RESUMEN
INTRODUCTION: Both systemic inflammation and dyslipidemia contribute to osteoarthritis (OA) development and have been suggested as a possible link between metabolic disease and OA development. Recently, the CANTOS trial showed a reduction in knee and hip replacements after inhibition of IL-1ß in patients with a history of cardiovascular disease and high inflammatory risk. In this light, we investigated whether inhibition of IL-1ß combined with cholesterol-lowering therapies can reduce OA development in dyslipidemic APOE∗3Leiden mice under pro-inflammatory dietary conditions. MATERIALS AND METHODS: Female ApoE3∗Leiden mice were fed a cholesterol-supplemented Western-Type diet (WTD) for 38 weeks. After 14 weeks, cholesterol-lowering and anti-inflammatory treatments were started. Treatments included atorvastatin alone or with an anti-IL1ß antibody, and atorvastatin combined with proprotein convertase subtilisin-kexin type 9 (PCSK9) inhibitor alirocumab without or with the anti-IL1ß antibody. Knee joints were analyzed for cartilage degradation, synovial inflammation and ectopic bone formation using histology at end point. RESULTS: Cholesterol-lowering treatment successfully decreased systemic inflammation in dyslipidemic mice, which was not further affected by inhibition of IL-1ß. Synovial thickening and cartilage degeneration were significantly decreased in mice that received cholesterol-lowering treatment combined with inhibition of IL-1ß (P < 0.01, P < 0.05, respectively) compared to mice fed a WTD alone. Ectopic bone formation was comparable between all groups. CONCLUSION: These results indicate that inhibition of IL-1ß combined with cholesterol-lowering therapy diminishes synovial thickening and cartilage degeneration in mice and may imply that this combination therapy could be beneficial in patients with metabolic inflammation.
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Dislipidemias , Osteoartritis , Sinovitis , Ratones , Femenino , Animales , Proproteína Convertasa 9 , Atorvastatina , Colesterol/metabolismo , Inflamación , Modelos Animales de Enfermedad , Osteoartritis/metabolismo , Cartílago/metabolismoRESUMEN
OBJECTIVE: Since the joint microenvironment and tissue homeostasis are highly dependent on synovial fluid, we aimed to compare the essential chondrocyte signaling signatures of non-osteoarthritic vs end-stage osteoarthritic knee synovial fluid. Moreover, we determined the phenotypic consequence of the distinct signaling patterns on articular chondrocytes. METHODS: Protein profiling of synovial fluid was performed using antibody arrays. Chondrocyte signaling and phenotypic changes induced by non-osteoarthritic and osteoarthritic synovial fluid were analyzed using a phospho-kinase array, luciferase-based transcription factor activity assays, and RT-qPCR. The origin of osteoarthritic synovial fluid signaling was evaluated by comparing the signaling responses of conditioned media from cartilage, synovium, infrapatellar fat pad and meniscus. Osteoarthritic synovial fluid induced pathway-phenotype relationships were evaluated using pharmacological inhibitors. RESULTS: Compared to non-osteoarthritic synovial fluid, osteoarthritic synovial fluid was enriched in cytokines, chemokines and growth factors that provoked differential MAPK, AKT, NFκB and cell cycle signaling in chondrocytes. Functional pathway analysis confirmed increased activity of these signaling events upon osteoarthritic synovial fluid stimulation. Tissue secretomes of osteoarthritic cartilage, synovium, infrapatellar fat pad and meniscus activated several inflammatory signaling routes. Furthermore, the distinct pathway signatures of osteoarthritic synovial fluid led to accelerated chondrocyte dedifferentiation via MAPK/ERK signaling, increased chondrocyte fibrosis through MAPK/JNK and PI3K/AKT activation, an elevated inflammatory response mediated by cPKC/NFκB, production of extracellular matrix-degrading enzymes by MAPK/p38 and PI3K/AKT routes, and enabling of chondrocyte proliferation. CONCLUSION: This study provides the first mechanistic comparison between non-osteoarthritic and osteoarthritic synovial fluid, highlighting MAPKs, cPKC/NFκB and PI3K/AKT as crucial OA-associated intracellular signaling routes.
