Optimisation of osteogenic and chondrogenic differentiation potential using clonal mesenchymal stem cell populations derived from synovial fat pad

Mesenchymal stem cells are a potential source of cells for the repair of bone and articular cartilage defects. We have previously demonstrated that the infrapatellar synovial fat pad is a rich source of mesenchymal stem cells and these cells are able to undergo osteogenic and chondrogenic differentiation. Although synovial fat pad derived mesenchymal stem cells may represent a heterogenous population, clonal populations derived from the synovial fat pad have not previously been studied

Gene expression and matrix turnover in overused and damaged tendons

Chronic, painful conditions affecting tendons, frequently known as tendinopathy, are very common types of sporting injury. The tendon extracellular matrix is substantially altered in tendinopathy, and these changes are thought to precede and underlie the clinical condition. The tendon cell response to repeated minor injuries or “overuse” is thought to be a major factor in the development of tendinopathy. Changes in matrix turnover may also be effected by the cellular response to physical load, altering the balance of matrix turnover and changing the structure and composition of the tendon. Matrix turnover is relatively high in tendons exposed to high mechanical demands, such as the supraspinatus and Achilles, and this is thought to represent either a repair or tissue maintenance function. Metalloproteinases are a large family of enzymes capable of degrading all of the tendon matrix components, and these are thought to play a major role in the degradation of matrix during development, adaptation and repair. It is proposed that some metalloproteinase enzymes are required for the health of the tendon, and others may be damaging, leading to degeneration of the tissue. Further research is required to investigate how these enzyme activities are regulated in tendon and altered in tendinopathy. A profile of all the metalloproteinases expressed and active in healthy and degenerate tendon is required and may lead to the development of new drug therapies for these common and debilitating sports injuries

In vitro suppression of the MMP-3 gene in normal and cytokine-treated human chondrosarcoma using small interfering RNA

<p>Abstract</p> <p>Background</p> <p>Matrix metalloproteinase (MMPs) synthesized and secreted from connective tissue cells have been thought to participate in degradation of the extracellular matrix. Increased MMPs activities that degrade proteoglycans have been measured in osteoarthritis cartilage. This study aims to suppress the expression of the <it>MMP-3 </it>gene in <it>in vitro </it>human chondrosarcoma using siRNA.</p> <p>Methods</p> <p>Cells were categorized into four groups: control (G.1); transfection solution treated (G.2); negative control siRNA treated (G.3); and <it>MMP-3 </it>siRNA treated (G.4). All four groups were further subdivided into two groups - treated and non-treated with IL-1β- following culture for 48 and 72 h. We observed the effects of gene suppression according to cell morphology, glycosaminoglycan (GAG) and hyaluronan (HA) production, and gene expression by using real-time polymerase chain reaction (PCR).</p> <p>Results</p> <p>In IL-1β treated cells the apoptosis rate in G.4 was found to be lower than in all other groups, while viability and mitotic rate were higher than in all other groups (<it>p </it>< 0.05). The production of GAG and HA in G.4 was significantly higher than the control group (<it>p </it>< 0.05). <it>MMP-3 </it>gene expression was downregulated significantly (<it>p </it>< 0.05).</p> <p>Conclusion</p> <p><it>MMP-3 </it>specific siRNA can inhibit the expression of <it>MMP-3 </it>in chondrosarcoma. This suggests that <it>MMP-3 </it>siRNA has the potential to be a useful preventive and therapeutic agent for osteoarthritis.</p

