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BACKGROUND: Recent findings support a connection between mitochondrial dysfunction and activation of inflammatory pathways in articular cells. This study investigates in vivo in an acute model whether intra-articular administration of oligomycin, an inhibitor of mitochondrial function, induces an oxidative and inflammatory response in rat knee joints. METHODS: Oligomycin was injected into the rat left knee joint on days 0, 2, and 5 before joint tissues were obtained on day 6. The right knee joint served as control. Results were evaluated by macroscopy and histopathology and by measuring cellular and mitochondrial reactive oxygen species (ROS), 4-hydroxy-2-nonenal (4-HNE, a marker of lipid peroxidation), nuclear factor erythroid 2-related factor 2 (Nrf2), and CD68 (macrophages) and chemokine levels. The marker of mitochondrial mass COX-IV was also evaluated. RESULTS: The macroscopic findings showed significantly greater swelling in oligomycin-injected knees than in control knees. Likewise, the histological score of synovial damage was also increased significantly. Immunohistochemical studies showed high expression of IL-8, coinciding with a marked infiltration of polymorphonuclears and CD68+ cells in the synovium. Mitochondrial mass was increased in the synovium of oligomycin-injected joints, as well as cellular and mitochondrial ROS production, and 4-HNE. Relatedly, expression of the oxidative stress-related transcription factor Nrf2 was also increased. As expected, no histological differences were observed in the cartilage; however, cytokine-induced neutrophil chemoattractant-1 mRNA and protein expression were up-regulated in this tissue. CONCLUSIONS: Mitochondrial failure in the joint is able to reproduce the oxidative and inflammatory status observed in arthritic joints.
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Artritis Experimental/patología , Inhibidores Enzimáticos/farmacología , Articulación de la Rodilla/patología , Mitocondrias/efectos de los fármacos , Osteoartritis/patología , Anciano , Anciano de 80 o más Años , Aldehídos/metabolismo , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Artritis Experimental/inducido químicamente , Cartílago Articular/patología , Quimiocina CXCL1/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Inyecciones Intraarticulares , Interleucina-8/metabolismo , Macrófagos/metabolismo , Persona de Mediana Edad , Mitocondrias/metabolismo , ATPasas de Translocación de Protón Mitocondriales/antagonistas & inhibidores , Factor 2 Relacionado con NF-E2/metabolismo , Oligomicinas/farmacología , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Membrana Sinovial/patologíaRESUMEN
OBJECTIVE: The present study aimed to determine the protective effects of dietary supplementation with resveratrol (RSV) in an acute antigen-induced arthritis (AIA) model. METHODS: Rats were randomly divided into three groups: control, AIA and RSV-treated AIA group. RSV (12.5 mg/kg/day) was given orally for 8 weeks before induction of AIA and until the end of the experiment (48 h after intra-articular injection). The control and AIA animals were administered 100 µl of water. Results were evaluated by macroscopic observation, histopathology and immunohistochemistry for anti-PCNA, macrophages (CD68), T lymphocytes (CD3), monocyte chemoattractant protein-1 and 8-oxo-7,8-dihydro-2'-deoxyguanine (a marker of DNA damage). Cytokine-induced neutrophil chemoattractant-1 in serum and peroxidase activity in synovial tissue were measured using commercial kits. RESULTS: At the end of the study, RSV significantly reduced knee swelling. Likewise, the histological score of synovial tissue also reduced significantly. The arthritis-protective effects were associated with a significant decrease in PCNA, CD68, CD3 and monocyte chemoattractant protein-1 staining, as well as a reduction in serum concentrations of cytokine-induced neutrophil chemoattractant-1. RSV treatment also decreased the level of the marker of DNA damage, 8-oxo-7,8-dihydro-2'-deoxyguanine. Accordingly, peroxidase activity in the synovial tissue was up-regulated. CONCLUSION: Dietary supplementation with RSV lowers the main pathological hallmarks of RA disease in an acute model of AIA. RSV may represent a promising strategy in controlling the severity of RA.
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Antioxidantes/farmacología , Artritis Experimental/tratamiento farmacológico , Estilbenos/farmacología , Membrana Sinovial/patología , Animales , Artritis Experimental/inmunología , Artritis Experimental/patología , Proliferación Celular/efectos de los fármacos , Citocinas/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Hiperplasia/inmunología , Hiperplasia/prevención & control , Inmunidad Celular , Peroxidasa/antagonistas & inhibidores , Distribución Aleatoria , Ratas Endogámicas Lew , Resveratrol , Membrana Sinovial/inmunologíaRESUMEN
In patients undergoing peritoneal dialysis (PD), chronic exposure to nonphysiologic PD fluids elicits low-grade peritoneal inflammation, leading to fibrosis and angiogenesis. Phenotype conversion of mesothelial cells into myofibroblasts, the so-called mesothelial-to-mesenchymal transition (MMT), significantly contributes to the peritoneal dysfunction related to PD. A number of factors have been described to induce MMT in vitro and in vivo, of which TGF-ß1 is probably the most important. The vasoconstrictor peptide endothelin-1 (ET-1) is a transcriptional target of TGF-ß1 and mediates excessive scarring and fibrosis in several tissues. This work studied the contribution of ET-1 to the development of peritoneal damage and failure in a mouse model of PD. ET-1 and its receptors were expressed in the peritoneal membrane and upregulated on PD fluid exposure. Administration of an ET receptor antagonist, either bosentan or macitentan, markedly attenuated PD-induced MMT, fibrosis, angiogenesis, and peritoneal functional decline. Adenovirus-mediated overexpression of ET-1 induced MMT in human mesothelial cells in vitro and promoted the early cellular events associated with peritoneal dysfunction in vivo. Notably, TGF-ß1-blocking peptides prevented these actions of ET-1. Furthermore, a positive reciprocal relationship was observed between ET-1 expression and TGF-ß1 expression in human mesothelial cells. These results strongly support a role for an ET-1/TGF-ß1 axis as an inducer of MMT and subsequent peritoneal damage and fibrosis, and they highlight ET-1 as a potential therapeutic target in the treatment of PD-associated dysfunction.
