RESUMEN
The extensive use of opioids for chronic pain management has contributed significantly to the current opioid epidemic. While many alternative nonopioid analgesics are available, opioids remain the most potent analgesics for moderate to severe pain management. In addition to the implementation of multimodal analgesia, there is a pressing need for the development of more effective and safer opioids. In this study, we developed a thermoresponsive N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer-based hydromorphone (HMP) prodrug (ProGel-HMP, HMP content = 16.2 wt %, in base form). The aqueous solution of ProGel-HMP was free-flowing at 4 °C but became a hydrogel when the temperature was raised to ≥37 °C, allowing sustained local retention when administered in vivo. When tested in the destabilization of the medial meniscus (DMM) mouse model of osteoarthritis (OA), ProGel-HMP was retained after intra-articular injection in the OA knee joint for at least 2 weeks postinjection, with low extra-articular distribution. ProGel-HMP was not detected in the central nervous system (CNS). A single dose of ProGel-HMP produced rapid and sustained joint pain resolution for greater than 14 days when compared to saline and dose-equivalent HMP controls, likely mediated through peripheral µ-opioid receptors in the knee joint. Systemic analgesia effect was absent in the DMM mice treated with ProGel-HMP, as evident in the lack of difference in tail flick response between the ProGel-HMP-treated mice and the controls (i.e., Healthy, Saline, and Sham). Repeated dosing of ProGel-HMP did not induce tolerance. Collectively, these data support the further development of ProGel-HMP as a potent, safe, long-acting and nonaddictive analgesic for better clinical pain management.
Asunto(s)
Analgesia , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Osteoartritis , Profármacos , Ratones , Animales , Hidromorfona , Manejo del Dolor , Profármacos/uso terapéutico , Dolor/tratamiento farmacológico , Analgésicos Opioides/efectos adversos , Analgésicos/uso terapéuticoRESUMEN
The relief of joint pain is one of the main objectives in the clinical management of arthritis. Although significant strides have been made in improving management of rheumatoid and related forms of inflammatory arthritis, there are still major unmet needs for therapies that selectively provide potent, sustained and safe joint pain relief, especially among patients with osteoarthritis (OA), the most common form of arthritis. We have recently developed ProGel-Dex, an N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer-based thermoresponsive dexamethasone (Dex) prodrug, which forms a hydrogel upon intra-articular administration and provides sustained improvement in pain-related behavior and inflammation in rodent models of arthritis. The focus of the present study was to investigate the impact of ProGel-Dex formulation parameters on its physicochemical properties and in vivo efficacy. The results of this study provide essential knowledge for the future design of ProGel-Dex that can provide more effective, sustained and safe relief of joint pain and inflammation.
RESUMEN
In this study, we aimed to assess the analgesic efficacy of a thermoresponsive polymeric dexamethasone (Dex) prodrug (ProGel-Dex) in a mouse model of osteoarthritis (OA). At 12 weeks post model establishment, the OA mice received a single intra-articular (IA) injection of ProGel-Dex, dose-equivalent Dex, or Saline. Comparing to Saline and Dex controls, ProGel-Dex provided complete and sustained pain relief for >15 weeks according to incapacitance tests. In vivo optical imaging confirmed the continuous presence of ProGel-Dex in joints for 15 weeks post-injection. According to micro-CT analysis, ProGel-Dex treated mice had significantly lower subchondral bone thickness and medial meniscus bone volume than Dex and Saline controls. Except for a transient delay of body weight increase and slightly lower endpoint liver and spleen weights, no other adverse effect was observed after ProGel-Dex treatment. These findings support ProGel-Dex's potential as a potent and safe analgesic candidate for management of OA pain.
Asunto(s)
Osteoartritis , Profármacos , Ratones , Animales , Dexametasona/farmacología , Dexametasona/uso terapéutico , Modelos Animales de Enfermedad , Profármacos/farmacología , Profármacos/uso terapéutico , Osteoartritis/tratamiento farmacológico , Artralgia/inducido químicamente , Artralgia/tratamiento farmacológico , Analgésicos/farmacología , Analgésicos/uso terapéuticoRESUMEN
The menisci exert a prominent role in joint stabilization and in the distribution of mechanical loading. Meniscal damage is associated with increased risk of knee OA. The aim of this study was to characterize the synovial membrane and meniscal tissues in patients undergoing arthroscopic partial meniscectomy for meniscal tear and to evaluate association with clinical outcomes. A total of 109 patients were recruited. Demographic and clinical data were collected. Visual Analogic Scale (VAS) measuring pain and Knee injury and Osteoarthritis Outcome Score (KOOS) were recorded at baseline and at 2-years follow-up. Histological and immunohistochemical characterizations were performed on synovial membranes and meniscal tissues. More than half of the patients demonstrated synovial mononuclear cell infiltration and hyperplasia. Synovial fibrosis was present in most of the patients; marked vascularity and CD68 positivity were observed. Inflammation had an impact on both pain and knee symptoms. Patients with synovial inflammation had higher values of pre-operative VAS and inflammation. Higher pre-operative pain was observed in patients with meniscal MMP-13 production. In conclusion, multivariate analysis showed that synovial inflammation was associated with pre-operative total KOOS scores, knee symptoms, and pain. Moreover, meniscal MMP-13 expression was found to be associated with pre-operative pain in multivariate analysis. Thus, targeting inflammation of the synovial membrane and meniscus might reduce clinical symptoms and dysfunction at the time of surgery.