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Cartílago Articular , Condrocitos , Condrocitos/metabolismo , Líquido Sinovial/metabolismo , Cartílago Articular/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Células Cultivadas , FenotipoRESUMEN
INTRODUCTION: The association between metabolic syndrome (MetS) and osteoarthritis (OA) development has become increasingly recognized. In this context, the exact role of cholesterol and cholesterol-lowering therapies in OA development has remained elusive. Recently, we did not observe beneficial effects of intensive cholesterol-lowering treatments on spontaneous OA development in E3L.CETP mice. We postulated that in the presence of local inflammation caused by a joint lesion, cholesterol-lowering therapies may ameliorate OA pathology. MATERIALS AND METHODS: Female ApoE3∗Leiden.CETP mice were fed a cholesterol-supplemented Western type diet. After 3 weeks, half of the mice received intensive cholesterol-lowering treatment consisting of atorvastatin and the anti-PCSK9 antibody alirocumab. Three weeks after the start of the treatment, OA was induced via intra-articular injections of collagenase. Serum levels of cholesterol and triglycerides were monitored throughout the study. Knee joints were analyzed for synovial inflammation, cartilage degeneration, subchondral bone sclerosis and ectopic bone formation using histology. Inflammatory cytokines were determined in serum and synovial washouts. RESULTS: Cholesterol-lowering treatment strongly reduced serum cholesterol and triglyceride levels. Mice receiving cholesterol-lowering treatment showed a significant reduction in synovial inflammation (P = 0.008, WTD: 95% CI: 1.4- 2.3; WTD + AA: 95% CI: 0.8- 1.5) and synovial lining thickness (WTD: 95% CI: 3.0-4.6, WTD + AA: 95% CI: 2.1-3.2) during early-stage collagenase-induced OA. Serum levels of S100A8/A9, MCP-1 and KC were significantly reduced after cholesterol-lowering treatment (P = 0.0005, 95% CI: -46.0 to -12.0; P = 2.8 × 10-10, 95% CI: -398.3 to -152.1; P = 2.1 × 10-9, -66.8 to -30.4, respectively). However, this reduction did not reduce OA pathology, determined by ectopic bone formation, subchondral bone sclerosis and cartilage damage at end-stage disease. CONCLUSION: This study shows that intensive cholesterol-lowering treatment reduces joint inflammation after induction of collagenase-induced OA, but this did not reduce end stage pathology in female mice.
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Cartílago Articular , Osteoartritis , Ratones , Femenino , Animales , Esclerosis/patología , Membrana Sinovial/metabolismo , Osteoartritis/inducido químicamente , Osteoartritis/tratamiento farmacológico , Osteoartritis/complicaciones , Inflamación/metabolismo , Colagenasas/toxicidad , Colagenasas/metabolismo , Colesterol/metabolismo , Modelos Animales de Enfermedad , Cartílago Articular/patologíaRESUMEN
OBJECTIVES: Alterations in the composition of synovial fluid have been associated with adverse effects on cartilage integrity and function. Here, we examined the phenotypic and proliferative behavior of human articular chondrocytes when cultured in vitro for 13 days with synovial fluid derived from end-stage osteoarthritis patients. MATERIALS AND METHODS: Chondrocyte proliferation and phenotypical changes induced by osteoarthritic synovial fluid were analyzed using DNA staining, RT-qPCR, immunostainings, and immunoblotting. The molecular mechanisms by which osteoarthritic synovial fluid induced fibrosis and proliferation were studied using a phospho-protein antibody array and luciferase-based transcription factor activity assays. Specific pathway inhibitors were used to probe the involvement of pathways in fibrosis and proliferation. RESULTS: Prolonged stimulation with osteoarthritic synovial fluid sustained chondrocyte proliferation and induced profound phenotypic changes, favoring a fibrotic over a chondrogenic or hypertrophic phenotype. A clear loss of chondrogenic markers at both the transcriptional and protein level was observed, while expression of several fibrosis-associated markers were upregulated over time. Phospho-kinase analysis revealed activation of MAPK and RhoGTPase signaling pathways by osteoarthritic synovial fluid, which was confirmed by elevated transcriptional activity of Elk-1 and SRF. Inhibitor studies revealed that ERK played a central role in the loss of chondrocyte phenotype, while EGFR and downstream mediators p38, JNK and Rac/Cdc42 were essential for fibrosis-associated collagen expression. Finally, we identified EGF signaling as a key activator of chondrocyte proliferation. CONCLUSIONS: Osteoarthritic synovial fluid promoted chondrocyte fibrosis and proliferation through EGF receptor activation and downstream MAPK and RhoGTPase signaling.
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Cartílago Articular , Osteoartritis , Cartílago Articular/patología , Proliferación Celular , Células Cultivadas , Condrocitos/metabolismo , Fibrosis , Humanos , Osteoartritis/metabolismo , Líquido Sinovial/metabolismoRESUMEN
OBJECTIVE: Metabolic dysfunction can cause IL-1ß mediated activation of the innate immune system, which could have important implications for the therapeutic efficacy of IL-1ß neutralizing drugs as treatment for OA in the context of metabolic syndrome (MetS). In the present study, we investigated whether early treatment with a single dose of IL-1ß blocking antibodies could prevent Western diet (WD) induced changes to systemic monocyte populations and their cytokine secretion profile and herewith modulate collagenase induced osteoarthritis (CiOA) pathology. METHODS: CiOA was induced in female C57Bl/6 mice fed either a standard diet (SD) or WD and treated with a single dose of either polyclonal anti-IL-1ß antibodies or control. Monocyte subsets and granulocytes in bone marrow and blood were analyzed with flow cytometry, and cytokine expression by bone marrow cells was analyzed using qPCR. Synovial cellularity, cartilage damage and osteophyte formation were assessed on histology. RESULTS: WD feeding of C57Bl/6 mice led to increased serum levels of low-density lipoprotein (LDL) and innate immune activation in the form of an increased number of Ly6Chigh cells in bone marrow and blood and increased cytokine expression of IL-6 and TNF-α by bone marrow cells. The increase in monocyte number and activity was ameliorated by anti-IL-1ß treatment. However, anti-IL-1ß treatment did not significantly affect synovial lining thickness, cartilage damage and ectopic bone formation during WD feeding. CONCLUSIONS: Single-dose systemic anti-IL-1ß treatment prevented WD-induced innate immune activation during early stage CiOA in C57Bl/6 mice, but did not ameliorate joint pathology.