Patients with rheumatoid arthritis have an altered circulatory aggrecan profile

<p>Abstract</p> <p>Background</p> <p>Rheumatoid arthritis (RA) is a chronic auto-immune disease with extensive articular cartilage destruction. Aggrecan depletion, mediated by aggrecanases is one of the first signs of early cartilage erosion. We investigated, whether measurement of aggrecan and fragments thereof in serum, could be used as biomarkers for joint-disease in RA patients and furthermore characterized the fragments found in the circulation.</p> <p>Methods</p> <p>The study consisted of 38 patients, 12 males (62.2 ± 16.0 years) and 26 females (59.8 ± 20.7 years) diagnosed with RA: 41.5 ± 27.5 mm/h erythrocyte sedimentation rate (ESR), 38.4 ± 34.7 mg/ml C-reactive protein (CRP) and 4.8 ± 1.7 disease activity score (DAS) and 108 healthy age-matched controls. Aggrecan levels were measured using two immunoassays, i.e. the ³⁷⁴ARGSVI-G2 sandwich ELISA measuring aggrecanase-mediated aggrecan degradation and the G1/G2 sandwich assay, detecting aggrecan molecules containing G1 and/or G2 (total aggrecan) We further characterized serum samples by western blots, by using monoclonal antibodies F-78, binding to G1 and G2, or by BC-3, detecting the aggrecanase-generated N-terminal ³⁷⁴ARGSVI neo-epitope.</p> <p>Results</p> <p>Total aggrecan levels in RA patients were significantly decreased from 824.8 ± 31 ng/ml in healthy controls to 570.5 ± 30 ng/ml (31% decrease, P < 0.0001), as measured by the G1/G2 ELISA. Western blot analysis with F-78 showed one strong band at 10 kDa, and weaker bands at 25 and 45 kDa in both healthy controls and RA patients. In contrast, staining for aggrecanase-activity revealed only one strong band in RA patients of 45 kDa.</p> <p>Conclusion</p> <p>This is the first study, which characterizes different aggrecan fragments in human serum. The data strongly suggests that total aggrecan levels, i.e. aggrecan molecules containing G1 and/or G2 are lower in RA patients, and that RA patients have at least one specific subpopulation of aggrecan fragments, namely aggrecanse generated ³⁷⁴ARGSVI fragments. Further clinical studies are needed to investigate the potential of G1/G2 as a structure-related biochemical marker in destructive joint-diseases.</p

Analysis of mass spectrometry data from the secretome of an explant model of articular cartilage exposed to pro-inflammatory and anti-inflammatory stimuli using machine learning

Background: Osteoarthritis (OA) is an inflammatory disease of synovial joints involving the loss and degeneration of articular cartilage. The gold standard for evaluating cartilage loss in OA is the measurement of joint space width on standard radiographs. However, in most cases the diagnosis is made well after the onset of the disease, when the symptoms are well established. Identification of early biomarkers of OA can facilitate earlier diagnosis, improve disease monitoring and predict responses to therapeutic interventions. Methods: This study describes the bioinformatic analysis of data generated from high throughput proteomics for identification of potential biomarkers of OA. The mass spectrometry data was generated using a canine explant model of articular cartilage treated with the pro-inflammatory cytokine interleukin 1 β (IL-1β). The bioinformatics analysis involved the application of machine learning and network analysis to the proteomic mass spectrometry data. A rule based machine learning technique, BioHEL, was used to create a model that classified the samples into their relevant treatment groups by identifying those proteins that separated samples into their respective groups. The proteins identified were considered to be potential biomarkers. Protein networks were also generated; from these networks, proteins pivotal to the classification were identified. Results: BioHEL correctly classified eighteen out of twenty-three samples, giving a classification accuracy of 78.3% for the dataset. The dataset included the four classes of control, IL-1β, carprofen, and IL-1β and carprofen together. This exceeded the other machine learners that were used for a comparison, on the same dataset, with the exception of another rule-based method, JRip, which performed equally well. The proteins that were most frequently used in rules generated by BioHEL were found to include a number of relevant proteins including matrix metalloproteinase 3, interleukin 8 and matrix gla protein. Conclusions: Using this protocol, combining an in vitro model of OA with bioinformatics analysis, a number of relevant extracellular matrix proteins were identified, thereby supporting the application of these bioinformatics tools for analysis of proteomic data from in vitro models of cartilage degradation

