Volatile vs Total intravenous Anaesthesia for major non-cardiac surgery: a pragmatic randomised triaL (VITAL).

BACKGROUND: Improving outcomes after surgery is a major public health research priority for patients, clinicians and the NHS. The greatest burden of perioperative complications, mortality and healthcare costs lies amongst the population of patients aged over 50 years who undergo major non-cardiac surgery. The Volatile vs Total Intravenous Anaesthesia for major non-cardiac surgery (VITAL) trial specifically examines the effect of anaesthetic technique on key patient outcomes: quality of recovery after surgery (quality of recovery after anaesthesia, patient satisfaction and major post-operative complications), survival and patient safety. METHODS: A multi-centre pragmatic efficient randomised trial with health economic evaluation comparing total intravenous anaesthesia with volatile-based anaesthesia in adults (aged 50 and over) undergoing elective major non-cardiac surgery under general anaesthesia. DISCUSSION: Given the very large number of patients exposed to general anaesthesia every year, even small differences in outcome between the two techniques could result in substantial excess harm. Results from the VITAL trial will ensure patients can benefit from the very safest anaesthesia care, promoting an early return home, reducing healthcare costs and maximising the health benefits of surgical treatments. TRIAL REGISTRATION: ISRCTN62903453. September 09, 2021.

Implementation of ReSPECT in acute hospitals: A retrospective observational study.

AIMS: To evaluate, in UK acute hospitals, the early implementation of the Recommended Summary Plan for Emergency Care and Treatment (ReSPECT), which embeds cardiopulmonary resuscitation (CPR) recommendations within wider emergency treatment plans. To understand for whom and how the process was being used and the quality of form completion. METHODS: A retrospective observational study evaluating emergency care and treatment planning approaches used in acute UK hospitals (2015-2019), and in six English hospital trusts the extent of ReSPECT use, patient characteristics and completion quality in a sample 3000 patient case notes. RESULTS: The use of stand-alone Do Not Attempt Cardiopulmonary Resuscitation forms fell from 133/186 hospitals in 2015 to 64/186 in 2019 (a 38% absolute reduction). ReSPECT accounted for 52% (36/69) of changes. In the six sites, ReSPECT was used for approximately 20% of patients (range 6%-41%). They tended to be older, to have had an emergency medical admission, to have cognitive impairment and a lower predicted 10 year survival. Most (653/706 (92%)) included a 'not for attempted resuscitation' recommendation 551/706 (78%) had at least one other treatment recommendation. Capacity was not recorded on 13% (95/706) of forms; 11% (79/706) did not record patient/family involvement. CONCLUSIONS: ReSPECT use accounts for 52% of the change, observed between 2015 and 2019, from using standalone DNACPR forms to approaches embedding DNACPR decisions within in wider emergency care plans in NHS hospitals in the UK. Whilst recommendations include other emergencies most still tend to focus on recommendations relating to CPR. Completion of ReSPECT forms requires improvement. STUDY REGISTRATION: https://www.isrctn.com/ISRCTN11112933.

99mTc-NTP 15-5 is a companion radiotracer for assessing joint functional response to sprifermin (rhFGF-18) in a murine osteoarthritis model.

With the emergence of disease modifying osteoarthritis drugs (DMOAD), imaging methods to quantitatively demonstrate their efficacy and to monitor osteoarthritis progression at the functional level are urgently needed. Our group showed that articular cartilage can be quantitatively assessed in nuclear medicine imaging by our radiotracer 99mTc-NTP 15-5 targeting cartilage proteoglycans. In this work, surgically induced DMM mice were treated with sprifermin or saline. We investigated cartilage remodelling in the mice knees by 99mTc-NTP 15-5 SPECT-CT imaging over 24 weeks after surgery, as wells as proteoglycan biochemical assays. OA alterations were scored by histology according to OARSI guidelines. A specific accumulation of 99mTc-NTP 15-5 in cartilage joints was evidenced in vivo by SPECT-CT imaging as early as 30 min post-iv injection. In DMM, 99mTc-NTP 15-5 accumulation in cartilage within the operated joints, relative to contralateral ones, was observed to initially increase then decrease as pathology progressed. Under sprifermin, 99mTc-NTP 15-5 uptake in pathological knees was significantly increased compared to controls, at 7-, 12- and 24-weeks, and consistent with proteoglycan increase measured 5 weeks post-surgery, as a sign of cartilage matrix remodelling. Our work highlights the potential of 99mTc-NTP 15-5 as an imaging-based companion to monitor cartilage remodelling in OA and DMOAD response.