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Anticuerpos Monoclonales/farmacología , Dieta Occidental/efectos adversos , Interleucina-1beta/inmunología , Osteoartritis/inmunología , Animales , Antígenos Ly/metabolismo , Artritis Experimental , Células de la Médula Ósea/metabolismo , Recuento de Células , Femenino , Humanos , Interleucina-6/metabolismo , Lipoproteínas LDL/sangre , Monocitos/metabolismo , Rodilla de Cuadrúpedos/patología , Membrana Sinovial/patología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
OBJECTIVE: Osteoarthritis (OA) development is strongly associated with ageing, possibly due to age-related changes in transforming growth factor-ß (TGF-ß) signaling in cartilage. Recently, we showed that TGF-ß suppresses interleukin (IL)-6 receptor (IL-6R) expression in chondrocytes. As IL-6 is involved in cartilage degeneration, we hypothesized that age-related loss of TGF-ß signaling results in increased IL-6R expression and signaling in ageing cartilage. DESIGN: Bovine articular cartilage was collected and immediately processed to study age-related changes in IL-6R expression using qPCR and IHC (age-range: 0.5-14 years). Moreover, cartilage from young and aged cows was stimulated with rhIL-6 and/or rhTGF-ß1 to measure IL-6-induced p-STAT3 using Western blot. Expression of STAT3-responsive genes was analyzed using qPCR. RESULTS: Expression of IL-6 receptor (bIL-6R) significantly increased in cartilage upon ageing (slope: 0.32, 95%CI: 0.20-0.45), while expression of glycoprotein 130 (bGP130) was unaffected. Cartilage stimulation with IL-6 showed increased induction of p-STAT3 upon ageing (slope: 0.14, 95%CI: 0.08-0.20). Furthermore, IL-6-mediated induction of STAT3-responsive genes like bSOCS3 and bMMP3 was increased in aged compared to young cartilage. Interestingly, the ability of TGF-ß to suppress bIL6R expression in young cartilage was lost upon ageing (slope: 0.21, 95%CI: 0.13-0.30). Concurrently, an age-related loss in TGF-ß-mediated suppression of IL-6-induced p-STAT3 and bSOCS3 expression was observed. CONCLUSIONS: Ageing results in enhanced IL-6R expression and subsequent IL-6-induced p-STAT3 signaling in articular cartilage. This is likely caused by age-related loss of protective TGF-ß signaling, resulting in loss of TGF-ß-mediated IL-6R suppression. Because of the detrimental role of IL-6 in cartilage, this mechanism may be involved in age-related OA development.
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Envejecimiento/fisiología , Cartílago Articular/metabolismo , Receptores de Interleucina-6/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/fisiología , Animales , Bovinos , Metaloproteinasa 3 de la Matriz/metabolismo , Fosforilación , Factor de Transcripción STAT3/metabolismo , Proteína 3 Supresora de la Señalización de Citocinas/metabolismoRESUMEN
Inflammatory changes are observed in affected joints of osteoarthritis (OA) patients and are thought to be involved in the pathology that develops along OA progression. This narrative review provides an overview of the various cell types that are present in the joint during OA and which alarmins, cytokines, chemokines, growth factors, and other mediators they produce. Moreover, the involvement of more systemic processes like inflammaging and its associated cellular senescence in the context of OA are discussed.
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Alarminas/inmunología , Citocinas/inmunología , Inmunidad Innata/inmunología , Inflamación/inmunología , Osteoartritis/inmunología , Senescencia Celular/inmunología , HumanosRESUMEN
OBJECTIVE: A hallmark of osteoarthritis (OA) is degradation of articular cartilage proteoglycans. In isolated human OA chondrocytes, the anti-inflammatory cytokine Interleukin-37 (IL-37) lowers the expression of the proteolytic MMP and ADAMTS enzymes, which mediate this degradation. Therefore, we investigated if IL-37 protects against proteoglycan loss in freshly obtained human OA explants. MATERIAL AND METHODS: Human OA cartilage explants were incubated with IL-37. Release of sulphated proteoglycans (sGAGs) was measured with the dimethylmethylene-blue assay. Production and degradation of newly synthesized proteoglycans was measured using 35S-sulphate. Proteoglycan and proteolytic enzyme expression were analyzed by qPCR and Western Blot. Proteolytic activity was determined by measuring MMP- and ADAMTS-generated aggrecan neo-epitopes with ELISA and by using MMP-3-, MMP-13- or ADAMTS-5-inhibitors. RESULTS: Over time, a linear release of sGAGs from OA cartilage was measured. IL-37 reduced this release by 87 µg/ml (24%) 95%CI [21.04-141.4]. IL-37 did not affect 35S-sulphate incorporation or proteoglycan gene expression. In contrast, IL-37 reduced loss of 35S-sulphate labeled GAGs and reduced MMP-3 protein expression, indicating that IL-37 inhibits proteoglycan degradation. Remarkably, we observed two groups of patients; one group in which MMP-3-inhibition lowered sGAG release, and one group in which ADAMTS5-inhibition had this effect. Remarkably, IL-37 was only functional in the group of patients that responded to MMP-3-inhibition. CONCLUSION: We identified a relationship between IL-37 and reduced sGAG loss in OA cartilage. Most likely, this effect is mediated by inhibition of MMP-3 expression. These results suggest that IL-37 could be applied as therapy in a subgroup of OA patients, in which cartilage degradation is mediated by MMP-3.