Asunto(s)
Menisco , Lesiones de Menisco Tibial , Humanos , Inflamación/patología , Metaloproteinasa 13 de la Matriz , Meniscectomía/efectos adversos , Meniscos Tibiales/patología , Meniscos Tibiales/cirugía , Menisco/cirugía , Dolor/patología , Lesiones de Menisco Tibial/complicaciones , Lesiones de Menisco Tibial/cirugíaRESUMEN
The aim of this study was to identify the molecules and pathways involved in the cross-talk between meniscus and synovium that may play a critical role in osteoarthritis (OA) pathophysiology. Samples of synovium and meniscus were collected from patients with early and end-stage OA and cultured alone or cocultured. Cytokines, chemokines, metalloproteases, and their inhibitors were evaluated at the gene and protein levels. The extracellular matrix (ECM) changes were also investigated. In early OA cultures, higher levels of interleukin-6 (IL-6) and IL-8 messenger RNA were expressed by synovium and meniscus in coculture compared with meniscus cultured alone. RANTES release was significantly increased when the two tissues were cocultured compared with meniscus cultured alone. Increased levels of matrix metalloproteinase-3 (MMP-3) and MMP-10 proteins, as well as increased release of glycosaminoglycans and aggrecan CS846 epitope, were observed when synovium was cocultured with meniscus. In end-stage OA cultures, increased levels of IL-8 and monocyte chemoattractant protein-1 (MCP-1) proteins were released in cocultures compared with cultures of meniscus alone. Chemokine (C-C motif) ligand 21 (CCL21) protein release was higher in meniscus cultured alone and in coculture compared with synovium cultured alone. Increased levels of MMP-3 and 10 proteins were observed when tissues were cocultured compared with meniscus cultured alone. Aggrecan CS846 epitope release was increased in cocultures compared with cultures of either tissue cultured alone. Our study showed the production of inflammatory molecules by synovium and meniscus which could trigger inflammatory signals in early OA patients, and induce ECM loss in the progressive and final stages of OA pathology.
Asunto(s)
Matriz Extracelular/patología , Menisco/metabolismo , Osteoartritis de la Rodilla/patología , Membrana Sinovial/metabolismo , Anciano , Anciano de 80 o más Años , Agrecanos/metabolismo , Células Cultivadas , Quimiocina CCL2/metabolismo , Quimiocina CCL21/metabolismo , Quimiocina CCL5/metabolismo , Técnicas de Cocultivo , Femenino , Glicosaminoglicanos/metabolismo , Humanos , Inflamación/patología , Interleucina-6/genética , Interleucina-8/genética , Masculino , Metaloproteinasa 10 de la Matriz/metabolismo , Metaloproteinasa 3 de la Matriz/metabolismo , Persona de Mediana EdadRESUMEN
Deoxynivalenol (DON) and T-2 toxin are prevalent mycotoxin contaminants in the food and feed stuffs worldwide, with non-negligible co-contamination and co-exposure conditions. Meanwhile, they are considerable risk factors for Kashin-Beck disease, a chronic endemic osteochondropathy. The aim of this study was to investigate the individual and combined cytotoxicity of DON and T-2 toxin on proliferating human C-28/I2 and newborn rat primary costal chondrocytes by MTT assay. Four molar concentration combination ratios of DON and T-2 toxin were used, 1:1 for R1 mixture, 10:1 for R10, 100:1 for R100 and 1000:1 for R1000. The toxicological interactions were quantified by the MixLow method. DON, T-2 toxin, and their mixtures all showed a clear dose-dependent toxicity for chondrocytes. The cytotoxicity of T-2 toxin was 285-fold higher than DON was in human chondrocytes, and 22-fold higher in the rat chondrocytes. The combination of DON and T-2 toxin was significantly synergistic at middle and high level concentrations of R10 mixtures in rat chondrocytes, but significantly antagonistic at the low concentrations of R100 mixtures in both cells and at the middle concentrations of R1000 mixtures in rat chondrocytes. These results indicated that the combined toxicity was influenced by the cell sensitivity for toxins, the difference between the combination ratio and equitoxic ratio, the concentrations and other factors.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Condrocitos/efectos de los fármacos , Toxina T-2/toxicidad , Tricotecenos/toxicidad , Animales , Supervivencia Celular/efectos de los fármacos , Condrocitos/patología , Relación Dosis-Respuesta a Droga , Antagonismo de Drogas , Sinergismo Farmacológico , Humanos , Cultivo Primario de Células , Ratas , Ratas Sprague-Dawley , Especificidad de la Especie , Toxina T-2/administración & dosificación , Tricotecenos/administración & dosificaciónRESUMEN
