteinases (MMPs) and catabolic factor expression in interleukin (IL)-1β- and lipopolysaccharide (LPS)-treated chondrocytes were analyzed by real-time quantitative reverse transcription-polymerase chain reaction and western blot. The effect of PEP-1-GRX1 on the mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light chain-enhancer of activated B cells (NF-κB) signaling pathway were also analyzed by western blot. Finally, the inhibitory effect of PEP-1-GRX-1 on MMP-13 production was measured in vivo in a mouse carrageenan-induced paw edema model. Results: PEP-1-GRX-1 significantly penetrated into human chondrocytes and mouse cartilage, whereas GRX-1 did not. PEP-1-GRX-1 significantly suppressed MMP-13 expression and nitric oxide (NO) production in LPS-stimulated chondrocytes, and NO production in IL-1β-stimulated chondrocytes, compared with GRX-1. In addition, PEP-1-GRX-1 decreased IL-1β- and LPS-induced activation of MAPK and NF-κB. In the mouse model of carrageenan-induced paw edema, PEP-1-GRX-1 significantly suppressed carrageenan-induced MMP-13 production as well as paw edema. Conclusion: These results demonstrate that PEP-1-GRX-1 can be transduced efficiently in vitro and in vivo into human chondrocytes and mouse cartilage tissue and downregulate catabolic responses in chondrocytes by inhibiting the MAPK and NF-κB pathway. PEP-1-GRX-1 thus has the potential to reduce catabolic responses in chondrocytes and cartilage.

S100A8 and S100A9 act as ‘primers’ of a catabolic response in chondrocytes but additional signals are required to activate cartilage degradation

S100A8 and S100A9 act as ‘primers’ of a catabolic response in chondrocytes but additional signals are required to activate cartilage degradation

A model for testing the effects of injury and repair using engineered cartilage tissue analogs

s / Osteoarthritis and Cartilage 20 (2012) S54–S296 S130 Conclusions: In this work, we showed for the first time that multiscale large field imaging (Macroscopy) combined to multimodality approaches (SHG-TCSPC) could be an innovative and non invasive technique to monitor the state of network collagen in biomedical studies such as cartilage tissue engineering. 247 A MODEL FOR TESTING THE EFFECTS OF INJURY AND REPAIR USING ENGINEERED CARTILAGE TISSUE ANALOGS B. Mohanraj, W.C. Strutz, R.L. Mauck, G.R. Dodge. Univ. of Pennsylvania, Philadelphia, PA, USA Purpose: Articular cartilage consists of unique extracellular matrix (ECM) which function to distribute loads in synovial joints. When injured, it cannot repair effectively and is increasingly thought that injury or trauma, even the smallest may initiate degenerative changes (i.e. OA). The goal was to devise a platform to test chondrocyte's response to injury using a cartilage tissue analogue (CTA) after a rapid blunt force trauma and/or laceration. The aimwas tomeasure biosynthetic and catabolic responses in chondrocytes, in a reproducible in vitro model, following injury. The long term goal was to provide a platform to study chondrocyte function and to test therapeutics. Methods: CTA PreparationChondrocytes were isolated from juvenile bovine (1-3 months old) knees and CTA generated. Briefly, cartilage was dissected and digested overnight with collagenase. The CTAmodel is based on using plates coated with hydrogel, poly (2-hydroxyethyl methacrylate), which prevents cell adherence. The cells coalesce and form a single mass which grows quickly and maintains a cartilage phenotype for long periods in culture. In these experiments chondrocytes were seeded at 1-2x106 cells/ well in 96 well plate and cultured in DMEM with 10% FBS for at least 6 -10 weeks to allow for substantial ECM production. Sample CTA Groups(1) Uninjured control, (2) Uninjured treated with 10 ng/mL IL-1a (positive control), (3) Cut CTAs, and (4) Impacted CTAs, were harvested 5 hours and 5 days post-injury. Injuryan Instron 5848 was used to determine the CTA height while resting on a flat surface. The impact injury regimen was 75% strain over 1 sec., held for 3 sec., then released. 3 full thickness lacerations were made through the CTA (‘Cut’). After impact or laceration, CTAs were transferred to medium for additional culture periods or immediately flash frozen for gene expression analysis. At corresponding harvest times CTA were rushed into a powder, and RNA was purified followed by qPCR using the Bio-Rad CFX384 and iQ Sybr GreenSupermix. Data was analyzed using 2T method to obtain relative expressionwith data presented as percent of untreated or un-injured controls. Results: The effects of impact injury on ECM gene expression over an extended period of three time points 0, 24, and 120 h was evaluated.Collagen and aggrecan were down-regulated at 24 h following impact and IL-1 treatment. However, at 120 h, collagen was markedly upregulated after injury, as was aggrecan but to a lesser extent. IL-1a treatment continued to down-regulate aggrecan at 120h, while collagen showed a slight increase (24%). Perlecan was up-regulated in response to injury at 120h (w30%) and to IL-1a at both time points. Comparisons between cut and impacted CTAs at 5h showed an upregulation of collagen and aggrecan expression w3-fold and w2.5-fold, respectively. (Fig 1). Ă However, this upregulation was diminished at 120h. In contrast, in cut CTAs, aggrecan and collagen II was only slightly up-regulated and remained at constant expression levels at 5 and 120h. The stress related gene iNOS was markedly up-regulated at 5h in impacted CTAs (w 4.5X), and this increasewas reduced nearly to uninjured at 120 h. In contrast, cut CTAs had low level expression of iNOS at 5h, but was up-regulated 2.5-fold at 120h. ADAM-TS4 expression was similarly unaffected at 5 h for both cut and impacted CTAs butwas up-regulated in impacted samples (w2.6X) at 120h. Conclusion: A single, rapid, uniaxial compressive load on an engineered cartilage construct was used to generate a model of injury and demonstrate the changes on chondrocyte gene expression. While proteases tested and stress indicators are involved in the cellular injury response after both injury types, chondrocytes appear to respond to compressive injury by increasing the biosynthesis of certain ECM genes at longer time points tested. Extending the recovery periods will reveal to what degree these mechanically-induced injuries heal or become degenerative. This model establishes a platform to test engineered cartilage surrogate's response to injury and ultimately identify pathways involved in repair and test therapeutics. 248 CARTILAGE REPAIR FOR STEROID-INDUCED OSTEONECROSIS OF THE KNEE JOINT H. Tsukiyama, M. Kobayasi, R. Arai, H. Harada, Y. Nakagawa. Kyoto Univ.

