Unknown,Transcriptomics,Genomics,Proteomics

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Osteoarthritis-related changes in gene expression in knee articular chondrocytes in a rat model of osteoarthritis.

ABSTRACT: Cartilage destruction in osteoarthritis (OA) results from disturbed chondrocyte metabolism. Here, we used microarrays to show that TGF alpha and CCL2 are simultaneously upregulated in a rat model of OA and cooperate to drive cartilage degradation. The goals of the experiments included here were to a) characterize gene expression in knee joint articular chondrocytes at various stages of development of OA (2 and 8 weeks after surgical induction of OA), and b) to establish trends in gene expression among groups of genes related to the TGF alpha-EGFR axis, over time, in OA. The model chosen to study these results has been previously validated (Appleton, CT et al, 2007, Arthritis Rheum) and used to describe similar gene expression results at a different time point (4 weeks) after induction of OA. The rat model of OA involves surgical destabilization of the knee joint, followed by forced low-intensity mobilization over several weeks; a sham surgery is used as the control (representing a healthy non-OA knee joint) wherein a surgical incision is made but not structural (i.e. ligamentous) modification is made to the joint. Altogether, our data indicate that TGF and CCL2 cooperate to drive cartilage degradation in osteoarthritis. A total of 12 samples were analyzed. 3 replicates were used per condition: OA surgery at 2 weeks, OA surgery at 8 weeks, Sham (control) surgery at 2 weeks and Sham surgery at 8 weeks. Expression of OA samples was assessed relaitve to Sham (control) expression levels.

ORGANISM(S): Rattus norvegicus

SUBMITTER: Tom Appleton

PROVIDER: E-GEOD-42295 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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:As the most common degenerative joint disease, osteoarthritis (OA) contributes significantly to pain and disability during aging. Several genes of interest involved in articular cartilage damage in OA have been identified. However, the direct causes of OA are poorly understood. Evaluating the public human RNA-seq dataset showed that Cbfβ, (subunit of a heterodimeric Cbfβ/Runx1,Runx2, or Runx3 complex) expression is decreased in the cartilage of patients with OA. Here, we found that the chondrocyte-specific deletion of Cbfβ in tamoxifen-induced Cbfβf/fCol2α1-CreERT mice caused a spontaneous OA phenotype, worn articular cartilage, increased inflammation, and osteophytes. RNA-sequencing analysis showed that Cbfβ deficiency in articular cartilage resulted in reduced cartilage regeneration, increased canonical Wnt signaling and inflammatory response, and decreased Hippo/YAP signaling and TGF-β signaling. Immunostaining and western blot validated these RNA-seq analysis results. ACLT surgery-induced OA decreased Cbfβ and Yap expression and increased active β-catenin expression in articular cartilage, while local AAV-mediated Cbfβ overexpression promoted Yap expression and diminished active β-catenin expression in OA lesions. Remarkably, AAV-mediated Cbfβ overexpression in knee joints of mice with OA showed the significant protective effect of Cbfβ on articular cartilage in the ACLT OA mouse model. Overall, this study, using loss-of-function and gain-of-function approaches, uncovered that low expression of Cbfβ may be the cause of OA. Moreover, Local admission of Cbfβ may rescue and protect OA through decreasing Wnt/β-catenin signaling, and increasing Hippo/Yap signaling and TGFβ/Smad2/3 signaling in OA articular cartilage, indicating that local Cbfβ overexpression could be an effective strategy for treatment of OA. Using unbiased genome-wide RNA-seq data from Cbfβf/f;Col2α1-Cre hip joint articular cartilage and Cbfβf/f;Aggrecan-cre knee joint articular cartilage and their controls, we examined Cbfβ-mediated transcriptional targets for articular cartilage regeneration in OA.

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.

Project description:OBJECTIVE: To analyze genome-wide changes in chondrocyte gene expression in a surgically induced model of early osteoarthritis (OA) in rats, to assess the similarity of this model to human OA, and to identify genes and mechanisms leading to OA pathogenesis. METHODS: OA was surgically induced in 5 rats by anterior cruciate ligament transection and partial medial meniscectomy. Sham surgery was performed in 5 additional animals, which were used as controls. Both groups underwent 4 weeks of forced mobilization, 3 times per week. RNA was extracted directly from articular chondrocytes in the OA (operated), contralateral, and sham-operated knees. Affymetrix GeneChip expression arrays were used to assess genome-wide changes in gene expression. Expression patterns of selected dysregulated genes, including Col2a1, Mmp13, Adamts5, Ctsc, Ptges, and Cxcr4, were validated by real-time polymerase chain reaction, immunofluorescence, or immunohistochemistry 2, 4, and 8 weeks after surgery. RESULTS: After normalization, comparison of OA and sham-operated samples showed 1,619 differentially expressed probe sets with changes in their levels of expression >/=1.5-fold, 722 with changes >/=2-fold, 135 with changes >/=4-fold, and 20 with changes of 8-fold. Dysregulated genes known to be involved in human OA included Mmp13, Adamts5, and Ptgs2, among others. Several dysregulated genes (e.g., Reln, Phex, and Ltbp2) had been identified in our earlier microarray study of hypertrophic chondrocyte differentiation. Other genes involved in cytokine and chemokine signaling, including Cxcr4 and Ccl2, were identified. Changes in gene expression were also observed in the contralateral knee, validating the sham operation as the appropriate control. CONCLUSION: Our results demonstrate that the animal model mimics gene expression changes seen in human OA, supporting the relevance of newly identified genes and pathways to early human OA. We propose new avenues for OA pathogenesis research and potential targets for novel OA treatments, including cathepsins and cytokine, chemokine, and growth factor signaling pathways, in addition to factors controlling the progression of chondrocyte differentiation. Experiment Overall Design: OA was surgically induced in 5 rats by anterior cruciate ligament transection and partial medial meniscectomy. Sham surgery was performed in 5 additional animals, which were used as controls. Both groups underwent 4 weeks of forced mobilization, 3 times per week. RNA was extracted directly from articular chondrocytes in the OA (operated), contralateral, and sham-operated knees, 28 days post-surgery. Affymetrix GeneChip expression arrays were used to assess genome-wide changes in gene expression.

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Osteoarthritis-related changes in gene expression in knee articular chondrocytes in a rat model of osteoarthritis.

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