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Endotelina-1/fisiología , Diálisis Peritoneal/efectos adversos , Fibrosis Peritoneal/patología , Adenoviridae/genética , Animales , Células Cultivadas , Endotelina-1/metabolismo , Células Epiteliales/metabolismo , Epitelio/metabolismo , Femenino , Fibrosis/metabolismo , Técnicas de Transferencia de Gen , Humanos , Ratones , Ratones Endogámicos C57BL , Peritoneo/metabolismo , Peritoneo/patología , Fenotipo , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
OBJECTIVES: In RA, synoviocytes cause increased oxidative stress, leading to mitochondrial alterations that may participate in the pathogenesis of RA. Here we investigated whether mitochondrial dysfunction induces inflammatory responses in cultured normal human synoviocytes, a hallmark of RA. METHODS: Mitochondrial dysfunction was induced with the inhibitor oligomycin. The effects of mitochondrial dysfunction on cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2) and IL-8 expression; cellular and mitochondrial reactive oxygen species (ROS) production; nuclear factor-κB (NF-κB) activation and p65 translocation were studied. ROS scavengers (N-acetylcysteine and mitoTEMPO) and an NF-κB inhibitor (BAY-117085) were used to investigate the pathways involved. The natural anti-inflammatory antioxidant resveratrol was also tested. RESULTS: Mitochondrial dysfunction per se significantly stimulated mitochondrial ROS production as well as low-grade expressions of COX-2, PGE2 and IL-8. Interestingly, mitochondrial dysfunction induced by pretreatment of synoviocytes with oligomycin synergized with IL-1ß to increase the expression of these inflammatory mediators. The inflammatory effects of mitochondrial damage appeared to be dependent on ROS production and NF-κB activation since the inflammatory response was counteracted by both N-acetylcysteine and mitoTEMPO and it was also reduced by BAY-117085. Antimycin A and paraquat (inhibitors of mitochondrial function) also induced inflammatory responses. Furthermore, resveratrol significantly reduced the inflammatory response by decreasing ROS production and NF-κB activation. CONCLUSION: These data suggest that mitochondrial dysfunction could induce an inflammatory response in normal human synoviocytes and sensitize these cells, causing a significant amplification of the inflammatory response induced by IL-1ß. Resveratrol may represent a promising strategy in controlling the synovial inflammatory response.
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Inhibidores Enzimáticos/farmacología , Inflamación/fisiopatología , Mitocondrias/efectos de los fármacos , Mitocondrias/fisiología , Oligomicinas/farmacología , Membrana Sinovial/patología , Membrana Sinovial/fisiopatología , Anciano , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Artritis Reumatoide/fisiopatología , Células Cultivadas , Ciclooxigenasa 2/metabolismo , Dinoprostona/metabolismo , Humanos , Inflamación/metabolismo , Interleucina-1beta/farmacología , Interleucina-8/metabolismo , Persona de Mediana Edad , FN-kappa B/metabolismo , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Especies Reactivas de Oxígeno/metabolismo , Resveratrol , Estilbenos/farmacología , Membrana Sinovial/efectos de los fármacosRESUMEN
OBJECTIVE: Alterations in mitochondria play a key role in the pathogenesis of osteoarthritis (OA). The role of inflammation in the progression of OA has also acquired important new dimensions. This study was undertaken to evaluate the potential role of mitochondrial dysfunction in increasing the inflammatory response of normal human chondrocytes to cytokines. METHODS: Mitochondrial dysfunction was induced by commonly used inhibitors. Interleukin-1ß (IL-1ß) and tumor necrosis factor α (TNFα) were used as inflammatory mediators. IL-8 and cyclooxygenase 2 (COX-2) protein and messenger RNA (mRNA) expression and prostaglandin E(2) (PGE(2) ) levels were assessed. The chemotactic activity of neutrophils was assayed. Additionally, inhibitors of reactive oxygen species (ROS) and NF-κB were used to identify possible inflammatory response pathways induced by mitochondrial dysfunction, and the effects of the natural antioxidant resveratrol were tested. RESULTS: Pretreatment with antimycin A or oligomycin (inhibitors of mitochondrial respiratory chain complexes III and V, respectively) triggered a strong potentiation of IL-1ß-induced IL-8 mRNA and protein expression (mean ± SEM at 18 hours 5,932 ± 1,995 pg/50,000 cells for IL-1ß alone versus 16,241 ± 5,843 pg/50,000 cells for antimycin A plus IL-1ß and 20,087 ± 5,407 pg/50,000 cells for oligomycin plus IL-1ß; P < 0.05). Similar results were observed with TNFα or when expression of the inflammatory mediator COX-2 or PGE(2) production was assessed. Mitochondrial dysfunction increased the chemotactic activity induced by cytokines, and ROS and NF-κB inhibitors decreased the production of IL-8. Resveratrol significantly reduced the inflammatory response. CONCLUSION: Our findings indicate that mitochondrial dysfunction could amplify the responsiveness to cytokine-induced chondrocyte inflammation through ROS production and NF-κB activation. This pathway might lead to the impairment of cartilage and joint function in OA.
