RNA sequencing of subchondral bone from patients that underwent a joint replacement surgery due to osteoarthritis.
ABSTRACT: Osteoarthritis (OA) has a considerable genetic component. Genetic research has resulted in many insights into underlying disease pathways. Nonetheless, follow up studies of identified susceptibility genes towards underlying biological mechanisms, preclinical studies on target discovery and eventually drug testing during have thus far focused on gene expression profiling in articular cartilage. Therefore, the aim of the current study was to characterize pathophysiological processes of subchondral bone in human osteoarthritis (OA) in large human cohort RNA sequencing.
Project description:INTRODUCTION:Matrix metalloproteinases (MMPs) and 'aggrecanase' a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs) are well established to play key roles in osteoarthritis (OA) through degradation of extracellular matrix (ECM) type II collagen and aggrecan, and are thus potential targets for development of OA therapies. OBJECTIVE:This paper aims to provide a comprehensive review of the expression and potential roles of other, lesser-known ADAMTSs and related adamalysins (or a disintegrin and metalloproteinases (ADAMs)) in cartilage, with a view to identifying potentially protective or homeostatic metalloproteinases in the joint and informing consequent selective inhibitor design. DESIGN:A comprehensive literature search was performed using PubMed terms 'osteoarthritis' and 'ADAMTS' or 'ADAM'. RESULTS:Several ADAMTSs and ADAMs were identified as having reportedly increased expression in OA. These include enzymes likely to play roles in cartilage matrix anabolism (e.g., the procollagen N-proteinases ADAMTS-2, ADAMTS-3 and ADAMTS-14), chondrocyte differentiation and proliferation (e.g., ADAM9, ADAM10, ADAM12), as well as enzymes contributing to cartilage catabolism (e.g., Cartilage oligomeric protein (COMP)-degrading ADAMTS-7 and ADAMTS-12). CONCLUSIONS:In addition to the well-characterised MMPs, ADAMTS-4 and ADAMTS-5, many other ADAMTSs and ADAMs are expressed in cartilage and several show significantly altered expression in OA. Studies aimed at elucidating the pathophysiological roles of these enzymes in cartilage will contribute to our understanding of OA pathogenesis and enable design of targeted inhibitors that effectively target metalloproteinase-mediated cartilage degradation while sparing cartilage repair pathways.
Project description:Osteoarthritis (OA) is a prevalent disease of articular joints and primarily characterized by degradation and calcification of articular cartilage. Presently, no effective treatment other than pain relief exists and patients ultimately need to undergo replacement surgery of the affected joint. During disease progression articular chondrocytes, the single cell type present in articular cartilage, show altered transcriptional profiles and undergo phenotypic changes that resemble the terminal differentiation route apparent in growth plate chondrocytes. Hence, given its prominent function in both regulating gene expression and maintaining cellular phenotypes, DNA methylation of CpG dinucleotides is intensively studied in the context of OA. An increasing number of studies have been published that employed a targeted approach on genes known to play a role in OA pathophysiology. As of such, it has become clear that OA responsive DNA methylation changes seem to mediate disease associated aberrant gene expression. Furthermore, established OA susceptibility alleles such as GDF5 and DIO2 appear to confer OA risk via DNA methylation and respective pathophysiological expression changes. In more recent years, genome wide profiling of DNA methylation in OA affected articular cartilage has emerged as a powerful tool to address the epigenetic changes in their entirety, which has resulted in the identification of putative patient subgroups as well as generic OA associated pathways.
Project description:Osteoarthritis (OA) is a destructive joint disease in which the initiation may be attributed to direct injury and mechanical disruption of joint tissues, but the progressive changes are dependent on active cell-mediated processes that can be observed or inferred during the generally long time-course of the disease. Based on clinical observations and experimental studies, it is now recognized a that it is possible for individual patients to exhibit common sets of symptoms and structural abnormalities due to distinct pathophysiological pathways that act independently or in combination. Recent research that has focused on the underlying mechanisms involving biochemical cross talk among the cartilage, synovium, bone, and other joint tissues within a background of poorly characterized genetic factors will be addressed in this review.
Project description:Osteoarthritis (OA) is a chronic degenerative disease that leads to joint failure with pain and disability. Gene regulations are implicated in driving an imbalance between the expression of catabolic and anabolic factors, leading eventually to osteoarthritic cartilage degeneration. The different stages of disease progression are described by the complex pattern of transcriptional regulations. The dynamics in pattern alterations were monitored in each individual animal during the time-course of OA progression. Overall design: Knee cartilage samples were collected from individual male Wistar rats with monosodium iodoacetate (MIA) induced OA (0, 2, 14, 28 days after the treatment). MIA-treated (3 mg) animals were compared to intact controls. Four replicate samples were analyzed per each experimental group. We used whole-genome microarrays (Affymetrix Rat Gene 2.0 ST) to identify OA-related molecular signatures in the knee cartilage.