BACKGROUND: The goal of this study was to determine if adenovirus-delivered LOXL2 protects against progressive knee osteoarthritis (OA), assess its specific mechanism of action; and determine if the overexpression of LOXL2 in transgenic mice can protect against the development of OA-related cartilage damage and joint disability. METHODS: Four-month-old Cho/+ male and female mice were intraperitoneally injected with either Adv-RFP-LOXL2 or an empty vector twice a month for four months. The proteoglycan levels and the expression of anabolic and catabolic genes were examined by immunostaining and qRT-PCR. The effect of LOXL2 expression on signaling was tested via the pro-inflammatory cytokine IL1ß in the cartilage cell line ATDC5. Finally; the OA by monosodium iodoacetate (MIA) injection was also induced in transgenic mice with systemic overexpression of LOXL2 and examined gene expression and joint function by treadmill tests and assessment of allodynia. RESULTS: The adenovirus treatment upregulated LOXL2; Sox9; Acan and Runx2 expression in both males and females. The Adv-RFP-LOXL2 injection; but not the empty vector injection increased proteoglycan staining and aggrecan expression but reduced MMP13 expression. LOXL2 attenuated IL-1ß-induced phospho-NF-κB/p65 and rescued chondrogenic lineage-related genes in ATDC5 cells; demonstrating one potential protective mechanism. LOXL2 attenuated phospho-NF-κB independent of its enzymatic activity. Finally; LOXL2-overexpressing transgenic mice were protected from MIA-induced OA-related functional changes; including the time and distance traveled on the treadmill and allodynia. CONCLUSION: Our study demonstrates that systemic LOXL2 adenovirus or LOXL2 genetic overexpression in mice can protect against OA. These findings demonstrate the potential for LOXL2 gene therapy for knee-OA clinical treatment in the future.
Asunto(s)
Envejecimiento/genética , Aminoácido Oxidorreductasas/genética , Osteoartritis de la Rodilla/etiología , Osteoartritis de la Rodilla/patología , Adenoviridae/genética , Aminoácido Oxidorreductasas/metabolismo , Animales , Artritis Experimental , Cartílago Articular/metabolismo , Cartílago Articular/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Expresión Génica , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Interleucina-1beta/metabolismo , Ratones , Ratones Transgénicos , FN-kappa B/metabolismo , Osteoartritis de la Rodilla/metabolismo , Transducción GenéticaRESUMEN
BACKGROUND/AIMS: The E74-like factor 3 (ELF3) is an inflammatory mediator that participates in cartilage destruction in osteoarthritis. Leptin and other adipokines negatively impact articular cartilage, triggering catabolic and inflammatory responses in chondrocytes. Here, we investigated whether leptin induces ELF3 expression in chondrocytes and the signaling pathway involved in this process. METHODS: We determined mRNA and protein levels of ELF3 by RT-qPCR and Western blotting using cultured human primary chondrocytes and the human T/C-28a2 chondrocyte cell line. Further, we measured luciferase activities of different reporter constructs, and we assessed the contribution of leptin to the induction of ELF3 mRNA by knocking down hLEPR gene expression using siRNA technology. RESULTS: Leptin synergizes with IL-1ß in inducing ELF3 expression in chondrocytes. We also found that PI3K, p38, and JAK2 signaling pathways are at play in the leptin-driven induction of ELF3. Moreover, we confirm the participation of NFΚB in the leptin/IL-1ß synergistic induction of ELF3. CONCLUSION: Here we show, for the first time, the regulation of ELF3 expression by leptin, suggesting that this transcription factor likely mediates the inflammatory responses triggered by leptin in articular chondrocytes.