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Cartílago Articular/efectos de los fármacos , Interleucina-1/farmacología , Metaloproteinasa 3 de la Matriz/metabolismo , Osteoartritis/metabolismo , Proteoglicanos/metabolismo , Cartílago Articular/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Interleucina-1/administración & dosificación , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Proteolisis/efectos de los fármacos , Proteínas Recombinantes/farmacología , Técnicas de Cultivo de TejidosRESUMEN
OBJECTIVE: To explore the associations between different histologically assessed, inflammatory synovial characteristics and subsequent clinical and structural aspects in knee osteoarthritis (OA). DESIGN: Knee OA patients, ranging in stage from early to advanced, were recruited from three different ongoing studies. Synovial tissue biopsies were taken and histologically assessed for six features (four inflammatory related aspects, fibrosis and fibrin deposition). Clinical aspects (WOMAC pain, functioning and stiffness and SF-36 vitality) and structural aspects (Kellgren and Lawrence (KL)-grade, joint space narrowing (JSN; 0-3) and osteophytes (0-3), and reception of total knee replacement (TKR)) were repeatedly assessed during follow-up. Associations between histology and clinical and structural aspects were analysed using linear mixed model analyses and cox proportional hazards analysis. RESULTS: Biopsies of 83 patients (median complaint duration: 5 [2-8] years) were analysed. Follow-up was a median of 1.4 [0.8-2.7] years for clinical and 1.8 [0.2-5.2] years for structural aspects. Fibrosis and fibrin deposition were inversely correlated with the inflammatory features. A higher fibrosis score was associated with a lower scores for KL-grade, JSN and osteophytes, while higher scores for perivascular oedema, synovial lining thickness and vascularisation were associated with higher scores for structural aspects during follow-up. No associations were found between each of the histological features and any of the clinical aspects or the chance for TKR during follow-up. CONCLUSIONS: Inflammatory related histological aspects are associated with subsequent increased radiological severity in knee OA, while fibrosis seems to protect against this, providing a potential therapeutic target for OA treatment.
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Articulación de la Rodilla/patología , Osteoartritis de la Rodilla/patología , Adulto , Anciano , Artroplastia de Reemplazo de Rodilla/estadística & datos numéricos , Artroscopía , Biopsia , Progresión de la Enfermedad , Femenino , Fibrosis , Humanos , Articulación de la Rodilla/diagnóstico por imagen , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Osteoartritis de la Rodilla/diagnóstico por imagen , Osteoartritis de la Rodilla/cirugía , Estudios Prospectivos , Radiografía , Índice de Severidad de la Enfermedad , Membrana Sinovial/patologíaRESUMEN
OBJECTIVE: Synovitis in collagenase-induced osteoarthritis (CiOA) is driven by locally released S100A8/A9 proteins and enhances joint destruction. S100A8/A9 can induce reactive oxygen species (ROS) release by phagocytes in OA synovium via neutrophil cytosolic factor-1 (Ncf1)-regulated NOX2 activation. In the present study we investigated whether NOX2-derived ROS affect joint pathology during CiOA. METHODS: CiOA was induced in knee joints of wild type (WT) and Ncf1-deficient (Ncf1**) mice. Synovial gene expression of NOX2-subunits was measured with quantitative real-time polymerase chain reaction (qRT-PCR). Joint pathology was assessed using histology and immunohistochemistry for aggrecan neo-epitope VDIPEN. Levels of inflammatory proteins were measured with Luminex or ELISA. Phagocytes in synovium, blood, bone marrow (BM) and spleen were analyzed with flow cytometry. ROS release by phagocytes was measured with a ROS detection kit. RESULTS: CiOA induction in knee joints of WT mice caused significantly increased synovial gene expression of NOX2 subunits. On day 7 of CiOA, cartilage damage and MMP activity, as measured by VDIPEN, were comparable between WT and Ncf1** mice. Synovial thickening, synovial S100A8/A9 levels and percentages of synovial macrophages, polymorphonuclear cells (PMNs), and monocytes were not different, as were levels of inflammatory mediators in serum and phagocyte percentages in blood, BM and spleen. On day 42 of CiOA, synovitis, cartilage damage, and osteophyte formation in Ncf1** mice were unaltered when compared to WT mice. ROS detection confirmed that Ncf1** PMNs lack functional NOX2, but in vitro macrophages showed ROS production, suggesting activation of compensatory mechanisms. CONCLUSIONS: Absence of Ncf1-mediated ROS production does not alter joint pathology in CiOA.