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Condrocitos/metabolismo , Interleucina-1beta/farmacología , Mitocondrias/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Anciano , Antimicina A/farmacología , Cartílago Articular/efectos de los fármacos , Cartílago Articular/metabolismo , Células Cultivadas , Quimiotaxis/efectos de los fármacos , Quimiotaxis/fisiología , Condrocitos/citología , Condrocitos/efectos de los fármacos , Ciclooxigenasa 2/metabolismo , Dinoprostona/metabolismo , Femenino , Humanos , Interleucina-8/metabolismo , Masculino , Persona de Mediana Edad , Mitocondrias/efectos de los fármacos , Oligomicinas/farmacologíaRESUMEN
OBJECTIVE: To determine the intracellular proteome of normal human chondrocytes stimulated with interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha) and to ascertain differences in the protein expression patterns of these 2 cytokines. METHODS: Normal human knee cartilage chondrocytes were incubated for 48 hours without stimulation or stimulated with IL-1beta (5 ng/ml) or with TNFalpha (10 ng/ml). For each culture condition, protein extracts from 4 normal subjects were pooled and resolved using 2-dimensional electrophoresis. Protein spots were visualized with Sypro stain, and qualitative and quantitative analyses were performed using PDQuest software. Protein spots were then identified by mass spectrometry, using matrix-assisted laser desorption ionization-time-of-flight/time-of-flight technology. RESULTS: We identified 37 spots by mass spectrometry (MS) or by MS/MS, corresponding to 35 different proteins. In IL-1beta-stimulated chondrocytes, IL-1beta was found to modulate 22 proteins, as compared with unstimulated chondrocytes. All of these proteins except connective tissue growth factor (CCND2) were up-regulated. Proteins involved in cellular metabolism and energy (23%) that were up-regulated or induced by IL-1beta included nicotinamide phosphoribosyltransferase, long-chain fatty acid-coenzyme A ligase 4, delta-aminolevulinic acid dehydratase, triosephosphate isomerase, and an isoform of glyceraldehyde-3-phosphate dehydrogenase. In TNFalpha-stimulated chondrocytes, TNFalpha was found to modulate 20 proteins, as compared with unstimulated chondrocytes. All of these except chitinase 3-like 1 (cartilage glycoprotein 39), proteasome activator complex subunit 2, and G3PDH, were up-regulated. Eighteen proteins were differently modulated by IL-1beta and TNFalpha. Of these, 45% were related to metabolism. CONCLUSION: IL-1beta and TNFalpha induce different profiles of intracellular protein expression in healthy human chondrocytes. Most of the proteins that are differently regulated are proteins that are implicated in the generation of cellular energy and in glycolysis.
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Condrocitos/química , Interleucina-1beta/farmacología , Proteoma/análisis , Factor de Necrosis Tumoral alfa/farmacología , Adolescente , Adulto , Anciano , Células Cultivadas , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Cartílago Auricular/citología , Electroforesis en Gel Bidimensional , Humanos , Espectrometría de Masas/métodos , Persona de Mediana Edad , Proteómica , Regulación hacia ArribaRESUMEN
BACKGROUND: Nitric oxide (NO) is a messenger implicated in the destruction and inflammation of joint tissues. Cartilage and synovial membrane from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) have high levels of NO. NO is known to modulate various cellular pathways and, thus, inhibit the activity of the mitochondrial respiratory chain (MRC) of chondrocytes and induce the generation of reactive oxygen species (ROS) and cell death in multiple cell types. For these reasons, and because of the importance of the synovial membrane in development of OA pathology, we investigated the effects of NO on survival, mitochondrial function, and activity of fibroblastic human OA synovial cells. METHODS: Human OA synovia were obtained from eight patients undergoing hip joint replacement. Sodium nitroprusside (SNP) was used as a NO donor compound and cell viability was evaluated by MTT assays. Mitochondrial function was evaluated by analyzing the mitochondrial membrane potential (Δψm) with flow cytometry using the fluorofore DePsipher. ATP levels were measured by luminescence assays, and the activities of the respiratory chain complexes (complex I: NADH CoQ1 reductase, complex II: succinate dehydrogenase, complex III: ubiquinol-cytochrome c reductase, complex IV: cytochrome c oxidase) and citrate synthase (CS) were measured by enzymatic assay. Protein expression analyses were performed by western blot. RESULTS: SNP at a concentration of 0.5 mM induced cell death, shown by the MTT method at different time points. The percentages of viable cells at 24, 48 and 72 hours were 86.11 ± 4.9%, 74.31 ± 3.35%, and 43.88 ± 1.43%, respectively, compared to the basal level of 100% (*p < 0.05). SNP at 0.5 mM induced depolarization of the mitochondrial membrane at 12 hours with a decrease in the ratio of polarized cells (basal = 2.48 ± 0.28; SNP 0.5 mM = 1.57 ± 0.11; *p < 0.01). The time course analyses of treatment with SNP at 0.5 mM demonstrated that treatment reliably and significantly reduced intracellular ATP production (68.34 ± 14.3% vs. basal = 100% at 6 hours; *p < 0.05). The analysis of the MRC at 48 hours showed that SNP at 0.5 mM increased the activity of complexes I (basal = 36.47 ± 3.92 mol/min/mg protein, SNP 0.5 mM = 58.08 ± 6.46 mol/min/mg protein; *p < 0.05) and III (basal = 63.87 ± 6.93 mol/min/mg protein, SNP 0.5 mM = 109.15 ± 30.37 mol/min/mg protein; *p < 0.05) but reduced CS activity (basal = 105.06 ± 10.72 mol/min/mg protein, SNP at 0.5 mM = 66.88 ± 6.08 mol/min/mg protein.; *p < 0.05), indicating a decrease in mitochondrial mass. Finally, SNP regulated the expression of proteins related to the cellular cycle; the NO donor decreased bcl-2, mcl-1 and procaspase-3 protein expression. CONCLUSIONS: This study suggests that NO reduces the survival of OA synoviocytes by regulating mitochondrial functionality, as well as the proteins controlling the cell cycle.