Asunto(s)
Condrocitos/metabolismo , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Inflamación/genética , Leptina/inmunología , Obesidad/genética , Proteínas Proto-Oncogénicas c-ets/genética , Factores de Transcripción/genética , Cartílago/inmunología , Cartílago/metabolismo , Línea Celular , Células Cultivadas , Condrocitos/inmunología , Proteínas de Unión al ADN/inmunología , Humanos , Inflamación/inmunología , Interleucina-1beta/inmunología , Leptina/genética , Obesidad/inmunología , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-ets/inmunología , Interferencia de ARN , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Receptores de Leptina/genética , Receptores de Leptina/inmunología , Factores de Transcripción/inmunología , Activación TranscripcionalRESUMEN
The aim of this study was to investigate the role of laminins and nidogen-2 in osteoarthritis (OA) and their potential to support chondrogenic differentiation. We applied immunohistochemistry, electron microscopy, siRNA, quantitative RT-PCR, Western blot, and proteome analysis for the investigation of cartilage tissue and isolated chondrocytes in three-dimensional culture obtained from patients with late-stage knee OA and nidogen-2 knockout mice. We demonstrate that subunits of laminins appear in OA cartilage and that nidogen-2-null mice exhibit typical osteoarthritic features. Chondrogenic progenitor cells (CPCs) produced high levels of laminin-α1, laminin-α5, and nidogen-2 in their pericellular matrix, and laminin-α1 enhanced collagen type II and reduced collagen type I expression by cultured CPCs. Nidogen-2 increased SOX9 gene expression. Knockdown of nidogen-2 reduced SOX9 expression, whereas it up-regulated RUNX2 expression. This study reveals that the influence of the pericellular matrix on CPCs is important for the expression of the major regulator transcription factors, SOX9 and RUNX2. Our novel findings that laminins and nidogen-2 drive CPCs toward chondrogenesis may help in the elucidation of new treatment strategies for cartilage tissue regeneration.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Diferenciación Celular/fisiología , Condrocitos/metabolismo , Matriz Extracelular/metabolismo , Laminina/metabolismo , Osteoartritis de la Rodilla/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Proteínas de Unión al Calcio , Condrogénesis/fisiología , Colágeno Tipo II/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Células Madre/metabolismoRESUMEN
AIM: We showed previously that E74-like factor 3 (ELF3) protein levels are increased in osteoarthritic (OA) cartilage, that ELF3 accounts for inflammatory cytokine-driven MMP13 gene expression, and that, upon induction by interleukin-1ß, ELF3 binds to the COL2A1 promoter and suppresses its activity in chondrocytes. Here, we aimed to further investigate the mechanism/s by which ELF3 represses COL2A1 transcription in chondrocytes. METHODS AND RESULTS: We report that ELF3 inhibits Sox9-driven COL2A1 promoter activity by interfering with the activator functions of CBP/300 and Sox9. Co-transfection of the pGL2B-COL2A1 (-577/+3428 bp) reporter construct with Sox9 and with Sox5 and/or Sox6 increased COL2A1 promoter activity, and ELF3 overexpression significantly reduced the promoter transactivation. Co-transfection of ELF3 with the pLuc 4x48 enhancer construct, containing the 89-bp COL2A1 promoter and lacking the previously defined ELF3 binding sites, decreased both basal and Sox9-driven promoter activity. Co-transfection of ELF3 with a Gal4 reporter construct also inhibited Gal4-Sox9-driven transactivation, suggesting that ELF3 directly interacts with Sox9. Using truncated Sox9 fragments, we found that ELF3 interacts directly with the HMG domain of Sox9. Importantly, overexpression of ELF3 significantly decreased Sox9/CBP-dependent HAT activity. Finally, we show evidence that increased ELF3 mRNA expression in OA chondrocytes correlates with hypermethylation of the proximal promoter, suggesting that ELF3 transcription is subjected to epigenetic control in OA disease. CONCLUSION: Our results highlight the contribution of ELF3 to transcriptional regulation of COL2A1 and its potential role in OA disease, and uncover epigenetic mechanisms at play in the regulation of ELF3 and its downstream targets in articular chondrocytes.
Asunto(s)
Condrocitos/metabolismo , Colágeno Tipo II/biosíntesis , Proteínas de Unión al ADN/metabolismo , Proteínas Proto-Oncogénicas c-ets/metabolismo , Factor de Transcripción SOX9/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética/fisiología , Factores de Transcripción p300-CBP/metabolismo , Línea Celular Transformada , Colágeno Tipo II/genética , Proteínas de Unión al ADN/genética , Humanos , Proteínas Proto-Oncogénicas c-ets/genética , Elementos de Respuesta/fisiología , Factor de Transcripción SOX9/genética , Factores de Transcripción/genética , Factores de Transcripción p300-CBP/genéticaRESUMEN
KEY POINTS: E74-like factor 3 (ELF3) is a transcription factor regulated by inflammation in different physio-pathological situations. Lipocalin-2 (LCN2) emerged as a relevant adipokine involved in the regulation of inflammation. In this study we showed for the first time the involvement of ELF3 in the control of LCN2 expression and its cooperation with nuclear factor-κB (NFκB). Our results will help to better understand of the role of ELF3, NFκB and LCN2 in the pathophysiology of articular cartilage. ABSTRACT: E74-like factor 3 (ELF3) is a transcription factor induced by inflammatory cytokines in chondrocytes that increases gene expression of catabolic and inflammatory mediators. Lipocalin 2 (LCN2) is a novel adipokine that negatively impacts articular cartilage, triggering catabolic and inflammatory responses in chondrocytes. Here, we investigated the control of LCN2 gene expression by ELF3 in the context of interleukin 1 (IL-1)-driven inflammatory responses in chondrocytes. The interaction of ELF3 and nuclear factor-κB (NFκB) in modulating LCN2 levels was also explored. LCN2 mRNA and protein levels, as well those of several other ELF3 target genes, were determined by RT-qPCR and Western blotting. Human primary chondrocytes, primary chondrocytes from wild-type and Elf3 knockout mice, and immortalized human T/C-28a2 and murine ATDC5 cell lines were used in in vitro assays. The activities of various gene reporter constructs were evaluated by luciferase assays. Gene overexpression and knockdown were performed using specific expression vectors and siRNA technology, respectively. ELF3 overexpression transactivated the LCN2 promoter and increased the IL-1-induced mRNA and protein levels of LCN2, as well as the mRNA expression of other pro-inflammatory mediators, in human and mouse chondrocytes. We also identified a collaborative loop between ELF3 and NFκB that amplifies the induction of LCN2. Our findings show a novel role for ELF3 and NFκB in the induction of the pro-inflammatory adipokine LCN2, providing additional evidence of the interaction between ELF3 and NFκB in modulating inflammatory responses, and a better understanding of the mechanisms of action of ELF3 in chondrocytes.
