Unknown,Transcriptomics,Genomics,Proteomics

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Differential gene expression in cartilage from the mouse DMM osteoarthritis model

ABSTRACT: Transcriptional profiling of mouse cartilage comparing destabilised medial meniscal operated knee joints to sham operated knee joints in both wild type C57Bl/6 and C57Bl/6 ADAMTS5Dcat mice at 1, 2 and 6 weeks post DMM surgery. The goal was to determine the differential gene expression with the onset of arthritis, both early, mid and late timepoints and to assess the gene expression differences when aggrecan loss is prevented in the ADAMTS5Dcat mouse. The overall design of the experiment is to compare DMM (right leg) to sham operated (left leg) cartilage from the same mouse. In order to identify genes involved in early and late arthritis, RNA was collected from cartilage samples from mice at 1, 2 and 6 weeks post surgery. In addition, to isolate genes differentially expressed downstream of aggrecan loss, the RNA as described was collected from C57Bl/6 wild type mice and ADAMTS5Dcat mice. Four mice were used at each timepoint with their left leg being the control Sham and their right leg the experimental DMM. One sample from the wild type 1wk post operation and one ADAMTS5Dcat dye swap were removed from the analysis for technical reasons.

ORGANISM(S): Mus musculus

SUBMITTER: Lynn Rowley

PROVIDER: E-GEOD-45793 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Transcriptomics of wild-type mice and mice lacking ADAMTS-5 activity identifies genes involved in osteoarthritis initiation and cartilage destruction.

Bateman John F JF Rowley Lynn L Belluoccio Daniele D Chan Ben B Bell Katrina K Fosang Amanda J AJ Little Christopher B CB

Arthritis and rheumatism 20130601 6

<h4>Objective</h4>To identify changes in gene expression in mice with osteoarthritis (OA) in order to explore the mechanisms of the disease.<h4>Methods</h4>Gene expression profiling was performed in cartilage from mice with surgically induced OA. We used wild-type (WT) mice and Adamts5Δcat mice, in which ADAMTS-5 activity is lacking and aggrecan loss and cartilage erosion are inhibited, to distinguish gene expression changes that are independent of ADAMTS-5 activity and cartilage breakdown. Mech ...[more]

PMID: 23436205

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Project description:Mechanical Stimuli are arguably the most important aetiolgical factors in osteoarthritis (OA) development. Not only do we see disease arising from joints where the cartilage has sustained direct (e.g. intraarticular fracture) or indirect (e.g. meniscal injury) trauma, but mechanical factors are considered, at least partly, to explain the disease associations with aging and obesity. It is now well established that OA is not simply due to repeated wear and tear, leading to attrition of the articular surfaces, but that it requires activation of a number of inflammatory genes, which drive catabolic protease activity in the joint. These enzymes lead to breakdown of the major extracellular matrix components of cartilage, namely type II collagen, and the proteoglycan, aggrecan. Although it is unclear precisely which enzymes are responsible for matrix breakdown in human OA, Glasson et al showed that deletion of the aggrecan degrading enzyme, ADAMTS5 substantially protected the joint from surgically induced murine OA suggesting that it is a major aggrecanase in the mouse. This study was part of a wider study using the surgical model of murine OA, induced by destabilising the medial meniscus (DMM), to confirm the dependence of joint loading on disease progression and to reveal mechanosensitive genes within the joint which may be involved in the development of OA. Using this model we, and others, have shown that there is a robust degradation of articular cartilage over 4-12wks in male C57B/6 mice. We identified the gene response in joints early following induction of OA, prior to cartilage degradation, by extracting RNA from whole joints (after skin and muscle had been removed) 6hrs, 3 days and 7 days following sham and DMM surgery. An Affymetrix ST1 gene array was performed. Significantly regulated genes (>1.4 fold above naive and sham samples at any timepoint), or those considered to have a putative role in OA pathogenesis were selected for quantitative validation using Taqman low density microfluidic card arrays (TLDA). Gene expression in whole knee joints at 3 early time points post DMM surgery (before cartilage loss), 6hrs, 3days and 7days was examined. Two controls were identified, the first control consisted of age matched naïve mice that had undergone 15 minutes of anaesthesia but were not operated on (0hrs post surgery). The second control included mice which had undergone sham surgery (capsulotomy alone). For this RNA was taken at the same time points post surgery as DMM samples (6hrs, 3days and 7days). There were three biological replicates per condition (total of 21 samples). The right (ipsilateral) knee joint was taken and RNA extracted and processed for microarray analysis using the Affymetrix Mouse Gene 1.0 st v1 platform.