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Mitocondrias/efectos de los fármacos , Mitocondrias/fisiología , Óxido Nítrico/farmacología , Osteoartritis de la Cadera/patología , Membrana Sinovial/patología , Adenosina Trifosfato/metabolismo , Ciclo Celular/efectos de los fármacos , Ciclo Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Potencial de la Membrana Mitocondrial/fisiología , Donantes de Óxido Nítrico/farmacología , Nitroprusiato/farmacología , Osteoartritis de la Cadera/fisiopatología , Membrana Sinovial/efectos de los fármacos , Membrana Sinovial/fisiopatologíaRESUMEN
Preservation of the peritoneal membrane is an essential determinant of the long-term outcome of peritoneal dialysis (PD). Epithelial-to-mesenchymal transition (EMT) plays a central role in the pathogenesis of PD-related peritoneal membrane injury. We hypothesized that mitochondria may be implicated in the mechanisms that initiate and sustain peritoneal membrane damage in this setting. Hence, we carried out ex vivo studies of effluent-derived human mesothelial cells, which disclosed a significant increase in mitochondrial reactive oxygen species (mtROS) production and a loss of mitochondrial membrane potential in mesothelial cells with a fibroblast phenotype, compared to those preserving an epithelial morphology. In addition, in vitro studies of omentum-derived mesothelial cells identified mtROS as mediators of the EMT process as mitoTEMPO, a selective mtROS scavenger, reduced fibronectin protein expression induced by TGF-ß1. Moreover, we quantified mitochondrial DNA (mtDNA) levels in the supernatant of effluent PD solutions, disclosing a direct correlation with small solute transport characteristics (as estimated from the ratio dialysate/plasma of creatinine at 240 min), and an inverse correlation with peritoneal ultrafiltration. These results suggest that mitochondria are involved in the EMT that human peritoneal mesothelial cells suffer in the course of PD therapy. The level of mtDNA in the effluent dialysate of PD patients could perform as a biomarker of PD-induced damage to the peritoneal membrane.
RESUMEN
SCOPE: Previous work reported that dietary supplementation with resveratrol lowers synovial hyperplasia, inflammatory and oxidative damage in an antigen-induced arthritis (AIA) model. Here, it is investigated whether resveratrol can regulate the abnormal synovial proliferation by inducing autophagy and controlling the associated inflammatory response. METHODS AND RESULTS: Animals treated with resveratrol 8 weeks before AIA induction show the highest significant signal for microtubule-associated protein 1 light chain 3 by confocal microscopy. Besides, resveratrol significantly reduces p62 expression, but it does not increase the signal of beclin-1. Also, active caspase-3 expression, as well as poly(ADP-ribose) polymerase, is upregulated in the AIA group, and is significantly reduced in resveratrol-treated AIA group. Resveratrol also mitigates angiopoietin-1 and vascular endothelial growth factor signals. Finally, resveratrol significantly reduces the serum levels of IL-1ß, C reactive protein, and prostaglandin E2, as well as nuclear factor κB synovial tissue expression, which shows a significant correlation with p62 expression. CONCLUSION: Dietary supplementation with resveratrol induces the noncanonical autophagy pathway and limits the cross-talk with inflammation, which in consequence modulates the synovial hyperplasia. Preventive strategies that incorporate dietary intervention with resveratrol may offer a potential therapeutic alternative to drugs to influence the risk of rheumatoid arthritis and influence its course.