Danger signals and inflammaging in osteoarthritis.

Osteoarthritis (OA) is the most common age-related chronic and disabling joint disease. Long considered to be a "wear and tear" disease, OA is now seen as a low-grade inflammation disease that affects all tissues of the joint, involving cartilage degradation, bone remodelling, osteophytes, and synovitis. The process, called inflammaging, is characterised by the association of low-grade inflammation, profound changes in intra-cellular mechanisms, and the decreased efficiency of the immune system with ageing. The activation of innate immunity plays a critical role in the development and progression of OA. Innate immunity, including inflammasome activation, is triggered by small endogenous molecules called alarmins or damage-associated molecular patterns (DAMPs). These molecules are released in the extracellular media after cell stress or damage, bind to pathogen-recognition receptors (PRRs), such as Toll-like receptors (TLRs) and the receptor for advanced glycation end products (RAGE), and activate the secretion of pro-inflammatory factors, leading to joint inflammation. Moreover, such sterile inflammation triggers cell senescence, characterised by a senescence-associated secretory phenotype (SASP). Understanding the substantial age-related changes of joint tissues that influence the pathogenesis of OA is critical to improving the quality of life of elderly people in the context of increased life expectancy. This review will focus on age-related sterile inflammation in OA and highlight the various innovative and promising therapies targeting the mechanisms of aging.

Impairment of glyoxalase-1, an advanced glycation end-product detoxifying enzyme, induced by inflammation in age-related osteoarthritis.

BACKGROUND: Accumulation of advanced glycation end-products (AGEs) is involved in age-related osteoarthritis (OA). Glyoxalase (Glo)-1 is the main enzyme involved in the removal of AGE precursors, especially carboxymethyl-lysine (CML). We aimed to investigate the expression of several AGEs and Glo-1 in human OA cartilage and to study chondrocytic Glo-1 regulation by inflammation, mediated by interleukin (IL)-1ß. METHODS: Ex vivo, we quantified AGEs (pentosidine, CML, methylglyoxal-hydroimidazolone-1) in knee cartilage from 30 OA patients. Explants were also incubated with and without IL-1ß, and we assessed Glo-1 protein expression and enzymatic activity. In vitro, primary cultured murine chondrocytes were stimulated with increasing concentrations of IL-1ß to assess Glo-1 enzymatic activity and expression. To investigate the role of oxidative stress in the IL-1ß effect, cells were also treated with inhibitors of mitochondrial oxidative stress or nitric oxide synthase. RESULTS: Ex vivo, only the human cartilage CML content was correlated with patient age (r = 0.78, p = 0.0031). No statistically significant correlation was found between Glo-1 protein expression and enzymatic activity in human cartilage and patient age. We observed that cartilage explant stimulation with IL-1ß decreased Glo-1 protein expression and enzymatic activity. In vitro, we observed a dose-dependent decrease in Glo-1 mRNA, protein quantity, and enzymatic activity in response to IL-1ß in murine chondrocytes. Inhibitors of oxidative stress blunted this downregulation. CONCLUSION: Glo-1 is impaired by inflammation mediated by IL-1ß in chondrocytes through oxidative stress pathways and may explain age-dependent accumulation of the AGE CML in OA cartilage.

Proactive and reactive inhibitory control in eating disorders.

Altered inhibitory control has been implicated in the development and maintenance of eating disorders (ED), however it is unclear how different types of inhibitory control are affected across the EDs. We explored whether individuals with bulimia nervosa (BN), binge eating disorder (BED) and anorexia nervosa (AN) differed from healthy individuals (HC) on two types of motor inhibitory control: proactive inhibition (related to the preparation/initiation of a response) and reactive inhibition (withholding a response in reaction to a signal). Ninety-four women (28 AN, 27 BN, 11 BED, 28 HC) completed two neuropsychological tasks (a cued reaction time task and a stop signal task), and questionnaires assessing clinical variables, mood, anxiety, and inhibitory control. Self-reported inhibitory control was poorer in women with BN compared to the HC and AN groups, but greater in women with AN compared to all other groups. However, no group differences in reactive inhibition were observed. Proactive inhibition was augmented in women with AN compared to HC, and this was related to self-reported intolerance of uncertainty. The findings suggest that proactive inhibition may be a relevant target for behavioural interventions for AN, and call for further research into the relationship between intolerance of uncertainty and proactive inhibition.