Peroxisome proliferator-activated receptor gamma in osteoarthritis

Osteoarthritis (OA) is among the most prevalent chronic human health disorders and the most common form of arthritis. It is a leading cause of disability in developed countries. This disease is characterized by cartilage deterioration, synovitis, and remodeling of the subchondral bone. There is not yet a satisfactory treatment to stop or arrest this disease process. Although several candidates for therapeutic approaches have been put forward, recent studies suggest that activation of the transcription factor peroxisome proliferator-activated receptor gamma (PPARγ) is an interesting target for this disease. PPARγ is a ligand-activated transcription factor and member of the nuclear receptor superfamily. Agonists of PPARγ inhibit inflammation and reduce synthesis of cartilage degradation products both invitro and invivo, and reduce the development/progression of cartilage lesions in OA animal models. This review will highlight the recent experimental studies on the presence of PPARγ in articular tissues and its effect on inflammatory and catabolic responses in chondrocytes and synovial fibroblasts, as well as the protective effects of PPARγ ligands in arthritis experimental models. Finally, the role of PPARγ polymorphism in the pathogenesis of OA and related musculoskeletal diseases will also be discussed.

The catabolic pathway mediated by Toll‐like receptors in human osteoarthritic chondrocytes

To examine the catabolic pathways mediated by Toll-like receptor (TLR) ligands in human osteoarthritic (OA) chondrocytes. The presence of TLRs in OA and non-OA articular cartilage was analyzed by immunohistochemistry. The regulation of TLR messenger RNA (mRNA) by interleukin-1 (IL-1) and tumor necrosis factor alpha (TNFalpha) was analyzed by reverse transcription-polymerase chain reaction. For stimulation of TLR-2 and TLR-4, chondrocytes were treated with Staphylococcus aureus peptidoglycan and lipopolysaccharides (LPS), respectively. Production of matrix metalloproteinases (MMPs) 1, 3, and 13 and prostaglandin E2 (PGE2) was evaluated by enzyme-linked immunosorbent assay. Production of nitric oxide (NO) was analyzed by the Griess reaction. Regulation of cyclooxygenase 2 protein and phosphorylation of MAPKs (p38, ERK, and JNK) were evaluated by Western blotting or solid-phase kinase assay. NF-kappaB activation was evaluated by electrophoretic mobility shift assay. Expression of TLRs 2 and 4 was up-regulated in lesional areas of OA cartilage. Treatment with IL-1, TNFalpha, peptidoglycan, and LPS all significantly up-regulated TLR-2 mRNA expression in cultured chondrocytes. Production of MMPs 1, 3, and 13 and of NO and PGE2 was significantly increased after treating chondrocytes with either of the TLR ligands. Prolonged culture of cartilage explants with TLR ligands also led to a significant increase in the release of proteoglycan and type II collagen degradation product. Treatment with TLR ligands led to phosphorylation of all 3 MAPKs and activation of NF-kappaB. We found that TLRs are increased in OA cartilage lesions. TLR-2 and TLR-4 ligands strongly induce catabolic responses in chondrocytes. Modulation of TLR-mediated signaling as a therapeutic strategy would require detailed elucidation of the signaling pathways involved.