Asunto(s)
Condrocitos/metabolismo , Proteínas de Unión al ADN/metabolismo , Lipocalina 2/metabolismo , FN-kappa B/metabolismo , Factores de Transcripción/metabolismo , Animales , Línea Celular , Humanos , Interleucina-1/genética , Interleucina-1/metabolismo , Lipocalina 2/genética , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
The role of DNA methylation in the regulation of catabolic genes such as MMP13 and IL1B, which have sparse CpG islands, is poorly understood in the context of musculoskeletal diseases. We report that demethylation of specific CpG sites at -110 bp and -299 bp of the proximal MMP13 and IL1B promoters, respectively, detected by in situ methylation analysis of chondrocytes obtained directly from human cartilage, strongly correlated with higher levels of gene expression. The methylation status of these sites had a significant impact on promoter activities in chondrocytes, as revealed in transfection experiments with site-directed CpG mutants in a CpG-free luciferase reporter. Methylation of the -110 and -299 CpG sites, which reside within a hypoxia-inducible factor (HIF) consensus motif in the respective MMP13 and IL1B promoters, produced the most marked suppression of their transcriptional activities. Methylation of the -110 bp CpG site in the MMP13 promoter inhibited its HIF-2α-driven transactivation and decreased HIF-2α binding to the MMP13 proximal promoter in chromatin immunoprecipitation assays. In contrast to HIF-2α, MMP13 transcriptional regulation by other positive (RUNX2, AP-1, ELF3) and negative (Sp1, GATA1, and USF1) factors was not affected by methylation status. However, unlike the MMP13 promoter, IL1B was not susceptible to HIF-2α transactivation, indicating that the -299 CpG site in the IL1B promoter must interact with other transcription factors to modulate IL1B transcriptional activity. Taken together, our data reveal that the methylation of different CpG sites in the proximal promoters of the human MMP13 and IL1B genes modulates their transcription by distinct mechanisms.
Asunto(s)
Islas de CpG , Metilación de ADN , Regulación Enzimológica de la Expresión Génica , Interleucina-1beta/metabolismo , Metaloproteinasa 13 de la Matriz/metabolismo , Regiones Promotoras Genéticas , Cartílago/metabolismo , Condrocitos/metabolismo , Epigénesis Genética , Perfilación de la Expresión Génica , Humanos , Interleucinas/metabolismo , Modelos Genéticos , Osteoartritis/metabolismo , Plásmidos/metabolismo , Mutación Puntual , Análisis de Secuencia de ADN , Activación TranscripcionalRESUMEN
OBJECTIVE: Alterations in the mechanical loading environment in joints may have both beneficial and detrimental effects on articular cartilage and subchondral bone, and may subsequently influence the development of osteoarthritis (OA). Using an in vivo tibial loading model, the aim of this study was to investigate the adaptive responses of cartilage and bone to mechanical loading and to assess the influence of load level and duration. METHODS: Cyclic compression at peak loads of 4.5N and 9.0N was applied to the left tibial knee joint of adult (26-week-old) C57BL/6 male mice for 1, 2, and 6 weeks. Only 9.0N loading was utilized in young (10-week-old) mice. Changes in articular cartilage and subchondral bone were analyzed by histology and micro-computed tomography. RESULTS: Mechanical loading promoted cartilage damage in both age groups of mice, and the severity of joint damage increased with longer duration of loading. Metaphyseal bone mass increased with loading in young mice, but not in adult mice, whereas epiphyseal cancellous bone mass decreased with loading in both young and adult mice. In both age groups, articular cartilage thickness decreased, and subchondral cortical bone thickness increased in the posterior tibial plateau. Mice in both age groups developed periarticular osteophytes at the tibial plateau in response to the 9.0N load, but no osteophyte formation occurred in adult mice subjected to 4.5N peak loading. CONCLUSION: This noninvasive loading model permits dissection of temporal and topographic changes in cartilage and bone and will enable investigation of the efficacy of treatment interventions targeting joint biomechanics or biologic events that promote OA onset and progression.
