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.
metalloproteinase-13 (MMP-13) level and collagen cleavage. In addition the ratio of the TGFb type 1 receptors, ALK1 and ALK5, increases with age. However, there was considerable cell-cell variability in the measured outcomes. The model shows that the main source of this cellular heterogeneity is due to the stochastic nature of cellular damage, as most variability in the model output was seen in the levels of oxidised proteins and AGE products. There was also considerable variability in the time at which damage starts to accrue, and this accounts for the wide variability in the activation of matrix degrading enzymes. The model showed that negative feedback loops in the system are the main contributor to the age-related increase in the ALK1/ALK5 receptor and this also contributes to the age-related increase in MMP-13 and collagen degradation. Conclusions: Age-related changes in cartilage are caused by complex molecular mechanisms responding to different stress signals. There is both a gradual increase in damaged components and a dysregulation in signalling responses with age. Computer modelling is a complementary approach to assist with unravelling this complexity.
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