Temporal Discounting and the Tendency to Delay Gratification across the Eating Disorder Spectrum.

Bulimia nervosa (BN) and binge eating disorder (BED) have been associated with poorer reward-related inhibitory control, reflected by a reduced tendency to delay gratification. The opposite has been reported in anorexia nervosa (AN), but differences have not been directly compared across eating disorders (EDs). This study investigated self-reported (Delaying Gratification Inventory) and task-based (temporal discounting) inhibitory control in 66 women with an ED and 28 healthy controls (HCs). Poorer task-based inhibitory control was observed in the BN compared with the AN group and poorer self-reported inhibitory control in the BN and in the BED groups compared with the AN and the HC groups, suggesting that reward-related inhibitory control varies across EDs. Symptom severity correlated with poorer self-reported (but not task-based) inhibitory control across the EDs. These data provide some support for transdiagnostic mechanisms and highlight the importance of addressing perceived loss of control in the treatment of EDs. Copyright © 2017 John Wiley & Sons, Ltd and Eating Disorders Association.

The danger from within: alarmins in arthritis.

Alarmins (also known as danger signals) are endogenous molecules that are released to the extracellular milieu after infection or tissue damage. Extracellular alarmins interact with specific receptors expressed by cells that are engaged in host defence to stimulate signalling pathways that result in initiation of innate and adaptive immune responses, triggering inflammation or tissue repair. Alarmins are considered to be markers of destructive processes that occur in degenerative joint diseases (primarily osteoarthritis (OA)) and chronic inflammatory joint diseases (such as rheumatoid arthritis, psoriatic arthritis and spondylarthropathy). In OA, high mobility group protein B1 (HMGB1) and S100 proteins, along with many other alarmins, are abundantly secreted by joint cells, promoting cartilage matrix catabolism, osteophyte formation, angiogenesis and hypertrophic differentiation. The involvement of alarmins in chronic inflammatory arthritides is suggested by their presence in serum at high levels in these conditions, and their expression within inflamed synovia and synovial fluid. S100 proteins, HMGB1, IL-33 and other endogenous molecules have deleterious effects on joints, and can recruit immune cells such as dendritic cells to inflamed synovia, initiating the adaptive immune response and perpetuating disease. Improving our understanding of the pathological mechanisms associated with these danger signals is important to enable the targeting of new therapeutic approaches for arthritis.

The pro-inflammatory cytokine 14-3-3ε is a ligand of CD13 in cartilage.

Osteoarthritis is a whole-joint disease characterized by the progressive destruction of articular cartilage involving abnormal communication between subchondral bone and cartilage. Our team previously identified 14-3-3ε protein as a subchondral bone soluble mediator altering cartilage homeostasis. The aim of this study was to investigate the involvement of CD13 (also known as aminopeptidase N, APN) in the chondrocyte response to 14-3-3ε. After identifying CD13 in chondrocytes, we knocked down CD13 with small interfering RNA (siRNA) and blocking antibodies in articular chondrocytes. 14-3-3ε-induced MMP-3 and MMP-13 was significantly reduced with CD13 knockdown, which suggests that it has a crucial role in 14-3-3ε signal transduction. Aminopeptidase N activity was identified in chondrocytes, but the activity was unchanged after stimulation with 14-3-3ε. Direct interaction between CD13 and 14-3-3ε was then demonstrated by surface plasmon resonance. Using labeled 14-3-3ε, we also found that 14-3-3ε binds to the surface of chondrocytes in a manner that is dependent on CD13. Taken together, these results suggest that 14-3-3ε might directly bind to CD13, which transmits its signal in chondrocytes to induce a catabolic phenotype similar to that observed in osteoarthritis. The 14-3-3ε-CD13 interaction could be a new therapeutic target in osteoarthritis.

Omics technologies provide new insights into the molecular physiopathology of equine osteochondrosis.