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Artritis Reumatoide/dietoterapia , Artritis Reumatoide/etiología , Autofagia/efectos de los fármacos , Resveratrol/farmacología , Animales , Artritis Reumatoide/patología , Artritis Reumatoide/prevención & control , Autofagia/fisiología , Proteína C-Reactiva/análisis , Suplementos Dietéticos , Dinoprostona/sangre , Modelos Animales de Enfermedad , Femenino , Ratas Endogámicas Lew , Líquido Sinovial/efectos de los fármacos , Líquido Sinovial/metabolismo , Membrana Sinovial/irrigación sanguínea , Membrana Sinovial/efectos de los fármacos , Membrana Sinovial/metabolismo , Factor de Transcripción ReIA/metabolismoRESUMEN
Rheumatic and musculoskeletal diseases are a heterogeneous group of disorders affecting joint tissues and in some cases even organs, some of them being among the most common diseases worldwide. Mitochondria are the organelles considered as powerhouse of cells providing energy to the organism mainly through oxidative phosphorylation. However, mitochondria are also involved in crucial pathways responsible for maintaining cell physiology, such as the activation of metabolic and survival signaling, and innate and adaptive immune response. As consequence of the pivotal role of mitochondria in cell homeostasis, an impairment of mitochondrial function has been associated with activation of pathological events, including oxidative stress and subsequently damaged protein and DNA, deregulation of programmed cell death, and over-activation of inflammatory responses modulated by redox-sensitive signaling or direct activation of the inflammasome. Thus, a growing amount of evidence emphasizes the role of mitochondria in aging and inflammatory-related diseases, including rheumatic disorders. In this regard, emerging findings suggest that targeting of the pathways involved in the maintenance of mitochondrial metabolism may control cell homeostasis, and in turn delay ageing and prevent or improve articular pathologies. In this review we will focus on the importance of mitochondria in metabolic homeostasis of articular cells, as well as their influence on the activation of pathological signaling pathways, as a result of a genetic predisposition, damage, decline or impairment of their function. Finally, we will discuss some of the most important evidences of involvement of mitochondria in the onset and progression of rheumatic diseases.
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Mitocondrias/fisiología , Enfermedades Reumáticas/etiología , Animales , Homeostasis , Humanos , Inmunidad , Inflamación/etiología , Proteína con Dominio Pirina 3 de la Familia NLR/fisiología , Enfermedades Reumáticas/inmunología , Enfermedades Reumáticas/metabolismo , Transducción de SeñalRESUMEN
In osteoarthritis (OA) a time or age dependent process leads to aberrant cartilage structure which is characterized by reduced number of chondrocytes, loss of existing cartilage extracellular matrix, the production of matrix with abnormal composition and pathologic matrix calcification. Because chondrocyte matrix synthesis and mineralization are modulated by the balance between ATP generation and consumption, the mechanism by which chondrocytes generate energy have been a topic of interest. The analysis of mitochondrial respiratory chain (MRC) activity in OA chondrocytes shows a significant decrease in complexes II and III compared to normal chondrocytes. On the other hand, mitochondrial mass is increased in OA, as demonstrated by a significant rise in CS activity. Furthermore, OA cells show a reduction in the mitochondrial membrane potential (deltapsim) as demonstrated by using the fluorescent probe JC-1. OA cartilage contains high number of apoptotic chondrocytes, and mitochondria play a key role in apoptosis. Interestingly, OA cartilages show markedly elevated Bcl-2 and caspasa-3 expression. This expression is also correlated with chondrocyte apoptosis and OA lesions. The pathogenesis of OA includes elaboration of increased amounts of NO as a consequence of up-regulation of chondrocyte-inducible NO synthase induced by IL-1, TNF-alpha and other factors. NO reduces chondrocyte survival and induces cell death with morphologic changes characteristic of chondrocyte apoptosis. NO reduces the activity of complex IV and decreases the deltapsim as measured as the ratio of red/green fluorescence. Furthermore, NO induces the mRNA expression of caspase-3 and -7, and it reduces the expression of mRNA bcl-2 and the bcl-2 protein synthesis. Some studies suggest that the chondrocyte mitochondria are specialized for calcium transport and are important in the calcification of the extracellular matrix. Mineral formation has been demonstrated in matrix vesicles (MV) and within mitochondria. Direct suppression of mitochondrial respiration promoted MV-mediated mineralization in chondrocytes. Regulation of MRC may be one of the signaling pathways by which NO modulates articular cartilage matrix biosynthesis and pathologic mineralization. After age 40, the incidence of OA in humans increases progressively with increasing age. Studies show a trend to statistic significance between the age and the reduction of complex I activity of human normal chondrocytes. However, the study of relation between age and deltapsim in normal chondrocytes do not demonstrate any significant correlation. It has been reported that as the number of population doublings increased, mitochondrial DNA was degraded and the number of mitochondria per chondrocyte decline. One approach for determining the role of mitochondria in OA is to determine the effects of the MRC inhibition and to compare them with the findings in OA. Inhibition of MRC with antimycin prevents the normal ability of TGFbeta to increase excretion of Pi, thereby worsening deposition of pathologic HA crystals. In chondrocytes, the inhibition of complex IV with NaN3 modified both the deltapsim and the survival of cells inducing apoptosis. Inhibition of complex I with rotenone increases the expression and synthesis of Bcl-2 and Cox-2, both effects are similar effects to produced by IL-1 in human chondrocytes.
