Heterogeneous accumulation of senescent cells expressing the senescence-associated secretory phenotype (SASP) affects tissue homeostasis which leads to diseases, such as osteoarthritis (OA). In... Show moreHeterogeneous accumulation of senescent cells expressing the senescence-associated secretory phenotype (SASP) affects tissue homeostasis which leads to diseases, such as osteoarthritis (OA). In this study, we set out to characterize heterogeneity of cellular senescence within aged articular cartilage and explored the presence of corresponding metabolic profiles in blood that could function as representative biomarkers. Hereto, we set out to perform cluster analyses, using a gene-set of 131 senescence genes (N = 57) in a previously established RNA sequencing dataset of aged articular cartilage and a generated metabolic dataset in overlapping blood samples. Using unsupervised hierarchical clustering and pathway analysis, we identified two robust cellular senescent endotypes. Endotype-1 was enriched for cell proliferating pathways, expressing forkhead box protein O4 (FOXO4), RB transcriptional corepressor like 2 (RBL2), and cyclin-dependent kinase inhibitor 1B (CDKN1B); the FOXO mediated cell cycle was identified as possible target for endotype-1 patients. Endotype-2 showed enriched inflammation-associated pathways, expressed by interleukin 6 (IL6), matrix metallopeptidase (MMP)1/3, and vascular endothelial growth factor (VEGF)C and SASP pathways were identified as possible targets for endotype-2 patients. Notably, plasma-based metabolic profiles in overlapping blood samples (N = 21) showed two corresponding metabolic clusters in blood. These non-invasive metabolic profiles could function as biomarkers for patient-tailored targeting of senescence in OA. Show less
ObjectivesIn osteoarthritis, methylation of lysine 79 on histone H3 (H3K79me), a protective epigenetic mechanism, is reduced. Histone methylation levels are dynamically regulated by histone... Show moreObjectivesIn osteoarthritis, methylation of lysine 79 on histone H3 (H3K79me), a protective epigenetic mechanism, is reduced. Histone methylation levels are dynamically regulated by histone methyltransferases and demethylases. Here, we aimed to identify which histone demethylases regulate H3K79me in cartilage and investigate whether their targeting protects against osteoarthritis. MethodsWe determined histone demethylase expression in human non-osteoarthritis and osteoarthritis cartilage using qPCR. The role of histone demethylase families and subfamilies on H3K79me was interrogated by treatment of human C28/I2 chondrocytes with pharmacological inhibitors, followed by western blot and immunofluorescence. We performed C28/I2 micromasses to evaluate effects on glycosaminoglycans by Alcian blue staining. Changes in H3K79me after destabilisation of the medial meniscus (DMM) in mice were determined by immunohistochemistry. Daminozide, a KDM2/7 subfamily inhibitor, was intra-articularly injected in mice upon DMM. Histone demethylases targeted by daminozide were individually silenced in chondrocytes to dissect their role on H3K79me and osteoarthritis. ResultsWe documented the expression signature of histone demethylases in human non-osteoarthritis and osteoarthritis articular cartilage. Inhibition of Jumonji-C demethylase family increased H3K79me in human chondrocytes. Blockade of KDM2/7 histone demethylases with daminozide increased H3K79me and glycosaminoglycans. In mouse articular cartilage, H3K79me decayed rapidly upon induction of joint injury. Early and sustained intra-articular treatment with daminozide enhanced H3K79me and exerted protective effects in mice upon DMM. Individual silencing of KDM7A/B demethylases in human chondrocytes demonstrated that KDM7A/B mediate protective effects of daminozide on H3K79me and osteoarthritis. ConclusionTargeting KDM7A/B histone demethylases could be an attractive strategy to protect joints against osteoarthritis. Show less
Tuerlings, M.; Janssen, G.M.C.; Boone, I.; Hoolwerff, M. van; Ruiz, A.R.; Houtman, E.; ... ; Meulenbelt, I. 2023
Objective: To explore the co-expression network of the osteoarthritis (OA) risk gene WWP2 in articular cartilage and study cartilage characteristics when mimicking the effect of OA risk allele... Show moreObjective: To explore the co-expression network of the osteoarthritis (OA) risk gene WWP2 in articular cartilage and study cartilage characteristics when mimicking the effect of OA risk allele rs1052429-A on WWP2 expression in a human 3D in vitro model of cartilage. Method: Co-expression behavior of WWP2 with genes expressed in lesioned OA articular cartilage (N = 35 samples) was explored. By applying lentiviral particle mediated WWP2 upregulation in 3D in vitro pellet cultures of human primary chondrocytes (N = 8 donors) the effects of upregulation on cartilage matrix deposition was evaluated. Finally, we transfected primary chondrocytes with miR-140 mimics to evaluate whether miR-140 and WWP2 are involved in similar pathways. Results: Upon performing Spearman correlations in lesioned OA cartilage, 98 highly correlating genes (| r| > 0.7) were identified. Among these genes, we identified GJA1, GDF10, STC2, WDR1, and WNK4. Sub-sequent upregulation of WWP2 on 3D chondrocyte pellet cultures resulted in a decreased expression of COL2A1 and ACAN and an increase in EPAS1 expression. Additionally, we observed a decreased expression of GDF10, STC2, and GJA1. Proteomics analysis identified 42 proteins being differentially expressed with WWP2 upregulation, which were enriched for ubiquitin conjugating enzyme activity. Finally, upregu-lation of miR-140 in 2D chondrocytes resulted in significant upregulation of WWP2 and WDR1. Conclusions: Mimicking the effect of OA risk allele rs1052429-A on WWP2 expression initiates detri-mental processes in the cartilage shown by a response in hypoxia associated genes EPAS1, GDF10, and GJA1 and a decrease in anabolic markers, COL2A1 and ACAN.