BACKGROUND: Osteochondrosis (OC(D)) is a juvenile osteo-articular disorder affecting several mammalian species. In horses, OC(D) is considered as a multifactorial disease and has been described as a focal disruption of endochondral ossification leading to the development of osteoarticular lesions. Nevertheless, OC(D) physiopathology is poorly understood. Affected horses may present joint swelling, stiffness and lameness. Thus, OC(D) is a major concern for the equine industry. Our study was designed as an integrative approach using omics technologies for the identification of constitutive defects in epiphyseal cartilage and/or subchondral bone associated with the development of primary lesions to further understand OC(D) pathology. This study compared samples from non-affected joints (hence lesion-free) from OC(D)-affected foals (n = 5, considered predisposed samples) with samples from OC-free foals (n = 5) considered as control samples. Consequently, results are not confounded by changes associated with the evolution of the lesion, but focus on altered constitutive molecular mechanisms. Comparative proteomics and micro computed tomography analyses were performed on predisposed and OC-free bone and cartilage samples. Metabolomics was also performed on synovial fluid from OC-free, OC(D)-affected and predisposed joints. RESULTS: Two lesion subtypes were identified: OCD (lesion with fragment) and OC (osteochondral defects). Modulated proteins were identified using omics technologies (2-DE proteomics) in cartilage and bone from affected foals compare to OC-free foals. These were associated with cellular processes including cell cycle, energy production, cell signaling and adhesion as well as tissue-specific processes such as chondrocyte maturation, extracellular matrix and mineral metabolism. Of these, five had already been identified in synovial fluid of OC-affected foals: ACTG1 (actin, gamma 1), albumin, haptoglobin, FBG (fibrinogen beta chain) and C4BPA (complement component 4 binding protein, alpha). CONCLUSION: This study suggests that OCD lesions may result from a cartilage defect whereas OC lesions may be triggered by both bone and cartilage defects, suggesting that different molecular mechanisms responsible for the equine osteochondrosis lesion subtypes and predisposition could be due to a defect in both bone and cartilage. This study will contribute to refining the definition of OC(D) lesions and may improve diagnosis and development of therapies for horses and other species, including humans.

Next-generation sequencing identifies equine cartilage and subchondral bone miRNAs and suggests their involvement in osteochondrosis physiopathology.

BACKGROUND: MicroRNAs (miRNAs) are an abundant class of small single-stranded non-coding RNA molecules ranging from 18 to 24 nucleotides. They negatively regulate gene expression at the post-transcriptional level and play key roles in many biological processes, including skeletal development and cartilage maturation. In addition, miRNAs involvement in osteoarticular diseases has been proved and some of them were identified as suitable biomarkers for pathological conditions. Equine osteochondrosis (OC) is one of the most prevalent juvenile osteoarticular disorders in horses and represents a major concern for animal welfare and economic reasons. Its etiology and pathology remain controversial and biological pathways as well as molecular mechanisms involved in the physiopathology are still unclear. This study aims to investigate the potential role of miRNAs in equine osteochondrosis (OC) physiopathology.Short-read NGS technology (SOLID™, Life Technologies) was used to establish a comprehensive repertoire of miRNA expressed in either equine cartilage or subchondral bone. Undamaged cartilage and subchondral bone samples from healthy (healthy samples) and OC-affected (predisposed samples) 10-month Anglo-Arabian foals were analysed. Samples were also subjected or not to an experimental mechanical loading to evaluate the role of miRNAs in the regulation of mechano-transduction pathways. Predicted targets of annotated miRNAs were identified using miRmap. RESULTS: Epiphyseal cartilage and subchondral bone miRNome were defined, including about 300 new miRNAs. Differentially expressed miRNAs were identified between bone and cartilage from healthy and OC foals, as well as after an experimental mechanical loading. In cartilage, functional annotation of their predicted targets suggests a role in the maintenance of cartilage integrity through the control of cell cycle and differentiation, energy production and metabolism as well as extracellular matrix structure and dynamics. In bone, miRNA predicited targets were associated with osteoblasts and osteoclasts differentiation, though the regulation of energy production, vesicle transport and some growth factor signaling pathways. CONCLUSION: Taken together, our results suggest a role of miRNAs in equine OC physiopathology and in the cellular response to biomechanical stress in cartilage and bone. In silico target prediction and functional enrichment analysis provides new insight into OC molecular physiopathology.