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Inflammation has been linked to multiple degenerative and acute diseases as well as the aging process. Moreover, mitochondrial alterations play a central role in these processes. Mitochondria have an important role in pro-inflammatory signaling; similarly, pro-inflammatory mediators may also alter mitochondrial function. Both of these processes increase mitochondrial oxidative stress, promoting a vicious inflammatory cycle. Additionally, damage-associated molecular patterns derived from mitochondria could contribute to inflammasome formation and caspase-1 activation, while alterations in mitochondrial autophagy may cause inflammation. Strategies aimed at controlling excessive oxidative stress within mitochondria may represent both preventive and therapeutic interventions in inflammation.
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Inflamación/patología , Mitocondrias/fisiología , Humanos , Mitocondrias/metabolismo , Estrés OxidativoRESUMEN
Osteoarthritis (OA) is characterized by cartilage degradation. The chondrocyte is the only cell type present in mature cartilage, and it is important in the control of cartilage integrity. The aim of this study was to analyze, by a proteomic approach, the changes that are characteristic of OA chondrocytes, and to identify new OA-related proteins. Chondrocytes were isolated from the cartilage of ten OA patients undergoing joint replacement and ten donors with no history of joint disease. Whole-cell proteins were resolved by 2-DE and stained with SYPRO Ruby. Protein expression patterns of 2-DE gels from OA and normal chondrocyte proteins were analyzed with PDQuest 7.3.1 software. OA-related proteins were identified by MALDI-TOF or MALDI-TOF/TOF MS. The results were validated for ANXA1, GSTO1, GRP78, and HSP90beta in cells by Western blotting and in tissue cartilage by immunohistochemistry. Results showed an average of 700 protein spots that were present in the 2-DE gels. Compared to normal chondrocytes, 19 protein spots were found to be significantly increased in OA cells (ratio OA:N> or =2.0, p<0.05), whereas nine were decreased in OA chondrocytes (ratio OA:N< or =0.5, p<0.05). Three stress response proteins were increased (HSP90beta, GRP78, and GRP94) and three proteins involved in glycolysis were decreased (enolase, glyceraldehyde 3-phosphate dehydrogenase, and fructose biphosphate aldolase). Functionally, almost all proteins could be classified as proteins involved in cellular metabolism (33%), structure (21%), or protein targeting (21%).
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Cartílago Articular/metabolismo , Condrocitos/metabolismo , Glucólisis/fisiología , Osteoartritis de la Rodilla/metabolismo , Proteoma/metabolismo , Cartílago Articular/patología , Células Cultivadas , Electroforesis en Gel Bidimensional/métodos , Chaperón BiP del Retículo Endoplásmico , Humanos , Compuestos Organometálicos/química , Osteoartritis de la Rodilla/patología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
OBJECTIVE: Mitochondrial alterations play a key role in the pathogenesis of osteoarthritis (OA). This study evaluated a potential role of mitochondrial respiratory chain (MRC) dysfunction in the inflammatory response of normal human chondrocytes. METHODS: Commonly used inhibitors of the MRC were utilized to induce mitochondrial dysfunction in normal human chondrocytes. Levels of prostaglandin E(2) (PGE(2)) protein and expression of cyclooxygenase 2 (COX-2) and COX-1 messenger RNA (mRNA) and protein were analyzed. To identify the underlying mechanisms responsible for PGE(2) liberation, reactive oxygen species (ROS) were measured. Inhibitors of ROS, including vitamin E, and inhibitors of mitochondrial Ca(2+) and NF-kappaB were used to test their effects on the MRC. RESULTS: Antimycin A and oligomycin (inhibitors of mitochondrial complexes III and V, respectively) significantly increased the levels of PGE(2) (mean +/- SEM 505 +/- 132 pg/50,000 cells and 288 +/- 104 pg/50,000 cells, respectively, at 24 hours versus a basal level of 29 +/- 9 pg/50,000 cells; P < 0.05) and increased the expression of COX-2 at both the mRNA and protein levels. Expression of COX-1 did not show any modulation with either inhibitor. Further experiments revealed that antimycin A and oligomycin induced a marked increase in the levels of ROS. Production of PGE(2) and expression of COX-2 protein were inhibited by antioxidants, vitamin E, and mitochondrial Ca(2+) and NF-kappaB inhibitors. The response to blockers of mitochondrial Ca(2+) movement showed that ROS production was dependent on mitochondrial Ca(2+) accumulation. CONCLUSION: These results strongly suggest that, in human chondrocytes, the inhibition of complexes III and V of the MRC induces an inflammatory response, which could be especially relevant in relation to PGE(2) production via mitochondrial Ca(2+) exchange, ROS production, and NF-kappaB activation. These data may prove valuable for a better understanding of the participation of mitochondria in the pathogenesis of OA.