Project description:Cartilage dyshomeostasis contributes to osteoarthritis (OA) pathogenesis, and tumor necrosis factor (TNF)-? has critical role in this process by driving inflammatory cascades and cartilage degradation. However, the negative regulation of TNF-?-mediated signaling remains undefined. Here we demonstrate the crucial role of miR-145 in the modulation of TNF-?-mediated signaling and cartilage matrix degradation. MicroRNA (miRNA) expression profiles of TNF-?-stimulated chondrocytes showed that miR-145 expression was rapidly downregulated by TNF-?. Moreover, miR-145 was directly repressed by p65 and was negatively correlated with TNF-? secretion during OA progression. Further, we found that miR-145 directly targeted mitogen-activated protein kinase kinase 4 (MKK4) and broadly restrained the production of several TNF-?-triggered matrix-degrading enzymes (MMP-3, MMP-13, and Adamts-5). Mechanistic studies unveiled that miR-145 negatively regulated TNF-?-mediated JNK and p38 activation, as well as the nuclear accumulation of p-c-Jun and p-ATF2, by inhibiting MKK4 phosphorylation, eventually resulting in the alteration of catabolic genes transcription. Indeed, p-ATF2 interacted with the promoter of Mmp-13, whereas p-c-Jun bound to promoters of Mmp-3 and Adamts-5. MKK4 was significantly elevated in OA cartilage. Eliminating MKK4 by short hairpin RNA resulted in obviously decreased matrix-degrading enzymes production, JNK and p38 inactivation, and an inhibition of cartilage degradation. On the contrary, MKK4 overexpression enhanced TNF-?-mediated signaling activation and transcription of downstream catabolic genes, and consequently worsened cartilage degradation. Moreover, intra-articular (IA) injection of miR-145 agonist to rat with surgery-induced OA alleviated cartilage destruction. Altogether, we elucidate a novel regulatory mechanism underlying TNF-?-triggered cartilage degradation and demonstrate the potential utility of miR-145 and MKK4 as therapy targets for OA.
Project description:To date, all of the prior osteoarthritic microarray studies in human tissue have focused on the overlying articular cartilage, meniscus, or synovium but not the underlying subchondral bone. In our previous study, our group developed a methodology for high quality RNA isolation from site-matched cartilage and bone from human knee joints, which allowed us to perform candidate gene expression analysis on the subchohndral bone (published on Osteoarthritis and Cartilage on Dec/5/2012 (doi: 10.1016/j.joca.2012.11.016). To the best of our knowledge, the current study is the first to successfully perform whole-genome microarray profiling analyses of human osteoarthritic subchondral bone. We believe our comprehensive microarray results can improve the understanding of the pathogenesis of osteoarthritis and could further contribute to the development of new biomarker and therapeutic strategies in osteoarthritis. Following histological assessment of the integrity of overlying cartilage and the severity of bone abnormality by microcomputed tomography, we isolated total RNA from regions of interest from human OA (n=20) and non-OA (n=5) knee lateral and medial tibial plateaus (LT and MT). A whole-genome profiling study was performed on an Agilent microarray platform and analyzed using Agilent GeneSpring GX11.5. Confirmatory quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis was performed on samples from nine OA individuals to confirm differential expression of 85 genes identified by microarray. Ingenuity Pathway Analysis (IPA) was used to investigate canonical pathways and immunohistochemical staining was performed to validate protein expression levels in samples.
Project description:Osteoarthritis (OA), characterized by insufficient extracellular matrix synthesis and cartilage degeneration, is known as an incurable disease because its pathogenesis is poorly elucidated. Thus far, limited information is available regarding the pathophysiological role of microRNAs (miRNAs) in OA. In this study, we investigated the specific function of miR-146a in OA pathophysiology using mouse OA models. We found that the articular cartilage degeneration of miR-146a knockout (KO) mice was alleviated compared with that of the wild-type (WT) mice in spontaneous and instability-induced OA models. We demonstrate that miR-146a aggravated pro-inflammatory cytokines induced suppressing the expression of cartilage matrix-associated genes. We further identified calcium/calmodulin-dependent protein kinase II delta (Camk2d) and protein phosphatase 3, regulatory subunit B, beta isoform (Ppp3r2, also known as calcineurin B, type II) were essential targets of miR-146a in regulating cartilage homeostasis. Moreover, we found that surgical-induced OA mice treated with a miR-146a inhibitor significantly alleviated the destruction of articular cartilage via targeting Camk2d and Ppp3r2. These results suggested that miR-146a has a crucial role in maintaining cartilage homeostasis. MiR-146a inhibition in chondrocytes can be a potential therapeutic strategy to ameliorate OA.