Protective role of frizzled-related protein B on matrix metalloproteinase induction in mouse chondrocytes.

INTRODUCTION: Our objective was to investigate whether a lack of frizzled-related protein B (FrzB), an extracellular antagonist of the Wnt signaling pathways, could enhance cartilage degradation by facilitating the expression, release and activation of matrix metalloproteinases (MMPs) by chondrocytes in response to tissue-damaging stimuli. METHODS: Cartilage explants from FrzB-/- and wild-type mice were challenged by excessive dynamic compression (0.5 Hz and 1 MPa for 6 hours). Load-induced glycosaminoglycan (GAG) release and MMP enzymatic activity were assessed. Interleukin-1ß (IL-1ß) (10, 100 and 1000 pg/mL for 24 hours) was used to stimulate primary cultures of articular chondrocytes from FrzB-/- and wild-type mice. The expression and release of MMP-3 and -13 were determined by RT-PCR, western blot and ELISA. The accumulation of ß-catenin was assessed by RT-PCR and western blot. RESULTS: Cartilage degradation, as revealed by a significant increase in GAG release (2.8-fold, P = 0.014) and MMP activity (4.5-fold, P = 0.014) by explants, was induced by an excessive load. Load-induced MMP activity appeared to be enhanced in FrzB-/- cartilage explants compared to wild-type (P = 0.17). IL-1ß dose-dependently induced Mmp-13 and -3 gene expression and protein release by cultured chondrocytes. IL-1ß-mediated increase in MMP-13 and -3 was slightly enhanced in FrzB-/- chondrocytes compared to wild-type (P = 0.05 and P = 0.10 at gene level, P = 0.17 and P = 0.10 at protein level, respectively). Analysis of Ctnn1b and Lef1 gene expression and ß-catenin accumulation at protein level suggests that the enhanced catabolic response of FrzB-/- chondrocytes to IL-1ß and load may be associated with an over-stimulation of the canonical Wnt/ß-catenin pathway. CONCLUSIONS: Our results suggest that FrzB may have a protective role on cartilage degradation and MMP induction in mouse chondrocytes by attenuating deleterious effects of the activation of the canonical Wnt/ß-catenin pathway.

Potential role of hyaluronic acid on bone in osteoarthritis: matrix metalloproteinases, aggrecanases, and RANKL expression are partially prevented by hyaluronic acid in interleukin 1-stimulated osteoblasts.

OBJECTIVE: To determine the effect of hyaluronic acid (HA) on proteolytic enzymes and bone remodeling mediators induced by interleukin 1ß (IL-1ß) and related to cartilage catabolism in murine osteoblasts. METHODS: Osteoblasts were obtained from Swiss mice and cultured for 3 weeks. HA-treated osteoblasts were incubated with 100 µg/ml HA during the last week of culture, then stimulated with IL-1ß (10 ng/ml) for 24 h. The expression of matrix metalloproteinases 3 and 13 (MMP-3 and MMP-13), ADAMTS-4 and ADAMTS-5, tissue inhibitor of metalloproteinases (TIMP), osteoprotegerin, and receptor activator of nuclear factor-κB ligand (RANKL) was determined by real-time polymerase chain reaction. MMP-3 and MMP-13 release was assessed by Western blot analysis. RESULTS: IL-1ß increased the mRNA levels of MMP-3 and MMP-13 and ADAMTS-4 and ADAMTS-5 and release of MMP-3 and MMP-13. Seven days of HA treatment significantly prevented the IL-1ß-increased mRNA levels of MMP-3 (-61%, p < 0.01), MMP-13 (-56%, p < 0.01), ADAMTS-4 (-58%, p < 0.05), ADAMTS-5 (-52%, p < 0.01), and RANKL (-49%, p < 0.05), but not TIMP. As well, IL-1ß-induced production of MMP-3 and MMP-13 was inhibited, by 27% (p < 0.01) and 40% (p < 0.01), respectively. CONCLUSION: In an inflammatory context in murine osteoblasts, HA can inhibit the expression of MMP and ADAMTS. Because HA can counteract the production of these mediators in chondrocytes, its beneficial effect in osteoarthritis may be due to its action on cartilage and subchondral bone.

