Unknown,Transcriptomics,Genomics,Proteomics

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Medial Tibial Plateau articular cartilage changes 2 ,4 and 8 weeks after DMM

ABSTRACT: 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).

ORGANISM(S): Mus musculus

SUBMITTER: Anastasios Chanalaris

PROVIDER: E-GEOD-53857 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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:Objective – Following destabilization of the medial meniscus (DMM), mice develop experimental osteoarthritis (OA) and associated pain behaviors that are dependent on the stage of disease. We aimed to describe changes in gene expression in knee-innervating dorsal root ganglia (DRG) after surgery, in order to identify molecular pathways associated with three pre-defined pain phenotypes: “post-surgical pain”, “early-stage OA pain”, and “persistent OA pain”. Design – We performed DMM or sham surgery in 10-week old male C57BL/6 mice and harvested L3-L5 DRG 4, 8, and 16 weeks after surgery or from age-matched naïve mice (n=3/group). RNA was extracted and an Affymetrix Mouse Transcriptome Array 1.0 was performed. Three pain phenotypes were defined: “post-surgical pain” (sham and DMM 4-week vs. 14-week old naïve), “early OA pain” (DMM 4-week vs. sham 4-week), and “persistent OA pain” (DMM 8- and 16-week vs. naïve and sham 8- and 16-week). ‘Top hit’ genes were defined as p<0.001. Pathway analysis (Ingenuity Pathway Analysis) was conducted using differentially expressed genes defined as p<0.05. In addition, we performed qPCR for Ngf and immunohistochemistry for F4/80+ macrophages in the DRG. Results – For each phenotype, top hit genes identified a small number of differentially expressed genes, some of which have been previously associated with pain (7/67 for “post-surgical pain”; 2/14 for “early OA pain”; 8/37 for “persistent OA pain”). Overlap between groups was limited, with 8 genes differentially regulated (p<0.05) in all three phenotypes. Pathway analysis showed that in the persistent OA pain phase many of the functions of differentially regulated genes are related to immune cell recruitment and activation. Genes previously linked to OA pain (CX3CL1, CCL2, TLR1, and NGF) were upregulated in this phenotype and contributed to activation of the neuroinflammation canonical pathway. In separate sets of mice, we confirmed that Ngf was elevated in the DRG 8 weeks after DMM (p=0.03), and numbers of F4/80+ macrophages were increased 16 weeks after DMM (p=0.002 vs. Sham). Conclusion- These transcriptomics findings support the idea that distinct molecular pathways discriminate early from persistent OA pain. Pathway analysis suggests neuroimmune interactions in the DRG contribute to initiation and maintenance of pain in OA. We grouped samples based on pain phenotype in the DMM mouse model of osteoarthritis. Group 1: post-surgical pain (DMM and sham +4 week samples); Group 2: post-surgical control (Naïve +4 week samples); Group 3: Early osteoarthritis pain (DMM +4 week samples); Group 4: Early controls (sham+4 and naive+4 week samples); Group 5: Persistent pain (DMM+8 and DMM+16 week samples); Group 6: Persistent controls (sham+8, naive+8, sham+16, and naive +16 samples). We compared Group 1 vs Group 2; Group 3 vs Group 4; and Group 5 vs Group 6 to draw the conclusions presented in our manuscript.

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Medial Tibial Plateau articular cartilage changes 2 ,4 and 8 weeks after DMM

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