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
Ruiz, A.R.; Hoolwerff, M. van; Sprangers, S.; Suchiman, E.; Schoenmaker, T.; Dibbets-Schneider, P.; ... ; Meulenbelt, I. 2022
Objectives: To study the mechanism by which the readthrough mutation in TNFRSF11B, encoding osteoprotegerin (OPG) with additional 19 amino acids at its C-terminus (OPG-XL), causes the... Show moreObjectives: To study the mechanism by which the readthrough mutation in TNFRSF11B, encoding osteoprotegerin (OPG) with additional 19 amino acids at its C-terminus (OPG-XL), causes the characteristic bidirectional phenotype of subchondral bone turnover accompanied by cartilage mineralization in chondrocalcinosis patients. Methods: OPG-XL was studied by human induced pluripotent stem cells expressing OPG-XL and two isogenic CRISPR/Cas9-corrected controls in cartilage and bone organoids. Osteoclastogenesis was studied with monocytes from OPG-XL carriers and matched healthy controls followed by gene expression characterization. Dual energy X-ray absorptiometry scans and MRI analyses were used to characterize the phenotype of carriers and non-carriers of the mutation. Results: Human OPG-XL carriers relative to sex- and age-matched controls showed, after an initial delay, large active osteoclasts with high number of nuclei. By employing hiPSCs expressing OPG-XL and isogenic CRISPR/Cas9-corrected controls to established cartilage and bone organoids, we demonstrated that expression of OPG-XL resulted in excessive fibrosis in cartilage and high mineralization in bone accompanied by marked downregulation of MGP, encoding matrix Gla protein, and upregulation of DIO2, encoding type 2 deiodinase, gene expression, respectively. Conclusions: The readthrough mutation at CCAL1 locus in TNFRSF11B identifies an unknown role for OPG-XL in subchondral bone turnover and cartilage mineralization in humans via DIO2 and MGP functions. Previously, OPG-XL was shown to affect binding between RANKL and heparan sulphate (HS) resulting in loss of immobilized OPG-XL. Therefore, effects may be triggered by deficiency in the immobilization of OPG-XL Since the characteristic bidirectional pathophysiology of articular cartilage calcification accompanied by low subchondral bone mineralization is also a hallmark of OA pathophysiology, our results are likely extrapolated to common arthropathies. Show less
Ruiz, A.R.; Tuerlings, M.; A. das; Almeida, R.C. de; Suchiman, H.E.D.; Nelissen, R.G.H.H.; ... ; Meulenbelt, I. 2022
Objectives OA is a complex genetic disease with different risk factors contributing to its development. One of the genes, TNFRSF11B, previously identified with gain-of-function mutation in a family... Show moreObjectives OA is a complex genetic disease with different risk factors contributing to its development. One of the genes, TNFRSF11B, previously identified with gain-of-function mutation in a family with early-onset OA with chondrocalcinosis, is among the highest upregulated genes in lesioned OA cartilage (RAAK-study). Here, we determined the role of TNFRSF11B overexpression in development of OA. Methods Human primary articular chondrocytes (9 donors RAAK study) were transduced using lentiviral particles with or without TNFRSF11B. Cells were cultured for 1 week in a 3 D in-vitro chondrogenic model. TNFRSF11B overexpression was confirmed by RT-qPCR, immunohistochemistry and ELISA. Effects of TNFRSF11B overexpression on cartilage matrix deposition, matrix mineralization, and genes highly correlated to TNFRSF11B in RNA-sequencing dataset (r >0.75) were determined by RT-qPCR. Additionally, glycosaminoglycans and collagen deposition were visualized with Alcian blue staining and immunohistochemistry (COL1 and COL2). Results Overexpression of TNFRSF11B resulted in strong upregulation of MMP13, COL2A1 and COL1A1. Likewise, mineralization and osteoblast characteristic markers RUNX2, ASPN and OGN showed a consistent increase. Among 30 genes highly correlated to TNFRSF11B, expression of only eight changed significantly, with BMP6 showing the highest increase (9-fold) while expression of RANK and RANKL remained unchanged indicating previously unknown downstream pathways of TNFRSF11B in cartilage. Conclusion Results of our 3D in vitro chondrogenesis model indicate that upregulation of TNFRSF11B in lesioned OA cartilage may act as a direct driving factor for chondrocyte to osteoblast transition observed in OA pathophysiology. This transition does not appear to act via the OPG/RANK/RANKL triad common in bone remodeling. Show less