Asunto(s)
Condrocitos/metabolismo , Ciclooxigenasa 2/metabolismo , Dinoprostona/metabolismo , Mitocondrias/fisiología , Adulto , Antibacterianos/farmacología , Antimicina A/farmacología , Calcio/metabolismo , Células Cultivadas , Condrocitos/citología , Condrocitos/efectos de los fármacos , Ciclooxigenasa 1/metabolismo , Transporte de Electrón/efectos de los fármacos , Transporte de Electrón/fisiología , Humanos , Interleucina-1beta/metabolismo , FN-kappa B/metabolismo , Oligomicinas/farmacología , Osteoartritis/etiología , Osteoartritis/metabolismo , Osteoartritis/fisiopatología , Especies Reactivas de Oxígeno/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Articular cartilage is optimised for bearing mechanical loads. Chondrocytes are the only cells present in mature cartilage and are responsible for the synthesis and integrity of the extracellular matrix. Appropriate joint loads stimulate chondrocytes to maintain healthy cartilage with a concrete protein composition according to loading demands. In contrast, inappropriate loads alter the composition of cartilage, leading to osteoarthritis (OA). Matrix metalloproteinases (MMPs) are involved in degradation of cartilage matrix components and have been implicated in OA, but their role in loading response is unclear. With this study, we aimed to elucidate the role of MMP-1 and MMP-3 in cartilage composition in response to mechanical load and to analyse the differences in aggrecan and type II collagen content in articular cartilage from maximum- and minimum-weight-bearing regions of human healthy and OA hips. In parallel, we analyse the apoptosis of chondrocytes in maximal and minimal load areas. Because human femoral heads are subjected to different loads at defined sites, both areas were obtained from the same hip and subsequently evaluated for differences in aggrecan, type II collagen, MMP-1, and MMP-3 content (enzyme-linked immunosorbent assay) and gene expression (real-time polymerase chain reaction) and for chondrocyte apoptosis (flow cytometry, bcl-2 Western blot, and mitochondrial membrane potential analysis). The results showed that the load reduced the MMP-1 and MMP-3 synthesis (p < 0.05) in healthy but not in OA cartilage. No significant differences between pressure areas were found for aggrecan and type II collagen gene expression levels. However, a trend toward significance, in the aggrecan/collagen II ratio, was found for healthy hips (p = 0.057) upon comparison of pressure areas (loaded areas > non-loaded areas). Moreover, compared with normal cartilage, OA cartilage showed a 10- to 20-fold lower ratio of aggrecan to type II collagen, suggesting that the balance between the major structural proteins is crucial to the integrity and function of the tissue. Alternatively, no differences in apoptosis levels between loading areas were found--evidence that mechanical load regulates cartilage matrix composition but does not affect chondrocyte viability. The results suggest that MMPs play a key role in regulating the balance of structural proteins of the articular cartilage matrix according to local mechanical demands.
Asunto(s)
Cartílago Articular/enzimología , Cartílago Articular/fisiología , Homeostasis/fisiología , Metaloproteinasa 1 de la Matriz/genética , Metaloproteinasa 3 de la Matriz/genética , Anciano , Apoptosis/fisiología , Cartílago Articular/citología , Supervivencia Celular/fisiología , Condrocitos/citología , Condrocitos/fisiología , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Humanos , Masculino , Metaloproteinasa 1 de la Matriz/metabolismo , Metaloproteinasa 3 de la Matriz/metabolismo , Persona de Mediana Edad , Osteoartritis de la Cadera/metabolismo , Osteoartritis de la Cadera/patología , Osteoartritis de la Cadera/fisiopatología , ARN Mensajero/metabolismo , Estrés Mecánico , Soporte de PesoRESUMEN
Articular cartilage is composed of cells and an extracellular matrix. The chondrocyte is the only cell type present in mature cartilage, and it is important in the control of cartilage integrity. There is currently a great lack of knowledge about the chondrocyte proteome. To solve this deficiency, we have obtained the first reference map of the human normal articular chondrocyte. Cells were isolated from cartilages obtained from autopsies without history of joint disease. Cultured cells were used to obtain protein extracts which were resolved by 2-DE and visualized by silver nitrate or CBB staining. Almost 200 spots were excised from the gels and analyzed using MALDI-TOF or MALDI-TOF/TOF MS. The analysis leads to the identification of 136 spots that represent 93 different proteins. A significant proportion of proteins are involved in cell organization (26%), energy (16%), protein fate (14%), metabolism (12%), and cell stress (12%). From all the identified proteins, annexins, vimentin, transgelin, destrin, cathepsin D, heat shock protein 47, and mitochondrial superoxide dismutase were more abundant in chondrocytes than in other types of mesenchymal cells such as Jurkat-T cells. As metabolic program of chondrocytes is altered in osteoarthritis and other rheumatic diseases, this proteomic map is an important tool for future studies on these pathologies.
Asunto(s)
Cartílago Articular/metabolismo , Osteoartritis/metabolismo , Proteómica/métodos , Enfermedades Reumáticas/metabolismo , Adolescente , Adulto , Anciano , Autopsia , Cartílago/metabolismo , Células Cultivadas , Condrocitos/citología , Condrocitos/metabolismo , Bases de Datos de Proteínas , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Matriz Extracelular/metabolismo , Humanos , Articulaciones/metabolismo , Células Jurkat , Espectrometría de Masas , Persona de Mediana Edad , Mapeo Peptídico , Proteoma , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The objective of this study was to evaluate the use of cultured porcine chondrocyte xenotransplantation for the repair of human chondral defects. Two-millimeter-diameter defects were drilled into explants of femoral cartilage from healthy adult donors. No cells were implanted in the chondral defects of the control group, while pig chondrocytes from normal femoral cartilage were deposited into the treated chondral defects. Cartilage explants were cultured for 4, 8, and 12 weeks. Tissue sections were processed for standard histologic staining and immunostaining with monoclonal antibodies against types I and II collagen, chondroitin-4-sulfate, chondroitin-6-sulfate, keratan sulfate, and integrin subunit beta1. The porcine origin of chondrocytes was confirmed using a specific pig monoclonal anti-CD46. Repair was only observed in the cell-treated defects. Mono- or bilayers of cells were detected after 4 culture weeks on the bottom of the defects, while after 8-12 weeks a repair tissue filled near 30-40 percent of the defect. At 8 weeks, the newly synthesized tissue was composed of a fibrous mesh including some cells. However, at 12 weeks it showed a hypercellular hyaline-like region. This hypercellular region showed excellent bonding with the native cartilage, cells were located in numerous lacunae, and a high content of proteoglycans as indicated by an intense toluidine blue stain was observed. The repaired tissue showed positive immunostaining for both type I and II collagen, as well as chondroitin-4-sulfate, chondroitin-6-sulfate, keratan sulfate, and integrin subunit beta1. Positive staining for porcine anti-CD46 was localized exclusively in the neo-synthesized tissue. We conclude that xenotransplantation of pig chondrocytes can repair, in an in vitro model, defects in human articular cartilage.