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Artritis Experimental/metabolismo , NADPH Oxidasa 2/metabolismo , Osteoartritis/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Artritis Experimental/patología , Cartílago Articular/lesiones , Cartílago Articular/patología , Colagenasas , Progresión de la Enfermedad , Femenino , Regulación de la Expresión Génica/fisiología , Macrófagos/metabolismo , Masculino , Ratones Endogámicos C3H , Ratones Mutantes , NADPH Oxidasa 2/genética , NADPH Oxidasas/deficiencia , NADPH Oxidasas/fisiología , Osteoartritis/patología , Membrana Sinovial/metabolismoRESUMEN
OBJECTIVE: Osteoarthritis (OA) is the most common joint disease but an effective pharmacological therapy has not been developed yet. To identify targets for treatment and ways to interfere with OA development and progression both spontaneous and induced OA models are still needed. In this narrative review it is discussed what variables can be identified that lead to variation in OA animal model studies. DESIGN: Literature was screened (Pubmed) with the following terms; OA animal models in combination with species, age, strain, gender/sex, housing, diet, fighting, circadian rhythm, transgenic. Relevant articles were selected and additional papers were searched for and read for specific subtopics. RESULTS: Studies with OA models are subject to a multitude of variables, stimuli and conditions that can influence the outcome of an animal experiment. Outcome will depend on amongst others; the model used, species and strain, age, gender, diet, housing conditions, circadian rhythm, timing of intervention, stress levels and activity. Variations in these variables can account for discrepancies between OA model experiments, intervention studies and conclusions. CONCLUSION: To improve OA animal model research, investigators should be aware of all the stimuli and conditions that can interfere with disease development and disease intervention and take these into account in their study design and execution.
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Modelos Animales de Enfermedad , Articulaciones/anatomía & histología , Osteoartritis/tratamiento farmacológico , Factores de Edad , Animales , Perros , Femenino , Cabras , Cobayas , Caballos , Vivienda para Animales , Humanos , Masculino , Ratones , Ratones Transgénicos , Conejos , Ratas , Factores Sexuales , Ovinos , Especificidad de la Especie , Resultado del TratamientoRESUMEN
This review highlights a selection of literature in the area of osteoarthritis biology published between the 2015 and 2016 Osteoarthritis Research Society International (OARSI) World Congress. Highlights were selected from a pubmed search covering cartilage, bone, inflammation and pain. A personal selection was made based, amongst other things, on topics presented during the 2015 conference. This covers circadian rhythm, TGF-ß signaling, autophagy, SIRT6, exercise, lubricin, TLR's, pain and NGF. Furthermore, in this review we have made an effort to connect these seemingly distant topics into one scheme of connections between them, revealing a theoretical big picture underneath.
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Osteoartritis/fisiopatología , Animales , Autofagia/fisiología , Ritmo Circadiano/fisiología , Ejercicio Físico/fisiología , Glicoproteínas/fisiología , Humanos , Osteoartritis/metabolismo , Sirtuinas/fisiología , Factor de Crecimiento Transformador beta/fisiologíaRESUMEN
OBJECTIVE: Increased Wisp1 expression was previously reported in experimental and human osteoarthritis (OA). Moreover, adenoviral overexpression of Wisp1 in naïve mouse knee joints resulted in early OA-like cartilage lesions. Here, we determined how the matricellular protein WISP1 is involved in the pathology that occurs in the complex osteoarthritic environment with aging and experimental OA in wild type (WT) and Wisp1-/- mice. METHODS: WT and Wisp1-/- mice were aged or experimental OA was induced with intraarticular collagenase injection, destabilization of the medial meniscus (DMM) or anterior cruciate ligament transection (ACLT). Joint pathology was assessed using histology and microCT. Protease expression was evaluated with qRT-PCR and activity was determined by immunohistochemical staining of the aggrecan neoepitope NITEGE. Protease expression in human end-stage OA synovial tissue was determined with qRT-PCR after stimulation with WISP1. RESULTS: With aging, spontaneous cartilage degeneration in Wisp1-/- was not decreased compared to their WT controls. However, we observed significantly decreased cartilage degeneration in Wisp1-/- mice after induction of three independent experimental OA models. While the degree of osteophyte formation was comparable between WT and Wisp1-/- mice, increased cortical thickness and reduced trabecular spacing was observed in Wisp1-/- mice. In addition, we observed decreased MMP3/9 and ADAMTS4/5 expression in Wisp1-/- mice, which was accompanied by decreased levels of NITEGE. In line with this, stimulation of human OA synovium with WISP1 increased the expression of various proteases. CONCLUSIONS: WISP1 plays an aggravating role in the development of post-traumatic experimental OA.