Project description:The purpose of this study was to characterize the histologic development of OA in a mouse model where OA is induced by destabilization of the medial meniscus (DMM model) and to identify genes regulated during different stages of the disease, using RNA isolated from the joint M-bM-^@M-^\organM-bM-^@M-^] and analyzed using microarrays.427 genes from the microarrays passed consistency and significance filters. There was an initial up-regulation at 2 and 4 weeks of genes involved in morphogenesis, differentiation, and development, including growth factor and matrix genes, as well as transcription factors including Atf2, Creb3l1, and Erg. Most genes were off or down-regulated at 8 weeks with the most highly down-regulated genes involved in cell division and the cytoskeleton. Gene expression increased at 16 weeks, in particular extracellular matrix genes including Prelp, Col3a1 and fibromodulin.The results support a phasic development of OA with early matrix remodelling and transcriptional activity followed by a more quiescent period that is not maintained. A group of 9 mice was used for collection of RNA at time 0 (before surgery) when the animals were 12 weeks old. For the other time points, 9 DMM and 9 sham controls were sacrificed at 2, 4, 8, and 16 weeks after surgery for RNA isolation. The tissue included tibial plateau and femoral condyle articular cartilage, subchondral bone with any osteophytes, meniscus, and the joint capsule with synovium was used for RNA isolation. The tissue was treated with RNAlaterM-BM-. (Invitrogen) prior to freezing and storage at -800 C. RNA was extracted by homogenization using the Precellys 24 tissue homogenizer (Bertin Technologies purchased from MO BIO) and the amount and quality of the RNA was determined using an Agilent 2100 Bioanalyzer. RNA was pooled prior to microarray analysis such that 3 randomly selected samples from each surgical group and time point were pooled to create each biological replicate. Because 9 mice were used for each experimental group, a total of three biological replicates per group were analyzed using the Affymetrix Mouse Genome 430 2.0 oligonucleotide arrays as described. One replicate pool, which was from week two DMM mice, did not meet the RNA integrity level needed for microarray analysis; thus, this pool was not analyzed further, leaving two pools for the week two DMM mice.

Project description:Osteoarthritis (OA) is a complex degenerative joint disease, which is not only a cartilage but also a bone disease. A better understanding of the early molecular mechanism changes of subchondral bone in vivo may contribute to elucidating the pathogenesis of OA. We used microarray technology to investigate the time-course molecular changes of subchondral bone just beneath damaged cartilage in early stage of experimental osteoarthritis, and found 2,234 differentially expressed (DE) genes at 1 week, 1,944 at 2 weeks and 1,517 at 4 weeks postsurgery.Further analysis of dysregulated genes indicated that subchondral bone remodeling occurred sequentially and in a time-dependent manner at the gene expression level. Some known dysregulated genes suspected roles in influencing bone development or bone remodeling, such as Alp, Igf1, Tgf M-NM-21, Postn, Mmp3, Tnfsf11, Acp5, Bmp5, Aspn and Ihh, were confirmed by real-time PCR, and results indicated that our microarray data could accurately reflect gene expression patterns of early OA. Subsequently, to validate the results of our microarray analysis at protein level, immunohistochemistry staining was introduced to investigate the translational level of genes Mmp3 and Aspn in tissue sections, and results showed that the level of Mmp3 protein expression was totally matched the results of microarray and real-time PCR analysis. Nevertheless, the expression of Aspn protein was not observed differentially expressed at any time point. Ninety 10-week-old male Sprague-Dawley rats, weighing 300-325g, were used in the study. Animals were equally divided into two groups: experimental group (E-Group) and sham-operated group (S-Group). The E-Group rats underwent open surgery, involved in both medial meniscectomy and medial collateral ligament (MCL) transaction with micro-scissors. The S-Group rats were carried out with a sham operation, via a similar incision, without operations of the medial meniscus and the medial collateral ligament.Animals were killed at 1, 2, and 4 weeks postsurgery, and 15 animals were put into use per-timepoint in each treatment group. 5 animals were used for histological analysis and immunohistochemistry, and others were used for microarray study and Real-time polymerase chain reaction (PCR) analysis equally at each timepoint.