Project description:Background:Osteoarthritis (OA) is the most common type of joint disease associated with cartilage breakdown. However, the role played by mitochondrial dysfunction in OA remains inadequately understood. Therefore, we investigated the role played by p66shc during oxidative damage and mitochondrial dysfunction in OA and the effects of p66shc downregulation on OA progression. Methods:Monosodium iodoacetate (MIA), which is commonly used to generate OA animal models, inhibits glycolysis and biosynthetic processes in chondrocytes, eventually causing cell death. To observe the effects of MIA and poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles, histological analysis, immunohistochemistry, micro-CT, mechanical paw withdrawal thresholds, quantitative PCR, and measurement of oxygen consumption rate and extracellular acidification rate were conducted. Results:p-p66shc was highly expressed in cartilage from OA patients and rats with MIA-induced OA. MIA caused mitochondrial dysfunction and reactive oxygen species (ROS) production, and the inhibition of p66shc phosphorylation attenuated MIA-induced ROS production in human chondrocytes. Inhibition of p66shc by PLGA-based nanoparticles-delivered siRNA ameliorated pain behavior, cartilage damage, and inflammatory cytokine production in the knee joints of MIA-induced OA rats. Conclusion:p66shc is involved in cartilage degeneration in OA. By delivering p66shc-siRNA-loaded nanoparticles into the knee joints with OA, mitochondrial dysfunction-induced cartilage damage can be significantly decreased. Thus, p66shc siRNA PLGA nanoparticles may be a promising option for the treatment of OA.
Project description:Objective: To assess the impact of osteoarthritis (OA) on the transcripts and biological process in the articular cartilage between patients with and without OA. Design: Patients undergoing arthroscopic partial meniscectomy (APM) without any evidence for OA and patients undergoing total knee arthroplasty (TKA) due to end-stage OA were consented. Healthy-appearing cartilage was garnered from the non-weight bearing site of the medial intercondylar notch. RNA preparation was subjected to SurePrint G3 human 8X60K RNA microarrays to probe differentially expressed (DE) transcripts followed by computational exploration of underlying biological processes and pathways. Real-time PCR was performed on selected transcripts to validate microarrays data. Results: We identified 603 transcripts significantly (FDR <0.05) differentially expressed (293 elevated, 310 repressed) between APM and TKA samples (1.5 fold). Among these, CFD, CSN1S1, TSPAN11, CSF1R and CD14 were the most prominent transcripts elevated in TKA group, CHI3L2, MEG3, HILPDA, COL3A1, COL27A and FGF2 were most highly repressed in TKA samples. Few long intergenic non-coding RNAs (linRNAs), and small nuclear RNAs (snoRNAs) were also differentially expressed between the two groups. Conclusions: Numerous transcripts with potential relevance to the pathogenesis of OA are DE in OA and non-OA cartilage. These data suggest an involvement of metabolic signaling and epigenetic markers (lincRNAs, snoRNAs) in cartilage. Overall design: We used Agilent microarrays to identify gene transcripts differentially expressed in cartilage tissues obtained from 12 patients with osteoarthritis and from 12 patients without osteoarthritis (arthroscopic partial meniscectomy). P4-010, P4-012, P4-104, and P4-108 were excluded from the final analysis.
Project description:Osteoarthritis (OA) is one of the most prevalent forms of joint disorder, associated with a tremendous socioeconomic burden worldwide. Various non-genetic and lifestyle-related factors such as aging and obesity have been recognized as major risk factors for OA, underscoring the potential role for epigenetic regulation in the pathogenesis of the disease. OA-associated epigenetic aberrations have been noted at the level of DNA methylation and histone modification in chondrocytes. These epigenetic regulations are implicated in driving an imbalance between the expression of catabolic and anabolic factors, leading eventually to osteoarthritic cartilage destruction. Cellular senescence and metabolic abnormalities driven by OA-associated risk factors appear to accompany epigenetic drifts in chondrocytes. Notably, molecular events associated with metabolic disorders influence epigenetic regulation in chondrocytes, supporting the notion that OA is a metabolic disease. Here, we review accumulating evidence supporting a role for epigenetics in the regulation of cartilage homeostasis and OA pathogenesis.