Asunto(s)
Artritis Experimental/patología , Cartílago/patología , Osteoartritis/patología , Tibia/patología , Adaptación Fisiológica , Animales , Artritis Experimental/diagnóstico por imagen , Fenómenos Biomecánicos , Cartílago/diagnóstico por imagen , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Endogámicos C57BL , Osteoartritis/diagnóstico por imagen , Estrés Mecánico , Tibia/diagnóstico por imagen , Tomografía Computarizada por Rayos XRESUMEN
OBJECTIVE: To investigate whether the abnormal expression of inducible nitric oxide synthase (iNOS) by osteoarthritic (OA) human chondrocytes is associated with changes in the DNA methylation status in the promoter and/or enhancer elements of iNOS. METHODS: Expression of iNOS was quantified by quantitative reverse transcriptase-polymerase chain reaction. The DNA methylation status of the iNOS promoter and enhancer regions was determined by bisulfite sequencing or pyrosequencing. The effect of CpG methylation on iNOS promoter and enhancer activities was determined using a CpG-free luciferase vector and a CpG methyltransferase. Cotransfections with expression vectors encoding NF-κB subunits were carried out to analyze iNOS promoter and enhancer activities in response to changes in methylation status. RESULTS: The 1,000-bp iNOS promoter has only 7 CpG sites, 6 of which were highly methylated in both control and OA samples. The CpG site at -289 and the sites in the starting coding region were largely unmethylated in both groups. The NF-κB enhancer region at -5.8 kb was significantly demethylated in OA samples compared with control samples. This enhancer element was transactivated by cotransfection with the NF-κB subunit p65, alone or together with p50. Critically, methylation treatment of the iNOS enhancer element significantly decreased its activity in a reporter assay. CONCLUSION: These findings demonstrate the association between demethylation of specific NF-κB-responsive enhancer elements and the activation of iNOS transactivation in human OA chondrocytes, consistent with the differences in methylation status observed in vivo in normal and human OA cartilage and, importantly, show association with the OA process.
Asunto(s)
Condrocitos/fisiología , Metilación de ADN/fisiología , Elementos de Facilitación Genéticos/fisiología , FN-kappa B/genética , Óxido Nítrico Sintasa de Tipo II/genética , Osteoartritis de la Cadera/genética , Anciano , Anciano de 80 o más Años , Cartílago Articular/citología , Cartílago Articular/fisiología , Condrocitos/citología , Islas de CpG/fisiología , Femenino , Regulación Enzimológica de la Expresión Génica/fisiología , Humanos , Masculino , Persona de Mediana Edad , FN-kappa B/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Osteoartritis de la Cadera/fisiopatología , Cultivo Primario de Células , Regiones Promotoras Genéticas/fisiologíaRESUMEN
OBJECTIVE: The pathophysiology of the most common joint disease, osteoarthritis (OA), remains poorly understood. Since synovial fluid (SF) bathes joint cartilage and synovium, we reasoned that a comparative analysis of its protein constituents in health and OA could identify pathways involved in joint damage. We undertook this study to perform a proteomic analysis of knee SF from OA patients and control subjects and to compare the results to microarray expression data from cartilage and synovium. METHODS: Age-matched knee SF samples from 10 control subjects, 10 patients with early-stage OA, and 10 patients with late-stage OA were compared using 2-dimensional difference-in-gel electrophoresis and mass spectrometry (MS). MS with a multiplexed peptide selected reaction monitoring assay was used to confirm differential expression of a subset of proteins in an independent OA patient cohort. Proteomic results were analyzed by Ingenuity Pathways Analysis and compared to published synovial tissue and cartilage messenger RNA profiles. RESULTS: Sixty-six proteins were differentially present in healthy and OA SF. Three major pathways were identified among these proteins: the acute-phase response signaling pathway, the complement pathway, and the coagulation pathway. Differential expression of 5 proteins was confirmed by selected reaction monitoring assay. A focused analysis of transcripts corresponding to the differentially present proteins indicated that both synovial and cartilage tissues may contribute to the OA SF proteome. CONCLUSION: Proteins involved in the acute-phase response signaling pathway, the complement pathway, and the coagulation pathway are differentially regulated in SF from OA patients, suggesting that they contribute to joint damage. Validation of these pathways and their utility as biomarkers or therapeutic targets in OA is warranted.