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Artritis Experimental/genética , Proteínas CCN de Señalización Intercelular/genética , Cartílago Articular/metabolismo , Osteoartritis de la Rodilla/genética , Péptido Hidrolasas/genética , Proteínas Proto-Oncogénicas/genética , Animales , Ligamento Cruzado Anterior/cirugía , Artritis Experimental/diagnóstico por imagen , Artritis Experimental/metabolismo , Artritis Experimental/patología , Cartílago Articular/diagnóstico por imagen , Cartílago Articular/patología , Colagenasas , Modelos Animales de Enfermedad , Humanos , Inyecciones Intraarticulares , Meniscos Tibiales/cirugía , Ratones , Ratones Noqueados , Osteoartritis de la Rodilla/diagnóstico por imagen , Osteoartritis de la Rodilla/metabolismo , Osteoartritis de la Rodilla/patología , Osteofito , Péptido Hidrolasas/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Membrana Sinovial/metabolismo , Vía de Señalización Wnt , Microtomografía por Rayos XRESUMEN
OBJECTIVE: Low-density lipoproteins (LDL) in inflamed synovium is oxidized and taken-up by synoviocytes. In this study, we investigate whether direct injection of oxidized LDL (oxLDL) into a normal murine knee joint induces joint pathology and whether synovial macrophages are involved in that process. DESIGN: Synovium was obtained from end-stage osteoarthritis (OA) patients in order to analyze LDL-uptake. Murine knee joints were injected five consecutive days with oxLDL, LDL, or vehicle (phosphate buffered saline (PBS)). This procedure was repeated in mice depleted of synovial macrophages by intra-articular injection of clodronate liposomes 7 days prior to the consecutive injections. Joint pathology was investigated by immunohistochemistry, flow cytometry (FCM) and synovial RNA expression and protein production. RESULTS: Synovial tissue of OA patients showed extensive accumulation of apolipoprotein B. Multiple injections of oxLDL in murine knee joints significantly increased TGF-ß activity in synovial wash-outs, but did not induce catabolic or inflammatory processes. In contrast, repeated injections of oxLDL in macrophage-depleted knee joints led to increased synovial thickening in combination with significantly upregulated protein and RNA levels of CCL2 and CCL3. FCM-analyses revealed increased presence of monocytes and neutrophils in the synovium, which was confirmed by immunohistochemistry. Also protein levels of S100A8/A9 were significantly increased in synovial wash-outs of oxLDL-injected joints, as was expression of aggrecanase-induced neo-epitopes. Interestingly, no raise in TGF-ß concentrations was measured in macrophage-depleted joints. CONCLUSIONS: OxLDL can affect joint pathology, since synovial macrophages promote anabolic processes after oxLDL injections. In absence of synovial macrophages, however, oxLDL induces production of pro-inflammatory mediators and aggrecanase activity combined with increased influx of monocytes and neutrophils.
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Calgranulina A/metabolismo , Calgranulina B/metabolismo , Lipoproteínas LDL/farmacología , Macrófagos/fisiología , Líquido Sinovial/citología , Factor de Crecimiento Transformador beta/fisiología , Animales , Humanos , Inyecciones Intraarticulares , Lipoproteínas LDL/administración & dosificación , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Osteoartritis/metabolismo , Líquido Sinovial/fisiologíaRESUMEN
OBJECTIVE: Interleukin-1 (IL-1) is an alleged important cytokine in osteoarthritis (OA), although the exact contribution of IL-1 to joint destruction remains unclear. Here we investigated the involvement of IL-1α and IL-1ß in joint pathology during collagenase-induced OA (CiOA). METHODS: CiOA was induced in wild type (WT) and IL-1αß-/- mice. Additionally, IL-1 signaling was inhibited in WT mice with CiOA using osmotic pumps containing IL-1RA. Joint pathology was assessed using histology. Activity of cartilage-degrading enzymes was determined using antibodies against aggrecan neo-epitopes VDIPEN and NITEGE. Synovial gene expression was analyzed using quantitative real-time polymerase chain reaction (qRT-PCR). Serum protein levels were measured with Luminex or enzyme-linked immunosorbent assay (ELISA). RESULTS: Synovial IL-1ß expression was strongly elevated 7 days after induction of CiOA in WT mice but decreased afterwards, whereas S100A8/A9, previously described to aggravate OA, remained elevated for 21 days. Remarkably, synovial inflammation was comparable between WT and IL-1αß-/- mice on day 7 of CiOA. In line, synovial mRNA expression of genes involved in IL-1 signaling and inflammatory mediators was comparable between WT and IL-1αß-/- mice, and serum levels for Keratinocyte Chemoattractant (KC)/IL-6/S100A8/S100A9/IL-10 were equal. Synovial matrix metalloproteinase (MMP)/aggrecanase expression and activity in cartilage was not different in WT and IL-1αß-/- mice on day 7 of CiOA. Cartilage destruction on day 42 was not different between WT and IL-1αß-/- mice, which was supported by our finding that IL-1RA treatment in WT mice with CiOA did not alter joint destruction. CONCLUSIONS: IL-1α and IL-1ß are not involved in synovial inflammation and cartilage destruction during CiOA, implicating that other mediators are responsible for the joint damage.
