molecules by a cell-based high-throughput screening (HTS) in human chondrocytes. Methods: To induce cellular senescence, immortalized human chondrocytes (TC28a2) were seeded (5000 cells/well) in 384 well plates, and treated with IL-6 (10 ng/ml) for 24 hours to induce cellular senescence or defective autophagy. Then, chondrocytes were incubated with Prestwick Chemical Library (1280 approved drugs with chemical and pharmacological diversity, as well as bioavailability and safety in humans) at 10 mM for 48 hours. Chondrocytes were incubated for 20 min with 4% formaldehyde and washed 3 times with PBS. Then, nuclei was stained with Hoechst 33342 (10 mM), while b-galactosidase subcellular structures and autophagy vacuoles were stained by using Imagene Green C12FDG substrate (10 mM) and Cyto-ID® (1 mM), respectively. Plates were imaged by using Operetta® High Content Screening (HCS) system in non-confocal mode using the 20x WD objective. For each well, 4 fields and 4 planes of bright field, Hoechst and fluorescein channels were obtained. Image analysis was performed by Harmony software. Relative intensity of C12FDG in cytoplasm and number of autophagosomes per area of cytoplasm were determined to quantitate b-galactosidase activity and autophagy flux respectively. Low signal of senescence was found for chloroquine (30 mM) and used as reference for anti-senescence activity. High signal of autophagy flux was found for Rapamycin (5 mM) and used as reference for proautophagy activity. Results: A primary screening was performed to identify anti-senescence compounds by measurement of senescence-associated bgalactosidase activity. From the total number of 1280 small molecules, 299 compounds with anti-senescence effects were identified by HTS. A secondary screening with the 299 compounds was performed for "cherry picking". Finally, 216 compounds were confirmed to have antisenescence activity. The anti-senescence compounds were analyzed by monitoring autophagy flux. 38 compounds with both anti-senescence and pro-autophagy effects were selected. For compound validation, a reporter cell line was generated by lentiviral transfection of pBABE-mCherry-EGFP-LC3 plasmid in TC28a2 chondrocytes. LC3 reporter chondrocytes can be used to determine autophagy activation by color changes upon stimulation. The preliminary results indicate a subset of compounds selectively induced LC3-associated autophagy activation. Conclusions: These observations provide a unique opportunity to study cartilage aging with the objective to explore the therapeutic potential of pharmacological prevention of chondrocyte senescence and autophagy as a strategy to slow or reverse aging-associated changes, prevent the onset of OA and provide benefits for its clinical management.
intermediate (inner lateral tibial plateaus, iLT) and late stage (inner medial plateaus, iMT) of OA in Japanese. Methods: Genome-wide DNA methylation analysis was performed using Illumina Infinium HumanMethylation450 BeadChip array on DNA extracted from the cartilage of oLT, iLT and iMT region of 8 patients. Raw IDAT files were analyzed using package "minfi" in R. Data was normalized through SWAN (subquantile normalization) method. Multidimensional scaling (MDS) was used to identify the outliers. Probes that locate on X/Y chromosomes were removed before applying F test to find out the differential methylated sites of iLT vs oLT and iMT vs oLT. Multidimensional scaling of the methylation beta values revealed significantclusters related to cartilage. F test with p value less than 0.05 and jDbj greater than 0.15 were used to identify the differential methylated sites. Genes with differentially methylated CpG sites were analyzed to identify gene ontologies, pathways, and upstream regulators. Results: We identified 310 differentially methylated sites (DMS) covering 147 genes in comparison of iMT vs. oLT region. Of these, 122(39%) DMS were hypermethylated and 188 (61%) were hypomethylated. However, in the comparison of iLT vs. oLT region, we only found 22 differential methylated sites (12 hypermethylated and 10 hypomethylated). The dramatic difference in DMS numbers of iLT and iMT suggests that methylation is highly involved only at the very late stage of OA. The DMS we found in iMT cartilage include genes reported related to OA, for instance, BMP6, COL14A1, NFATC1, and SPOCK1. Enrichment analysis of the genes with DMS revealed significant enrichment of HOX family genes and development pathways.Conclusions: In the current study, we have identified the methylation changes in both the iLT region and iMT region, which represent the intermediat and late stage of OA disease. Our data suggested that the significant changes in methylation occurred at during the late stages of OA. The development pathways and HOX family genes enriched at the late stage of OA implicated new aspect of cartilage maintenance in adulthood. The further study of the underlying mechanism may supply a direction for future cartilage regeneration approaches.
Sporadic Inclusion Body Myositis (IBM) remains without proven treatment, reflecting a previous lack of preclinical disease models on which to evaluate potential therapies. We developed a panel of pathological outcome measures, using primary satellite cell cultures, that reflects the multifaceted pathogenesis of IBM. This allowed initial assessment of Heat Shock Response manipulation as a new therapeutic strategy for IBM. Over-expression of β-Amyloid Precursor Protein or exposure to inflammatory mediators IL1β, TNFα and IFNγ reproduced salient features of the cellular environment found in IBM. Myotubes demonstrated ubiquitinated intracellular inclusions, increased expression of MHC Class I, mitochondrial dysfunction and cytoplasmic translocation of TDP-43. ER stress and activation of the NFκB cascade, proposed to be central pathogenic mechanisms in IBM, were also observed. We developed quantifiable measures of these changes, allowing the impact of drugs to be tested. The effects of heat shock response augmentation using Arimoclomol, a co-inducer of the transcription factor HSF-1 that drives expression of cytoprotective endogenous Heat Shock Proteins, were examined on this model. Arimoclomol treatment ameliorated several IBM-relevant features, represented by reduced inclusion formation, reduced ER stress, inhibition of the NFκB cascade, reduced cytoplasmic translocation of TDP-43 and improved mitochondrial function. We have advanced Arimoclomol into a Phase IIa study in IBM patients.
expression (MMP3, MMP13, ADAMTS-4, ADAMTS-5) compared to controls. We found that the expression of S1P receptors 1À3 is increased in chondrocytes cultured with Oc-CM, as well as the activation of their signaling pathway (MAPK). However, only the inhibition of the receptor S1PR2 by JTE-013 and RNA silencing abolished Oc-CM effect on MMP-3 and -13 in primary chondrocytes, but not those of S1PR1 and 3. This inhibition was confirmed in femoral head explants as JTE-013 reduced loss of proteoglycan and extracellular matrix degradation initially induced by Oc-CM. The specificity of Oc-CM was assessed using interleukin 1 beta know to induce MMPs expression. To demonstrate the involvement of S1P signaling in OA development in mice, we tested the effects of systemic and intra-articular injection of JTE-013. OA score was reduced after the local administration of JTE-013 (6.25 ± 0.63 vs 4.14 ± 0.26, p<0.05). Conclusions: These data demonstrate that osteoclast-secreted factors disrupt the balance of chondrocyte metabolism. The activation of S1P signaling in chondrocytes by osteoclasts promotes chondrocyte catabolism. Therefore, subchondral bone manipulations may affect chondrocyte function and OA.
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