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
The aim of this thesis was to combine transcriptomics, genetics and human disease modelling to obtain further insight into molecular processes underlying osteoarthritis. More specifically, we aimed... Show moreThe aim of this thesis was to combine transcriptomics, genetics and human disease modelling to obtain further insight into molecular processes underlying osteoarthritis. More specifically, we aimed to elucidate the role of long noncoding RNAs expression changes as aberrant epigenetic mechanism in regulating gene expression in chondrocytes. We identified previously unknown long noncoding RNAs associated with the osteoarthritic process and showed enrichment for cis¬-regulation of these long noncoding RNAs with target messenger RNAs.To provide insight in the etiology of osteoarthritis, causal pathways can be identified by unravelling the substantial genetic component. To this end, we investigated the biological functionality of the high-impact, pathogenic mutation identified in the gene fibronectin1 in an early-onset osteoarthritis family. We demonstrated that the identified causal missense mutation in the gelatin-binding domain of the extracellular matrix protein fibronectin resulted in significant decreased binding capacity to collagen type II.Finally, the common function of fibronectin1 was investigated in cartilage and what changes occur at the transcript level of fibronectin1 with osteoarthritis. Down-regulation of full-length fibronectin was unbeneficial for in vitro chondrogenesis, we hypothesize that this was caused by decreased availability of the classical integrin binding site of fibronectin. Show less
Houtman, E.; Hoolwerff, M. van; Lakenberg, N.; Suchiman, E.H.D.; Zwaag, E.V.V. van der van der; Nelissen, R.G.H.H.; ... ; Meulenbelt, I. 2021
Introduction: Likely due to ignored heterogeneity in disease pathophysiology, osteoarthritis (OA) has become the most common disabling joint disease, without effective disease-modifying treatment... Show moreIntroduction: Likely due to ignored heterogeneity in disease pathophysiology, osteoarthritis (OA) has become the most common disabling joint disease, without effective disease-modifying treatment causing a large social and economic burden. In this study we set out to explore responses of aged human osteochondral explants upon different OA-related perturbing triggers (inflammation, hypertrophy and mechanical stress) for future tailored biomimetic human models.Methods: Human osteochondral explants were treated with IL-1 beta (10 ng/ml) or triiodothyronine (T3; 10 nM) or received 65% strains of mechanical stress (65% MS). Changes in chondrocyte signalling were determined by expression levels of nine genes involved in catabolism, anabolism and hypertrophy. Breakdown of cartilage was measured by sulphated glycosaminoglycans (sGAGs) release, scoring histological changes (Mankin score) and mechanical properties of cartilage.Results: All three perturbations (IL-1 beta, T3 and 65% MS) resulted in upregulation of the catabolic genes MMP13 and EPAS1. IL-1 beta abolished COL2A1 and ACAN gene expression and increased cartilage degeneration, reflected by increased Mankin scores and sGAGs released. Treatment with T3 resulted in a high and significant upregulation of the hypertrophic markers COL1A1, COL10A1 and ALPL. However, 65% MS increased sGAG release and detrimentally altered mechanical properties of cartilage.Conclusion: We present consistent and specific output on three different triggers of OA. Perturbation with the pro-inflammatory IL-1 beta mainly induced catabolic chondrocyte signalling and cartilage breakdown, while T3 initiated expression of hypertrophic and mineralization markers. Mechanical stress at a strain of 65% induced catabolic chondrocyte signalling and changed cartilage matrix integrity. The major strength of our ex vivo models was that they considered aged, preserved, human cartilage of a heterogeneous OA patient population. As a result, the explants may reflect a reliable biomimetic model prone to OA onset allowing for development of different treatment modalities. Show less