Hoolwerff, M. van; Ruiz, A.R.; Bouma, M.; Suchiman, H.E.D.; Koning, R.I.; Jost, C.R.; ... ; Meulenbelt, I. 2021
Osteoarthritis is the most prevalent joint disease worldwide, yet progress in development of effective disease-modifying treatments is slow because of lack of insight into the underlying disease... Show moreOsteoarthritis is the most prevalent joint disease worldwide, yet progress in development of effective disease-modifying treatments is slow because of lack of insight into the underlying disease pathways. Therefore, we aimed to identify the causal pathogenic mutation in an early-onset osteoarthritis family, followed by functional studies in human induced pluripotent stem cells (hiPSCs) in an in vitro organoid cartilage model. 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. Further analyses of formed hiPSC-derived neo-cartilage tissue highlighted that mutated fibronectin affected chondrogenic capacity and propensity to a procatabolic osteoarthritic state. Together, we demonstrate that binding of fibronectin to collagen type II is crucial for fibronectin downstream gene expression of chondrocytes. We advocate that effective treatment development should focus on restoring or maintaining proper binding between fibronectin and collagen type II. Show less
Ruiz, A.R.; Dicks, A.; Tuerlings, M.; Schepers, K.; Pel, M. van; Nelissen, R.G.H.H.; ... ; Ramos, Y.F.M. 2021
Cartilage has little intrinsic capacity for repair, so transplantation of exogenous cartilage cells is considered a realistic option for cartilage regeneration. We explored whether human-induced... Show moreCartilage has little intrinsic capacity for repair, so transplantation of exogenous cartilage cells is considered a realistic option for cartilage regeneration. We explored whether human-induced pluripotent stem cells (hiPSCs) could represent such unlimited cell sources for neo-cartilage comparable to human primary articular chondrocytes (hPACs) or human bone marrow-derived mesenchymal stromal cells (hBMSCs). For this, chondroprogenitor cells (hiCPCs) and hiPSC-derived mesenchymal stromal cells (hiMSCs) were generated from two independent hiPSC lines and characterized by morphology, flow cytometry, and differentiation potential. Chondrogenesis was compared to hBMSCs and hPACs by histology, immunohistochemistry, and RT-qPCR, while similarities were estimated based on Pearson correlations using a panel of 20 relevant genes. Our data show successful differentiations of hiPSC into hiMSCs and hiCPCs. Characteristic hBMSC markers were shared between hBMSCs and hiMSCs, with the exception of CD146 and CD45. However, neo-cartilage generated from hiMSCs showed low resemblances when compared to hBMSCs (53%) and hPACs (39%) characterized by lower collagen type 2 and higher collagen type 1 expression. Contrarily, hiCPC neo-cartilage generated neo-cartilage more similar to hPACs (65%), with stronger expression of matrix deposition markers. Our study shows that taking a stepwise approach to generate neo-cartilage from hiPSCs via chondroprogenitor cells results in strong similarities to neo-cartilage of hPACs within 3 weeks following chondrogenesis, making them a potential candidate for regenerative therapies. Contrarily, neo-cartilage deposited by hiMSCs seems more prone to hypertrophic characteristics compared to hPACs. We therefore compared chondrocytes derived from hiMSCs and hiCPCs with hPACs and hBMSCs to outline similarities and differences between their neo-cartilage and establish their potential suitability for regenerative medicine and disease modelling. Show less
Almeida, R.C. de; Ramos, Y.F.M.; Mahfouz, A.; Hollander, W. den; Lakenberg, N.; Houtman, E.; ... ; Meulenbelt, I. 2019