Asunto(s)
Cartílago/metabolismo , Osteoartritis de la Rodilla/metabolismo , Proteoma/análisis , ARN Mensajero/análisis , Líquido Sinovial/metabolismo , Membrana Sinovial/metabolismo , Proteínas de Fase Aguda/genética , Proteínas de Fase Aguda/metabolismo , Reacción de Fase Aguda/metabolismo , Anciano , Factores de Coagulación Sanguínea/genética , Factores de Coagulación Sanguínea/metabolismo , Estudios de Casos y Controles , Proteínas del Sistema Complemento/genética , Proteínas del Sistema Complemento/metabolismo , Electroforesis en Gel Bidimensional , Femenino , Perfilación de la Expresión Génica , Humanos , Articulación de la Rodilla/metabolismo , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Osteoartritis de la Rodilla/genética , Líquido Sinovial/químicaRESUMEN
Expression of the pro-angiogenic vascular endothelial growth factor (VEGF) stimulates angiogenesis and correlates with the progression of osteoarthritis. Mechanical joint loading seems to contribute to this cartilage pathology. Cyclic equibiaxial strains of 1% to 16% for 12 h, respectively, induced expression of VEGF in human chondrocytes dose- and frequency-dependently. Stretch-mediated VEGF induction was more prominent in the human chondrocyte cell line C-28/I2 than in primary articular chondrocytes. Twelve hours of 8% stretch induced VEGF expression to 175% of unstrained controls for at least 24 h post stretching, in promoter reporter and enzyme-linked immunosorbent assay (ELISA) studies. High affinity soluble VEGF-receptor, sVEGFR-1/sFlt-1 was less stretch-inducible than its ligand, VEGF-A, in these cells. ELISA assays demonstrated, for the first time, a stretch-mediated suppression of sVEGFR-1 secretion 24 h after stretching. Overall, strained chondrocytes activate their VEGF expression, but in contrast, strain appears to suppress the secretion of the major VEGF decoy receptor (sVEGFR-1/sFlt-1). The latter may deplete a biologically relevant feedback regulation to inhibit destructive angiogenesis in articular cartilage. Our data suggest that mechanical stretch can induce morphological changes in human chondrocytes in vitro. More importantly, it induces disturbed VEGF signaling, providing a molecular mechanism for a stress-induced increase in angiogenesis in cartilage pathologies.
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Condrocitos/metabolismo , Regulación de la Expresión Génica , Estrés Mecánico , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Receptor 1 de Factores de Crecimiento Endotelial Vascular/biosíntesis , Cartílago Articular/citología , Línea Celular , Forma de la Célula , Células Cultivadas , Condrocitos/ultraestructura , Ensayo de Inmunoadsorción Enzimática , Genes Reporteros , Humanos , Técnicas In Vitro , Microscopía Electrónica de Rastreo , Cultivo Primario de Células , Regiones Promotoras Genéticas , Factores de Tiempo , Factor A de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
Osteoarthritis (OA) treatment is limited by the lack of effective nonsurgical interventions to slow disease progression. Here, we examined the contributions of the subchondral bone properties to OA development. We used parathyroid hormone (PTH) to modulate bone mass before OA initiation and alendronate (ALN) to inhibit bone remodeling during OA progression. We examined the spatiotemporal progression of joint damage by combining histopathological and transcriptomic analyses across joint tissues. The additive effect of PTH pretreatment before OA initiation and ALN treatment during OA progression most effectively attenuated load-induced OA pathology. Individually, PTH directly improved cartilage health and slowed the development of cartilage damage, whereas ALN primarily attenuated subchondral bone changes associated with OA progression. Joint damage reflected early transcriptomic changes. With both treatments, the structural changes were associated with early modulation of immunoregulation and immunoresponse pathways that may contribute to disease mechanisms. Overall, our results demonstrate the potential of subchondral bone-modifying therapies to slow the progression of OA.
