P.E. Slagboom

Knee and hip articular cartilage have distinct epigenomic landscapes: implications for future cartilage regeneration approaches

Objectives To elucidate the functional epigenomic landscape of articular cartilage in osteoarthritis (OA) affected knee and hip joints in relation to gene expression. Methods Using Illumina Infinium HumanMethylation450 BeadChip arrays, genome-wide DNA methylation was measured in 31 preserved and lesioned cartilage sample pairs (14 knees and 17 hips) from patients who underwent a total joint replacement due to primary OA. Using previously published genome-wide expression data of 33 pairs of cartilage samples, of which 13 pairs were overlapping with the current methylation dataset, we assessed gene expression differences in differentially methylated regions (DMRs). Results Principal component analysis of the methylation data revealed distinct clustering of knee and hip samples, irrespective of OA pathophysiology. A total of 6272 CpG dinucleotides were differentially methylated between the two joints, comprising a total of 357 DMRs containing 1817 CpGs and 245 unique genes. Enrichment analysis of genes proximal of the DMRs revealed significant enrichment for developmental pathways and homeobox (HOX) genes. Subsequent transcriptomic analysis of DMR genes exposed distinct knee and hip expression patterns. Conclusions Our findings reveal consistent DMRs between knee and hip articular cartilage that marked transcriptomic differences among HOX genes, which were not reflecting the temporal sequential HOX expression pattern during development. This implies distinct mechanisms for maintaining cartilage integrity in adulthood, thereby contributing to our understanding of cartilage homeostasis and future tissue regeneration approaches.

The role of plasma cytokine levels, CRP and Selenoprotein S gene variation in OA

OBJECTIVEnInvestigating the association between plasma levels of cytokines and chemokines, Selenoprotein S (SELS) gene variation and osteoarthritis (OA) subtypes.nnnMETHODSnThe genetics of osteoarthritis and progression (GARP) study consists of 191 sibling pairs with symptomatic OA at multiple joint sites. We have measured plasma levels of 17 cytokines and chemokines and genetic variation at the SELS gene.nnnRESULTSnNine out of 17 serum markers could be assessed quantitatively, whereas eight markers were assessed qualitatively. Principal component analysis (PCA) on the quantitatively assessed markers and serum high sensitive C-reactive protein (S-HsCRP) revealed that three components underlie 61% of the total plasma variation. Three single nucleotide polymorphisms (SNPs) in the SELS gene revealed four common haplotypes, one of which, GAG (frequency 3.5%) showed significant association to an anti-inflammatory (P=0.019) and acute phase related (P=0.036) component. OA subtype analysis showed that one component (mainly representing chemokine variation) was significantly associated to hand OA and disc degeneration (P=0.029 and P=0.010 respectively) as well as a physical component score (PCS) (P=0.042). The CRP related component also showed a strong association to the PCS (P=0.007). SELS haplotypes showed no association to OA subtypes in the GARP study.nnnCONCLUSIONnGenetic variation in the SELS gene associates to components representing inflammatory signaling. Another component, representing chemokine variation, showed association to hand OA and disc degeneration in the GARP study indicating chemokines may contribute to OA pathogenesis.

Neo-cartilage engineered from primary chondrocytes is epigenetically similar to autologous cartilage, in contrast to using mesenchymal stem cells

OBJECTIVESnTo compare the epigenetic landscape of 3D cell models of human primary articular chondrocytes (hPACs) and human bone-marrow derived mesenchymal stem cells (hBMSCs) and their respective autologous articular cartilage.nnnDESIGNnUsing Illumina Infinium HumanMethylation450 BeadChip arrays, the DNA methylation landscape of the different cell sources and autologous cartilage was determined. Pathway enrichment was analyzed using DAVID.nnnRESULTSnPrincipal Component Analysis (PCA) of methylation data revealed separate clustering of hBMSC samples. Between hBMSCs and autologous cartilage 86,881 cytosine-phosphate-guanine dinucleotides (CpGs) (20.2%), comprising 3,034 differentially methylated regions (DMRs; Δβxa0>xa00.1; with the same direction of effect), were significantly differentially methylated. In contrast, between hPACs and autologous cartilage only 5,706 CpGs (1.33%) were differentially methylated. Of interest was the finding of the transcriptionally active, hyper-methylation of a Cartilage Intermediate Layer Protein (CILP) annotated DMR (Δβxa0=xa00.16) in PAC-cartilage, corresponding to a profound decrease in CILP expression after inxa0vitro culturing of hPACs as compared to autologous cartilage.nnnCONCLUSIONSnInxa0vitro engineered neo-cartilage tissue from primary chondrocytes, hPACs, exhibits a DNA methylation landscape that is almost identical (99% similarity) to autologous cartilage, in contrast to neo-cartilage engineered from bone marrow-derived mesenchymal stem cells (MSCs). Although hBMSCs are widely used for cartilage engineering purposes the effects of these vast differences on cartilage regeneration and long term consequences of implantation, are not known. The use of hBMSCs or hPACs for future cartilage tissue regeneration purposes should therefore be investigated in more depth in future endeavors to better understand the consequences of the differential methylome on neo-cartilage.