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Cartílago/patología , Colagenasas/metabolismo , Interleucina-1/metabolismo , Osteoartritis/metabolismo , Sinovitis/metabolismo , Animales , Femenino , Interleucina-1alfa/metabolismo , Interleucina-1beta/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Osteoartritis/etiología , Osteoartritis/patología , Reacción en Cadena en Tiempo Real de la Polimerasa , Membrana Sinovial/metabolismo , Sinovitis/etiología , Sinovitis/patología , TranscriptomaRESUMEN
OBJECTIVE: Alarmins S100A8 and S100A9 are major products of activated macrophages regulating cartilage damage and synovial activation during murine and human osteoarthritis (OA). In the current study, we investigated whether S100A8 and S100A9 are involved in osteophyte formation during experimental OA and whether S100A8/A9 predicts osteophyte progression in early human OA. METHODS: OA was elicited in S100A9-/- mice in two experimental models that differ in degree of synovial activation. Osteophyte size, S100A8, S100A9 and VDIPEN neoepitope was measured histologically. Chondrogenesis was induced in murine mesenchymal stem cells in the presence of S100A8. Levels of S100A8/A9 were determined in plasma of early symptomatic OA participants of the Cohort Hip and Cohort Knee (CHECK) cohort study and osteophytes measured after 2 and 5 years. RESULTS: Osteophyte size was drastically reduced in S100A9-/- mice in ligaments and at medial femur and tibia on days 21 and 42 of collagenase-induced OA, in which synovial activation is high. In contrast, osteophyte size was not reduced in S100A9-/- mice during destabilised medial meniscus OA, in which synovial activation is scant. S100A8 increased expression and activation of matrix metalloproteinases during micromass chondrogenesis, thereby possibly increasing cartilage matrix remodelling allowing for larger osteophytes. Interestingly, early symptomatic OA participants of the CHECK study with osteophyte progression after 2 and 5 years had elevated S100A8/A9 plasma levels at baseline, while C-reactive protein, erythrocyte sedimentation rate and cartilage oligomeric matrix protein were not elevated at baseline. CONCLUSIONS: S100A8/A9 aggravate osteophyte formation in experimental OA with high synovial activation and may be used to predict osteophyte progression in early symptomatic human OA.
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Artritis Experimental/metabolismo , Calgranulina A/fisiología , Calgranulina B/fisiología , Osteoartritis/metabolismo , Osteofito/metabolismo , Animales , Artritis Experimental/complicaciones , Artritis Experimental/patología , Biomarcadores/metabolismo , Calgranulina A/deficiencia , Cartílago Articular/enzimología , Cartílago Articular/fisiopatología , Condrogénesis/fisiología , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Metaloproteinasas de la Matriz/biosíntesis , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Osteoartritis/complicaciones , Osteoartritis/patología , Osteofito/etiología , Osteofito/patología , Membrana Sinovial/metabolismo , Regulación hacia Arriba/fisiologíaRESUMEN
OBJECTIVE: Mechanical signals control key cellular processes in articular cartilage. Previously we have shown that mechanical compression is an important ALK5/Smad2/3P activator in cartilage explants. However, age-related changes in the cartilage are known to affect tissue mechanosensitivity and also ALK5/Smad2/3P signaling. We have investigated whether ageing of cartilage is associated with an altered response to mechanical compression. DESIGN: Articular cartilage explants of two different age groups (young-6-36 months old, aged-6 - 13 years old) were subjected to dynamic mechanical compression with 3 MPa (physiological) or 12 MPa (excessive) load. Subsequently, essential cartilage extracellular matrix (ECM) components and tissue growth factors gene expression was measured in young and aged cartilage by QPCR. Furthermore, the ability of young and aged cartilage, to activate the Smad2/3P signaling in response to compression was analyzed and compared. This was done by immunohistochemical (IH) Smad2P detection and Smad3-responsive gene expression analysis. RESULTS: Aged cartilage showed a highly reduced capacity for mechanically-mediated activation of Smad2/3P signaling when compared to young cartilage. Compression of aged cartilage, induced collagen type II (Col2a1) and fibronectin (Fn1) expression to a far lesser extent than in young cartilage. Additionally, in aged cartilage no mechanically mediated up-regulation of bone morphogenetic protein 2 (Bmp2) and connective tissue growth factor (Ctgf) was observed. CONCLUSIONS: We identified age-related changes in cellular responses to mechanical stimulation of articular cartilage. We propose that these changes might be associated with age-related alterations in cartilage functioning and can underlie mechanisms for development of age-related cartilage diseases like osteoarthritis (OA).