Asunto(s)
Cartílago Articular , Osteoartritis , Hormona Paratiroidea , Animales , Ratones , Alendronato/farmacología , Alendronato/uso terapéutico , Huesos , Cartílago Articular/efectos de los fármacos , Cartílago Articular/metabolismo , Cartílago Articular/patología , Osteoartritis/tratamiento farmacológico , Osteoartritis/metabolismo , Osteoartritis/patología , Hormona Paratiroidea/farmacología , Hormona Paratiroidea/uso terapéutico , Remodelación Ósea/efectos de los fármacos , Soporte de PesoRESUMEN
Matrix metalloproteinase (MMP)-13 has a pivotal, rate-limiting function in cartilage remodeling and degradation due to its specificity for cleaving type II collagen. The proximal MMP13 promoter contains evolutionarily conserved E26 transformation-specific sequence binding sites that are closely flanked by AP-1 and Runx2 binding motifs, and interplay among these and other factors has been implicated in regulation by stress and inflammatory signals. Here we report that ELF3 directly controls MMP13 promoter activity by targeting an E26 transformation-specific sequence binding site at position -78 bp and by cooperating with AP-1. In addition, ELF3 binding to the proximal MMP13 promoter is enhanced by IL-1ß stimulation in chondrocytes, and the IL-1ß-induced MMP13 expression is inhibited in primary human chondrocytes by siRNA-ELF3 knockdown and in chondrocytes from Elf3(-/-) mice. Further, we found that MEK/ERK signaling enhances ELF3-driven MMP13 transactivation and is required for IL-1ß-induced ELF3 binding to the MMP13 promoter, as assessed by chromatin immunoprecipitation. Finally, we show that enhanced levels of ELF3 co-localize with MMP13 protein and activity in human osteoarthritic cartilage. These studies define a novel role for ELF3 as a procatabolic factor that may contribute to cartilage remodeling and degradation by regulating MMP13 gene transcription.
Asunto(s)
Cartílago Articular/metabolismo , Condrocitos/metabolismo , Proteínas de Unión al ADN/metabolismo , Metaloproteinasa 13 de la Matriz/biosíntesis , Osteoartritis/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Animales , Cartílago Articular/patología , Condrocitos/patología , Proteínas de Unión al ADN/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/genética , Humanos , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Interleucina-1beta/farmacología , Metaloproteinasa 13 de la Matriz/genética , Ratones , Ratones Noqueados , Osteoartritis/genética , Osteoartritis/patología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-ets , Elementos de Respuesta/genética , Estrés Fisiológico/efectos de los fármacos , Estrés Fisiológico/genética , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismo , Factores de Transcripción/genéticaRESUMEN
Osteoarthritis and rheumatoid arthritis are destructive joint diseases that involve the loss of articular cartilage. Degradation of cartilage extracellular matrix is believed to occur due to imbalance between the catabolic and anabolic processes of resident chondrocytes. Previous work has suggested that various lysosomal cysteine cathepsins participate in cartilage degeneration; however, their exact roles in disease development and progression have not been elucidated. In order to study degradation processes under conditions resembling the in vivo milieu of the cartilage, we cultivated chondrocytes on a type II collagen-containing matrix. Stimulation of the cultivated chondrocytes with interleukin-1α and/or tumor necrosis factor α resulted in a time-dependent increase in cathepsin S expression and induced its secretion into the conditioned media. Using a novel bioluminescent activity-based probe, we were able to demonstrate a significant increase in proteolytic activity of cathepsin S in the conditioned media of proinflammatory cytokine-stimulated chondrocytes. For the first time, cathepsin S was demonstrated to be secreted from chondrocytes upon stimulation with the proinflammatory cytokines, and displayed proteolytic activity in culture supernatants. Its stability at neutral pH and potent proteolytic activity on extracellular matrix components mean that cathepsin S may contribute significantly to cartilage degradation and may thus be considered a potential drug target in joint diseases.
Asunto(s)
Catepsinas/biosíntesis , Catepsinas/metabolismo , Condrocitos/metabolismo , Inflamación/metabolismo , Interleucina-1alfa/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Células Cultivadas , Humanos , ProteolisisRESUMEN
OBJECTIVES: Tumour necrosis factor inhibition plus methotrexate is believed to inhibit radiographic progression independent of inflammation. This analysis assessed whether these protective effects are exerted on bone (joint erosion; JE) and/or cartilage (joint space narrowing; JSN), and what the independent effects of JE/JSN progression are on longer-term patient-reported outcomes. METHODS: PREMIER was a 2-year, randomised, controlled trial of adalimumab plus methotrexate (ADA+MTX) versus the monotherapies. The impact of treatment on the relationships between time-averaged disease activity (TA-DAS28(CRP)) and changes in JE/JSN and associations of JE/JSN with the disability index of the health assessment questionnaire (HAQ-DI) at baseline and weeks 52 and 104 were assessed through non-parametric approaches of analysis of variance and quantile regression. JE/JSN association with employment status was evaluated at baseline and weeks 52 and 104 through logistic regression. RESULTS: Increasing tertiles of TA-DAS28(CRP) were associated with JE and JSN progression in the monotherapy groups, a phenomenon largely absent in ADA+MTX-treated patients. Although JSN was not associated with HAQ-DI at baseline, it was at 52 and 104 weeks. In contrast, JE was not associated with HAQ-DI at any time point examined. Odds of being employed at baseline, 52 weeks and 104 weeks were significantly associated with lower JSN, but not JE, scores. CONCLUSIONS: ADA+MTX inhibited both JE and JSN progression independently of disease activity. JSN played a more prominent role in patient-reported outcomes than JE. Preventing the onset or worsening of JSN probably represents a critical aspect of effective disease management of early rheumatoid arthritis patients.