Expression and function of visfatin (Nampt), an adipokine-enzyme involved in inflammatory pathways of osteoarthritis.

INTRODUCTION: Visfatin is an adipokine that may be involved in intertissular joint communication in osteoarthritis (OA). With a homodimeric conformation, it exerts nicotinamide phosphoribosyltransferase (Nampt) enzymatic activity, essential for nicotinamide adenine dinucleotide biosynthesis. We examined the tissular origin and conformation of visfatin/Nampt in human OA joints and investigated the role of visfatin/Nampt in chondrocytes and osteoblasts by studying Nampt enzymatic activity. METHODS: Synovium, cartilage and subchondral bone from human OA joints were used for protein extraction or incubated for 24 hours in serum-free media (conditioned media), and synovial fluid was obtained from OA patients. Visfatin/Nampt expression in tissular extracts and conditioned media was evaluated by western blot and enzyme-linked immunosorbent assay (ELISA), respectively. Nampt activity was assessed in OA synovium by colorimetric assay. Primary cultures of murine chondrocytes and osteoblasts were stimulated with visfatin/Nampt and pretreated or not with APO866, a pharmacologic inhibitor of Nampt activity. The effect on cytokines, chemokines, growth factors and hypertrophic markers expression was examined by quantitative reverse transcriptase polymerase chain reaction and/or ELISA. RESULTS: In tissular explants, conditioned media and synovial fluid, visfatin/Nampt was found as a homodimer, corresponding to the enzymatically active conformation. All human OA joint tissues released visfatin/Nampt (synovium: 628 ± 106 ng/g tissue; subchondral bone: 195 ± 26 ng/g tissue; cartilage: 152 ± 46 ng/g tissue), with significantly higher level for synovium (P <0.0005). Nampt activity was identified ex vivo in synovium. In vitro, visfatin/Nampt significantly induced the expression of interleukin 6, keratinocyte chemoattractant and monocyte chemoattractant protein 1 in chondrocytes and osteoblasts. APO866 decreased the mRNA and protein levels of these pro-inflammatory cytokines in the two cell types (up to 94% and 63% inhibition, respectively). Levels of growth factors (vascular endothelial growth factor, transforming growth factor ß) and hypertrophic genes were unchanged with treatment. CONCLUSION: Visfatin/Nampt is released by all human OA tissues in a dimeric enzymatically active conformation and mostly by the synovium, which displays Nampt activity. The Nampt activity of visfatin is involved in chondrocyte and osteoblast activation, so targeting this enzymatic activity to disrupt joint tissue interactions may be novel in OA therapy.

Identification of soluble 14-3-3∊ as a novel subchondral bone mediator involved in cartilage degradation in osteoarthritis.

OBJECTIVE: Mechanical stress plays an important role in cartilage degradation and subchondral bone remodeling in osteoarthritis (OA). The remodeling of the subchondral bone could initiate cartilage loss in OA through the interplay of bone and cartilage. The aim of this study was to identify soluble mediators released by loaded osteoblasts/osteocytes that could induce the release of catabolic factors by chondrocytes. METHODS: Murine osteoblasts/osteocytes were subjected to cyclic compression, and then conditioned medium from either compressed (CCM) or uncompressed (UCM) cells was used to stimulate mouse chondrocytes. Chondrocyte expression of matrix metalloproteinase 3 (MMP-3), MMP-13, type II collagen, and aggrecan was assessed by reverse transcription-polymerase chain reaction, Western blotting, and enzyme-linked immunosorbent assay. Soluble mediators released by compressed osteoblasts/osteocytes were identified using iTRAQ (isobaric tags for relative and absolute quantification), a differential secretome analysis. Subchondral bone and cartilage samples were isolated from OA patients, and culture medium conditioned with OA subchondral bone or cartilage was used to stimulate human chondrocytes. RESULTS: Stimulation of mouse chondrocytes with CCM strongly induced the messenger RNA (mRNA) expression and protein release of MMP-3 and MMP-13 and inhibited the mRNA expression of type II collagen and aggrecan. Differential secretome analysis revealed that 10 proteins were up-regulated in compressed osteoblasts/osteocytes. Among them, soluble 14-3-3∊ (s14-3-3∊) dose-dependently induced the release of catabolic factors by chondrocytes, mimicking the effects of cell compression. Addition of a 14-3-3∊ blocking antibody greatly attenuated the CCM-mediated induction of MMP-3 and MMP-13 expression. Furthermore, in human OA subchondral bone, s14-3-3∊ was strongly released, and in cultures of human OA chondrocytes, s14-3-3∊ stimulated MMP-3 expression. CONCLUSION: The results of this study identify s14-3-3∊ as a novel soluble mediator critical in the communication between subchondral bone and cartilage in OA. Thus, s14-3-3∊ may be a potential target for future therapeutic or prognostic applications in OA.