Transcriptional Response of Zebrafish Embryos Exposed to Neurotoxic Compounds Reveals a Muscle Activity Dependent hspb11 Expression

Acetylcholinesterase (AChE) inhibitors are widely used as pesticides and drugs. Their primary effect is the overstimulation of cholinergic receptors which results in an improper muscular function. During vertebrate embryonic development nerve activity and intracellular downstream events are critical for the regulation of muscle fiber formation. Whether AChE inhibitors and related neurotoxic compounds also provoke specific changes in gene transcription patterns during vertebrate development that allow them to establish a mechanistic link useful for identification of developmental toxicity pathways has, however, yet not been investigated. Therefore we examined the transcriptomic response of a known AChE inhibitor, the organophosphate azinphos-methyl (APM), in zebrafish embryos and compared the response with two non-AChE inhibiting unspecific control compounds, 1,4-dimethoxybenzene (DMB) and 2,4-dinitrophenol (DNP). A highly specific cluster of APM induced gene transcripts was identified and a subset of strongly regulated genes was analyzed in more detail. The small heat shock protein hspb11 was found to be the most sensitive induced gene in response to AChE inhibitors. Comparison of expression in wildtype, ache and sopfixe mutant embryos revealed that hspb11 expression was dependent on the nicotinic acetylcholine receptor (nAChR) activity. Furthermore, modulators of intracellular calcium levels within the whole embryo led to a transcriptional up-regulation of hspb11 which suggests that elevated intracellular calcium levels may regulate the expression of this gene. During early zebrafish development, hspb11 was specifically expressed in muscle pioneer cells and Hspb11 morpholino-knockdown resulted in effects on slow muscle myosin organization. Our findings imply that a comparative toxicogenomic approach and functional analysis can lead to the identification of molecular mechanisms and specific marker genes for potential neurotoxic compounds

Selective Regulation of NR2B by Protein Phosphatase-1 for the Control of the NMDA Receptor in Neuroprotection

An imbalance between pro-survival and pro-death pathways in brain cells can lead to neuronal cell death and neurodegeneration. While such imbalance is known to be associated with alterations in glutamatergic and Ca2+ signaling, the underlying mechanisms remain undefined. We identified the protein Ser/Thr phosphatase protein phosphatase-1 (PP1), an enzyme associated with glutamate receptors, as a key trigger of survival pathways that can prevent neuronal death and neurodegeneration in the adult hippocampus. We show that PP1α overexpression in hippocampal neurons limits NMDA receptor overactivation and Ca2+ overload during an excitotoxic event, while PP1 inhibition favors Ca2+ overload and cell death. The protective effect of PP1 is associated with a selective dephosphorylation on a residue phosphorylated by CaMKIIα on the NMDA receptor subunit NR2B, which promotes pro-survival pathways and associated transcriptional programs. These results reveal a novel contributor to the mechanisms of neuroprotection and underscore the importance of PP1-dependent dephosphorylation in these mechanisms. They provide a new target for the development of potential therapeutic treatment of neurodegeneration

New technologies for examining neuronal ensembles in drug addiction and fear

Correlational data suggest that learned associations are encoded within neuronal ensembles. However, it has been difficult to prove that neuronal ensembles mediate learned behaviours because traditional pharmacological and lesion methods, and even newer cell type-specific methods, affect both activated and non-activated neurons. Additionally, previous studies on synaptic and molecular alterations induced by learning did not distinguish between behaviourally activated and non-activated neurons. Here, we describe three new approaches—Daun02 inactivation, FACS sorting of activated neurons and c-fos-GFP transgenic rats — that have been used to selectively target and study activated neuronal ensembles in models of conditioned drug effects and relapse. We also describe two new tools — c-fos-tTA mice and inactivation of CREB-overexpressing neurons — that have been used to study the role of neuronal ensembles in conditioned fear