(c) 2022 The Author(s). Published by Elsevier Ltd on behalf of Osteoarthritis Research Society International. This is an open access article under the CC BY license (http://creativecommons.org/ licenses/by/4.0/). Show less
Houtman, E.; Tuerlings, M.; Riechelman, J.; Suchiman, E.H.E.D.; Wal, R.J.P. van der; Nelissen, R.G.H.H.; ... ; Meulenbelt, I. 2021
Background Failing of intrinsic chondrocyte repair after mechanical stress is known as one of the most important initiators of osteoarthritis. Nonetheless, insight into these early mechano... Show moreBackground Failing of intrinsic chondrocyte repair after mechanical stress is known as one of the most important initiators of osteoarthritis. Nonetheless, insight into these early mechano-pathophysiological processes in age-related human articular cartilage is still lacking. Such insights are needed to advance clinical development. To highlight important molecular processes of osteoarthritis mechano-pathology, the transcriptome-wide changes following injurious mechanical stress on human aged osteochondral explants were characterized. Methods Following mechanical stress at a strain of 65% (65%MS) on human osteochondral explants (n(65%MS) = 14 versus n(control) = 14), RNA sequencing was performed. Differential expression analysis between control and 65%MS was performed to determine mechanical stress-specific changes. Enrichment for pathways and protein-protein interactions was analyzed with Enrichr and STRING. Results We identified 156 genes significantly differentially expressed between control and 65%MS human osteochondral explants. Of note, IGFBP5 (FC = 6.01; FDR = 7.81 x 10(-3)) and MMP13 (FC = 5.19; FDR = 4.84 x 10(-2)) were the highest upregulated genes, while IGFBP6 (FC = 0.19; FDR = 3.07 x 10(-4)) was the most downregulated gene. Protein-protein interactions were significantly higher than expected by chance (P = 1.44 x 10(-15) with connections between 116 out of 156 genes). Pathway analysis showed, among others, enrichment for cellular senescence, insulin-like growth factor (IGF) I and II binding, and focal adhesion. Conclusions Our results faithfully represent transcriptomic wide consequences of mechanical stress in human aged articular cartilage with MMP13, IGF binding proteins, and cellular senescence as the most notable results. Acquired knowledge on the as such identified initial, osteoarthritis-related, detrimental responses of chondrocytes may eventually contribute to the development of effective disease-modifying osteoarthritis treatments. Show less
Houtman, E.; Almeida, R.C. de; Tuerlings, M.; Suchiman, H.E.D.; Broekhuis, D.; Nelissen, R.G.H.H.; ... ; Meulenbelt, I. 2021
Objective: We here aimed to characterize changes of Matrix Gla Protein (MGP) expression in relation to its recently identified OA risk allele rs1800801-T in OA cartilage, subchondral bone and human... Show moreObjective: We here aimed to characterize changes of Matrix Gla Protein (MGP) expression in relation to its recently identified OA risk allele rs1800801-T in OA cartilage, subchondral bone and human ex vivo osteochondral explants subjected to OA related stimuli. Given that MGP function depends on vitamin K bioavailability, we studied the effect of frequently prescribed vitamin K antagonist warfarin. Methods: Differential (allelic) mRNA expression of MGP was analyzed using RNA-sequencing data of human OA cartilage and subchondral bone. Human osteochondral explants were used to study exposures to interleukin one beta (IL-1b; inflammation), triiodothyronine (T3; Hypertrophy), warfarin, or 65% mechanical stress (65%MS) as function of rs1800801 genotypes. Results: We confirmed that the MGP risk allele rs1800801-T was associated with lower expression and that MGP was significantly upregulated in lesioned as compared to preserved OA tissues, mainly in risk allele carriers, in both cartilage and subchondral bone. Moreover, MGP expression was downregulated in response to OA like triggers in cartilage and subchondral bone and this effect might be reduced in carriers of the rs1800801-T risk allele. Finally, warfarin treatment in cartilage increased COL10A1 and reduced SOX9 and MMP3 expression and in subchondral bone reduced COL1A1 and POSTN expression. Discussion & conclusions: Our data highlights that the genetic risk allele lowers MGP expression and upon OA relevant triggers may hamper adequate dynamic changes in MGP expression, mainly in carti-lage. The determined direct negative effect of warfarin on human explant cultures functionally un-derscores the previously found association between vitamin K deficiency and OA. (c) 2021 The Authors. Published by Elsevier Ltd on behalf of Osteoarthritis Research Society International. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Show less