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Envejecimiento/genética , Cartílago Articular/metabolismo , Osteoartritis/genética , Presión , Proteína Smad2/genética , Proteína smad3/genética , Agrecanos/genética , Agrecanos/metabolismo , Envejecimiento/metabolismo , Envejecimiento/fisiología , Animales , Proteína Morfogenética Ósea 2/genética , Proteína Morfogenética Ósea 2/metabolismo , Cartílago Articular/fisiología , Bovinos , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Factor de Crecimiento del Tejido Conjuntivo/genética , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Matriz Extracelular , Fibronectinas/genética , Fibronectinas/metabolismo , Perfilación de la Expresión Génica , Proteoglicanos de Heparán Sulfato/genética , Proteoglicanos de Heparán Sulfato/metabolismo , Osteoartritis/metabolismo , Inhibidor 1 de Activador Plasminogénico/genética , Inhibidor 1 de Activador Plasminogénico/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
OBJECTIVE: Recently it was shown that loading of articular cartilage explants activates TGFß signaling. Here we investigated if in vivo chondrocytes express permanently high TGFß signaling, and the consequence of the loss of compressive loading-mediated TGFß signaling on chondrocyte function and phenotype. METHOD: Bovine articular cartilage explants were collected within 10 min post mortem and stained immediately and after 30, 60 and 360 min for phosphorylated-Smad2, indicating active TGFß signaling. Explants were unloaded for 48 h and subsequently repeatedly loaded with a compressive load of 3 MPa. In addition, explants were cultured unloaded for 2 weeks and the effect of loading or exogenous TGFß on proteoglycan level and chondrocyte phenotype (Col10a1 mRNA expression) was analyzed. RESULTS: Unloading of articular cartilage results in rapid loss of TGFß signaling while subsequent compressive loading swiftly restored this. Loading and exogenous TGFß enhanced expression of TGFß1 and ALK5. Unloading of explants for 2 weeks resulted in proteoglycan loss and increased Col10a1 expression. Both loading and exogenous TGFß inhibited elevated Col10a1 expression but not proteoglycan loss. CONCLUSION: Our data might imply that in vivo regular physiological loading of articular cartilage leads to enduring TGFß signaling and TGFß-induced gene expression. We propose a hypothetical model in which loading activates a self-perpetuating system that prevents hypertrophic differentiation of chondrocytes and is crucial for cartilage homeostasis.
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Cartílago Articular , Animales , Bovinos , Condrocitos , Fenotipo , Proteoglicanos , Factor de Crecimiento Transformador betaRESUMEN
OBJECTIVE: Ageing is the main risk factor for osteoarthritis (OA). We investigated if expression of transforming growth factor ß (TGFß)-family components, a family which is crucial for the maintenance of healthy articular cartilage, is altered during ageing in cartilage. Moreover, we investigated the functional significance of selected age-related changes. DESIGN: Age-related changes in expression of TGFß-family members were analysed by quantitative PCR in healthy articular cartilage obtained from 42 cows (age: ¾-10 years). To obtain functional insight of selected changes, cartilage explants were stimulated with TGFß1 or bone morphogenetic protein (BMP) 9, and TGFß1 and BMP response genes were measured. RESULTS: Age-related cartilage thinning and loss of collagen type 2a1 expression (â¼256-fold) was observed, validating our data set for studying ageing in cartilage. Expression of the TGFß-family type I receptors; bAlk2, bAlk3, bAlk4 and bAlk5 dropped significantly with advancing age, whereas bAlk1 expression did not. Of the type II receptors, expression of bBmpr2 decreased significantly. Type III receptor expression was unaffected by ageing. Expression of the ligands bTgfb1 and bGdf5 also decreased with age. In explants, an age-related decrease in TGFß1-response was observed for the pSmad3-dependent gene bSerpine1 (P = 0.016). In contrast, ageing did not affect BMP9 signalling, an Alk1 ligand, as measured by expression of the pSmad1/5 dependent gene bId1. CONCLUSIONS: Ageing negatively affects both the TGFß-ALK5 and BMP-BMPR signalling routes, and aged chondrocytes display a lowered pSmad3-dependent response to TGFß1. Because pSmad3 signalling is essential for cartilage homeostasis, we propose that this change contributes to OA development.
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Envejecimiento , Animales , Receptores de Proteínas Morfogenéticas Óseas , Cartílago Articular , Bovinos , Condrocitos , Transducción de Señal , Factor de Crecimiento Transformador betaRESUMEN
OBJECTIVE: A relation between osteoarthritis (OA) and increased cholesterol levels is apparent. In the present study we investigate OA pathology in apolipoprotein E (ApoE)(-)(/-) mice with and without a cholesterol-rich diet, a model for high systemic low density lipoprotein (LDL) cholesterol levels independent of weight. METHOD: Wild type (WT), Apoe(-)(/-), S100a9(-/-) and Apoe(-)(/-)S100a9(-/-) mice (C57BL/6 background) received a standard or cholesterol-rich diet. Experimental OA was induced by intra-articular injection of collagenase and animals were sacrificed at day 10 and day 36. RESULTS: Although minimal differences in cartilage damage were found between the WT and ApoE(-)(/-) mice, increased synovial thickening was found in the latter. Thirty-six days after OA-induction, ApoE(-)(/-) mice on a standard diet showed increased ectopic bone formation, particularly at the medial collateral ligament, compared with OA in WT mice. Furthermore, a significant increase in synovial gene expression of both S100a8 and S100a9 and S100A8/S100A9 protein levels was found in ApoE(-)(/-) mice, suggesting an activated inflammatory status of synovial cells. In both ApoE(-)(/-) and WT mice, addition of a cholesterol-rich diet resulted in excessive bone formation in the medial collateral ligament at late-time-point OA. Interestingly, at the early time point, proteoglycan deposition was already significantly increased in ApoE(-)(/-) mice compared with WT mice. Mice deficient for both ApoE and S100a9 also showed increased ectopic bone formation, but not synovial activation, suggesting a role for S100-proteins in cholesterol-mediated synovial activation. CONCLUSIONS: Increased cholesterol levels strongly elevate synovial activation and ectopic bone formation in early-stage collagenase-induced OA.