Stress-induced cartilage degradation does not depend on the NLRP3 inflammasome in human osteoarthritis and mouse models.

OBJECTIVE: The main feature of osteoarthritis (OA) is degradation and loss of articular cartilage. Interleukin-1ß (IL-1ß) is thought to have a prominent role in shifting the metabolic balance toward degradation. IL-1ß is first synthesized as an inactive precursor that is cleaved to the secreted active form mainly in the "inflammasome," a complex of initiators (including NLRP3), adaptor molecule ASC, and caspase 1. The aim of this study was to clarify the roles of IL-1ß and the inflammasome in cartilage breakdown. METHODS: We assessed IL-1ß release by cartilage explants from 18 patients with OA. We also evaluated the lipopolysaccharide (LPS)-, IL-1α-, and tumor necrosis factor α (TNFα)-induced activity of matrix metalloproteinase 3 (MMP-3), MMP-9, and MMP-13 in NLRP3-knockout mice and wild-type mice and the inhibition of caspase 1 with Z-YVAD-FMK and the blockade of IL-1ß with IL-1 receptor antagonist (IL-1Ra). Cartilage explants from NLRP3-knockout mice and IL-1R type I (IL-1RI)-knockout mice were subjected to excessive dynamic compression (0.5 Hz, 1 MPa) to trigger degradation, followed by assessment of load-induced glycosaminoglycan (GAG) release and MMP enzymatic activity. RESULTS: Despite the expression of NLRP3, ASC, and caspase 1, OA cartilage was not able to produce active IL-1ß. LPS, IL-1α, and TNFα dose-dependently increased MMP-3, MMP-9, and MMP-13 activity in cultured chondrocytes and in NLRP3(-/-) chondrocytes, and this effect was not changed by inhibiting caspase 1 or IL-1ß. The load-induced increase in GAG release and MMP activity was not affected by knockout of NLRP3 or IL-1RI in cartilage explants. CONCLUSION: OA cartilage may be degraded independently of any inflammasome activity, which may explain, at least in part, the lack of effect of IL-1ß inhibitors observed in previous trials.

Proinflammatory actions of visfatin/nicotinamide phosphoribosyltransferase (Nampt) involve regulation of insulin signaling pathway and Nampt enzymatic activity.

Visfatin (also termed pre-B-cell colony-enhancing factor (PBEF) or nicotinamide phosphoribosyltransferase (Nampt)) is a pleiotropic mediator acting on many inflammatory processes including osteoarthritis. Visfatin exhibits both an intracellular enzymatic activity (nicotinamide phosphoribosyltransferase, Nampt) leading to NAD synthesis and a cytokine function via the binding to its hypothetical receptor. We recently reported the role of visfatin in prostaglandin E(2) (PGE(2)) synthesis in chondrocytes. Here, our aim was to characterize the signaling pathways involved in this response in exploring both the insulin receptor (IR) signaling pathway and Nampt activity. IR was expressed in human and murine chondrocytes, and visfatin triggered Akt phosphorylation in murine chondrocytes. Blocking IR expression with siRNA or activity using the hydroxy-2-naphthalenyl methyl phosphonic acid tris acetoxymethyl ester (HNMPA-(AM)(3)) inhibitor diminished visfatin-induced PGE(2) release in chondrocytes. Moreover, visfatin-induced IGF-1R(-/-) chondrocytes released higher concentration of PGE(2) than IGF-1R(+/+) cells, a finding confirmed with an antibody that blocked IGF-1R. Using RT-PCR, we found that visfatin did not regulate IR expression and that an increased insulin release was also unlikely to be involved because insulin was unable to increase PGE(2) release. Inhibition of Nampt activity using the APO866 inhibitor gradually decreased PGE(2) release, whereas the addition of exogenous nicotinamide increased it. We conclude that the proinflammatory actions of visfatin in chondrocytes involve regulation of IR signaling pathways, possibly through the control of Nampt enzymatic activity.