Project description:Objective: To Identify gene changes in the medial tibial plateau of articular cartilage at 2, 4 and 8 weeks after destabilisation of the medial meniscus (DMM) in mice and compare our data set with previously published sets to ascertain dysregulated pathways and genes in osteoarthritis. Materials and methods: RNA was extracted from the ipsilateral and contralateral medial tibial plateaus, amplified, labelled and hybridized on Illumina WG_v2 microarrays. Results were confirmed by RT-PCR for selected genes. Results: Transcriptional analysis and network reconstruction revealed changes in extracellular matrix (ECM) and cytoskeletal genes induced by DMM. TGFalpha signalling pathway and complement and coagulation cascade genes were regulated at 2 weeks. Fibronectin (FN1) is a hub in a reconstructed network of potential protein-protein interactions at 2 weeks. Regulated genes decrease over time and by eight weeks fibromodulin (FMOD) and tenascin N (TNN) are the only dysregulated genes present in the DMM operated knees. Comparison with human and rodent published gene sets discovered genes overlapping between our array and 6 others. Conclusions: Cartilage contributes a minute percentage to the RNA extracted from the whole joint (>0.2%), yet is sensitive to changes in gene expression post-DMM. The post-DMM transcriptional reprogramming wanes over time and dissipates by 8 weeks. Common pathways between published gene sets include focal adhesion, regulation of actin cytoskeleton and TGFalpha. Common genes include Jagged 1 (Jag1), Tetraspanin 2 (Tspan2), neuroblastoma, suppression of tumourigenicity 1 (Nbl1) and N-myc downstream regulated gene 2 (Ndrg2). The concomitant genes and pathways we identify are novel and warrant further investigation as biomarkers or modulators of osteoarthritis. In total 18 samples. Each sample was a pool of the medial tibial plateau of 8 mice. Two categories (Treated and untreated) with three biological replicates each and three time points (2, 4 and 8 weeks post-surgery).

Project description:Fetal cartilage fully regenerates following injury while in adult mammals cartilage injury leads to osteoarthritis (OA). OA is characterized by cartilage breakdown and joint inflammation and associated with significant pain and socioeconomic costs. As no clinically satisfactory treatment is available to date, disease-modifying therapies aimed to achieve cartilage regeneration are urgently required. The inherent regeneration potential of fetal individuals may hold answers to this unmet need. Therefore, to characterize the differences in fetal and adult response to cartilage injury, we carried out histology and comprehensive proteome analyses on fetal (day 80/150-day gestation) and adult cartilage samples one (fetal samples) and three (adult and fetal samples) days after surgical induction of a full-thickness cartilage lesion. In addition, proteins secreted by inflamed fetal MSCs in vitro were compared with the in vivo response to injury to evaluate their therapeutic potential. Histology of synovial samples revealed the presence of neutrophils one day post injury (p.i.) and an influx of macrophages into the subsynovial tissue on day 3 p.i. in fetal samples. In contrast, adult synovial samples showed invasion of neutrophils on day 3 p.i. Activation and migration of Iba1+- macrophages of the synovial lining was observed both in fetal and adult animals. Comparative mass spectrometry revealed 57 proteins significantly up-regulated (> 2FC, FDR<0.05), and 67 proteins significantly down-regulated (<-2 FC) upon injury in adults. Neutrophil-related proteins and acute phase proteins were the two major upregulated protein groups in adult cartilage following injury compared to fetal sheep. In contrast, several immunomodulating proteins and growth factors were significantly higher expressed in the fetus than the adult. Comparison of the in vitro MSCs with the in vivo fetal proteome revealed shared upregulation of 17 proteins, which were considered to be of potential therapeutic interest. The results of this study support our molecular understanding of successful fetal cartilage healing and new therapeutic strategies to induce regeneration in adult articular cartilage by modulating the inflammatory environment. The shared protein upregulation in fetal cartilage in vivo and in fetal MSCS during in vitro inflammation supports the possible therapeutic potential of these factors in specific and fetal MSCs in general.

Dataset Information

Differential gene expression in cartilage from the mouse DMM osteoarthritis model

Publications

Transcriptomics of wild-type mice and mice lacking ADAMTS-5 activity identifies genes involved in osteoarthritis initiation and cartilage destruction.

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