Asunto(s)
Cartílago Articular/lesiones , Condrocitos/trasplante , Trasplante Heterólogo , Cicatrización de Heridas/fisiología , Anciano , Animales , Cartílago Articular/patología , Humanos , Inmunohistoquímica , Persona de Mediana EdadRESUMEN
Articular cartilage has only a limited ability to regenerate. The transplantation of autologous chondrocytes is currently used to treat focal defects in human articular cartilage, although use of organs, tissues, or cells from different species is being investigated as an alternative treatment. The object of this study was to use xeno-transplantation of cultured pig chondrocytes for the repair of rabbit chondral defects, and to analyze the significance of tissue rejection in this animal model. Partial chondral defects, including removal of cartilage tissue and a part of the subchondral bone, were created in the lateral femoral condyles of 30 adult New Zealand White rabbits. A periosteal flap was sutured to the native cartilage with the cambium layer facing the defect. As a control, culture medium was injected into the defect void of one group of rabbits while in a treatment group, chondrocytes, isolated from normal femoral pig cartilage, were injected into the defect void. All rabbits were killed by 24 weeks. Macroscopic changes of the cartilage were analyzed using Mankin's score. The distal femoral portion was studied histologically using hematoxylin and eosin, alcian blue, toluidine blue, and Mason's trichrome. Pig cells and pig genetic material were detected in the neo-synthesized tissue by immunohistochemical detection of SLA-II-DQ and polymerase chain reaction analysis of the gene SLA-II-DQB. The synovial membrane was studied histologically by hematoxylin and eosin staining. In the control group, on average, less than 25 percent of the chondral defect was filled. The repair tissue had an irregular surface with few cells similar to chondrocytes or fibroblasts and a minimal formation of extracellular matrix. In the treatment group, the chondral defect was approximately 90 percent filled with good integration between the neo-synthesized cartilage and the native cartilage. The repair tissue had a smooth surface with cells similar to chondrocytes and a hyaline-like extracellular matrix. The neo-synthesized cartilage was morphologically similar to hyaline cartilage. Importantly, there were no signs of graft-vs.-host rejections or infiltration by immune cells. In the neo-synthesized tissue, pig genetic material was detected in 27 +/- 5 percent of all cells. These cells containing pig genetic material were distributed throughout the neo-synthesized cartilage. We conclude that the xeno-transplantation of chondrocytes could be an alternative method for the repair of articular cartilage defects.
Asunto(s)
Cartílago Articular/trasplante , Trasplante Heterólogo/métodos , Animales , Cartílago Articular/citología , Cartílago Articular/lesiones , Cartílago Articular/fisiología , Células Cultivadas , Rechazo de Injerto/fisiopatología , Modelos Animales , Conejos , Regeneración/fisiología , PorcinosRESUMEN
OBJECTIVE: Osteoarthritis (OA) is a degenerative rheumatic disease that is associated with extracellular matrix degradation and chondrocyte apoptosis in the articular cartilage. The role of mitochondria in degenerative diseases is widely recognized. We undertook this study to evaluate mitochondrial function in normal and OA chondrocytes and to examine age-related changes in mitochondria. METHODS: Mitochondrial function was evaluated by analyzing respiratory chain enzyme complexes and citrate synthase (CS) activities as well as changes in mitochondrial membrane potential (Delta Psi m). The activities of mitochondrial respiratory chain complexes (complex I: rotenone-sensitive NADH-coenzyme Q(1) reductase; complex II: succinate dehydrogenase; complex III: antimycin-sensitive ubiquinol cytochrome c reductase; and complex IV: cytochrome c oxidase) and CS were measured in human articular chondrocytes isolated from OA and normal cartilage. Delta Psi m was measured by JC-1 using flow cytometry. Statistical analysis was performed using the Mann-Whitney U test and Student's t-test as well as several models of multiple linear regression. RESULTS: OA articular chondrocytes had reduced activities of complexes II and III compared with cells from normal cartilage. However, the mitochondrial mass was increased in OA. Cultures of OA chondrocytes contained a higher proportion of cells with de-energized mitochondria. We found no relationship between mitochondrial function and donor age either in normal or in OA chondrocytes. CONCLUSION: These findings suggest the involvement of mitochondrial function in the pathophysiology of OA. Cartilage degradation by OA and cartilage aging may be two different processes.