Induction of the chemokine IL-8/Kc by the articular cartilage: possible influence on osteoarthritis.

PURPOSE: IL-8 and its murine equivalent keratinocyte chemoattractant (Kc), chemokines produced by chondrocytes, contribute to the pathophysiology of osteoarthritis. However, the mechanisms leading to their production are poorly known. We aimed to investigate whether mechanical (compression), inflammatory (IL-1ß) and metabolic (visfatin) stresses may induce the release of Kc when applied on murine cartilage. METHODS: Mouse cartilage explants were subjected to intermittent compression for 4, 6 and 24h. Primary cultures of immature murine articular chondrocytes were obtained by enzymatic digestion of articular cartilage from 6-days-old newborns mice. The effect of compression, IL-1ß (10, 50, 100pg/mL) and of visfatin (5µg/mL) on the release of Kc was assessed by ELISA. IL-8 levels in conditioned media from human OA joint tissues (cartilage or synovium) were also assessed. RESULTS: In comparison with non-compressed explants, loading increased Kc release of 3.2-, 1.9- and 2.0-fold at 4, 6 and 24h respectively (P<0.004, n=9). IL-1ß triggered an increase of Kc release by primary cultured chondrocytes of 4.1-, 15.5- and 35.2-fold at 10, 50 and 100pg/mL of IL-1ß respectively (P<0.05, n=4). Likewise, visfatin (5µg/mL) induced an increase in Kc release of 56.5±25.2 fold (P=0.002, n=6). IL-8 was released in conditioned media by synovium as well as by cartilage. CONCLUSION: We show for the first time that IL-8/Kc is highly responsive to mechanical, inflammatory and metabolic stresses, strengthening the hypothesis that IL-8/Kc could be added to the cytokines which may have a deleterious impact in osteoarthritis.

Inhibition of matrix metalloproteinase-3 and -13 synthesis induced by IL-1beta in chondrocytes from mice lacking microsomal prostaglandin E synthase-1.

Joint destruction in arthritis is in part due to the induction of matrix metalloproteinase (MMP) expression and their inhibitors, especially MMP-13 and -3, which directly degrade the cartilage matrix. Although IL-1ß is considered as the main catabolic factor involved in MMP-13 and -3 expression, the role of PGE(2) remains controversial. The goal of this study was to determine the role of PGE(2) on MMP synthesis in articular chondrocytes using mice lacking microsomal PGE synthase-1 (mPGES-1), which catalyses the rate-limiting step of PGE(2) synthesis. MMP-3 and MMP-13 mRNA and protein expressions were assessed by real-time RT-PCR, immunoblotting, and ELISA in primary cultures of articular chondrocytes from mice with genetic deletion of mPGES-1. IL-1ß-induced PGE(2) synthesis was dramatically reduced in mPGES-1(-/-) and mPGES-1(+/-) compared with mPGES-1(+/+) chondrocytes. A total of 10 ng/ml IL-1ß increased MMP-3 and MMP-13 mRNA, protein expression, and release in mPGES-1(+/+) chondrocytes in a time-dependent manner. IL-1ß-induced MMP-3 and MMP-13 mRNA expression, protein expression, and release decreased in mPGES-1(-/-) and mPGES-1(+/-) chondrocytes compared with mPGES-1(+/+) chondrocytes from 8 up to 24 h. Otherwise, MMP inhibition was partially reversed by addition of 10 ng/ml PGE(2) in mPGES-1(-/-) chondrocytes. Finally, in mPGES-1(-/-) chondrocytes treated by forskolin, MMP-3 protein expression was significantly decreased compared with wild-type, suggesting that PGE(2) regulates MMP-3 expression via a signaling pathway dependent on cAMP. These results demonstrate that PGE(2) plays a key role in the induction of MMP-3 and MMP-13 in an inflammatory context. Therefore, mPGES-1 could be considered as a critical target to counteract cartilage degradation in arthritis.