Osteoarthritis, a degenerative disease of the articular joints : towards the implementation of functional genomics in OA

Osteoarthritis (OA) is an age related disorder of the joints characterized by pain, crepitus, and stiffness resulting in decreased mobility. Pathophysiology consists of cartilage degeneration and bone remodeling, however, knowledge of OA etiology is still limited. Due to the growing population of elderly, OA prevalence rapidly increases. The fact that no reliable clinical markers are available for diagnosis, monitoring and progression is a major impediment in OA disease management and incurs high costs in drug development and clinical trials. Molecular markers were studied in OA affected cartilage compared to unaffected cartilage of the same joint (chapter 2) and in blood of OA patients (chapter 3). Perturbation of the application of traditional biochemical markers sCOMP and uCTX2 in the clinic due to genetic factors that, independent of OA, affect innate levels was investigated (chapter 4). Furthermore, we have tried to go beyond the results of molecular epidemiological studies to increase insights into underlying mechanisms (chapter 6 & 7). This shows how functional genomics can be achieved by combining genetic and functional data and will facilitate translation of knowledge of genetic variants to the needs of OA patients and thus to application in the clinic.UBL - phd migration 201

Circulating microRNAs highly correlate to expression of cartilage genes potentially reflecting OA susceptibility: towards identification of applicable early OA biomarkers

Objective: To identify and validate circulating micro RNAs (miRNAs) that mark gene expression changes in articular cartilage early in osteoarthritis (OA) pathophysiology process. Methods: Within the ongoing RAAK study, human preserved OA cartilage and plasma (N = 22 paired samples) was collected for RNA sequencing (respectively mRNA and miRNA). Spearman correlation was determined for 114 cartilage genes consistently and significantly differentially expressed early in osteoarthritis and 384 plasma miRNAs. Subsequently, the minimal number of circulating miRNAs serving to discriminate between progressors and non-progressors was assessed by regression analysis and area under receiver operating curves (AUC) was calculated with progression data and plasma miRNA sequencing from the GARP study (N = 71). Results: We identified strong correlations (rho >= |0.7|) among expression levels of 34 unique plasma miRNAs and 21 genes, including 4 genes that correlated with multiple miRNAs. The strongest correlation was between let-7d-5p and EGFLAM (rho = -0.75, P = 6.9 x 10(-5)). Regression analysis of the 34 miRNAs resulted in a set of 7 miRNAs that, when applied to the GARP study, demonstrated clinically relevant predictive value with AUC > 0.8 for OA progression over 2 years and near-clinical value for progression over 5 years- (AUC = 0.8). Conclusions: We show that plasma miRNAs levels reflect gene expression levels in cartilage and can be exploited to represent ongoing pathophysiological processes in articular cartilage. We advocate that identified signature of 7 plasma miRNAs can contribute to direct further studies toward early biomarkers predictive for progression of osteoarthritis over 2 and 5 years.Molecular Epidemiolog

Characterization of dynamic changes in Matrix Gla Protein (MGP) gene expression as function of genetic risk alleles, osteoarthritis relevant stimuli, and the vitamin K inhibitor warfarin

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 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/).Molecular Epidemiolog

Hyper-physiologic mechanical cues, as an osteoarthritis disease-relevant environmental perturbation, cause a critical shift in set points of methylation at transcriptionally active CpG sites in neo-cartilage organoids

Background Osteoarthritis (OA) is a complex, age-related multifactorial degenerative disease of diarthrodial joints marked by impaired mobility, joint stifness, pain, and a signifcant decrease in quality of life. Among other risk factors, such as genetics and age, hyper-physiological mechanical cues are known to play a critical role in the onset and progression of the disease (Guilak in Best Pract Res Clin Rheumatol 25:815–823, 2011). It has been shown that postmitotic cells, such as articular chondrocytes, heavily rely on methylation at CpG sites to adapt to environmental cues and maintain phenotypic plasticity. However, these long-lasting adaptations may eventually have a negative impact on cellular performance. We hypothesize that hyper-physiologic mechanical loading leads to the accumulation of altered epigenetic markers in articular chondrocytes, resulting in a loss of the tightly regulated balance of gene expression that leads to a dysregulated state characteristic of the OA disease state. Results We showed that hyper-physiological loading evokes consistent changes in CpGs associated with expression changes (ML-tCpGs) in ITGA5, CAV1, and CD44, among other genes, which together act in pathways such as anatomical structure morphogenesis (GO:0009653) and response to wound healing (GO:0042060). Moreover, by comparing the ML-tCpGs and their associated pathways to tCpGs in OA pathophysiology (OA-tCpGs), we observed a modest but particular interconnected overlap with notable genes such as CD44 and ITGA5. These genes could indeed represent lasting detrimental changes to the phenotypic state of chondrocytes due to mechanical perturbations that occurred earlier in life. The latter is further suggested by the association between methylation levels of ML-tCpGs mapped to CD44 and OA severityMolecular Epidemiolog

Human osteochondral explants: reliable biomimetic models to investigate disease mechanisms and develop personalized treatments for osteoarthritis

Introduction: 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.Orthopaedics, Trauma Surgery and Rehabilitatio

Mechanical stress and inflammation have opposite effects on Wnt signaling in human chondrocytes

Dysregulation of Wingless and Int-1 (Wnt) signaling has been strongly associated with development and progression of osteoarthritis (OA). Here, we set out to investigate the independent effects of either mechanical stress (MS) or inflammation on Wnt signaling in human neocartilage pellets, and to relate this Wnt signaling to OA pathophysiology. OA synovium-conditioned media (OAS-CM) was collected after incubating synovium from human end-stage OA joints for 24 h in medium. Cytokine levels in the OAS-CM were determined with a multiplex immunoassay (Luminex). Human neocartilage pellets were exposed to 20% MS, 2% OAS-CM or 1 ng/mL Interleukin-1 & beta; (IL-1 & beta;). Effects on expression levels of Wnt signaling members were determined by reverse transcription-quantitative polymerase chain reaction. Additionally, the expression of these members in articular cartilage from human OA joints was analyzed in association with joint space narrowing (JSN) and osteophyte scores. Protein levels of IL-1 & beta;, IL-6, IL-8, IL-10, tumor necrosis factor & alpha;, and granulocyte-macrophage colony-stimulating factor positively correlated with each other. MS increased noncanonical WNT5A and FOS expression. In contrast, these genes were downregulated upon stimulation with OAS-CM or IL-1 & beta;. Furthermore, Wnt inhibitors DKK1 and FRZB decreased in response to OAS-CM or IL-1 & beta; exposure. Finally, expression of WNT5A in OA articular cartilage was associated with increased JSN scores, but not osteophyte scores. Our results demonstrate that MS and inflammatory stimuli have opposite effects on canonical and noncanonical Wnt signaling in human neocartilage. Considering the extent to which MS and inflammation contribute to OA in individual patients, we hypothesize that targeting specific Wnt pathways offers a more effective, individualized approach.Orthopaedics, Trauma Surgery and Rehabilitatio

High-impact FN1 mutation decreases chondrogenic potential and affects cartilage deposition via decreased binding to collagen type II

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 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.Microscopic imaging and technolog

Elucidating mechano-pathology of osteoarthritis: transcriptome-wide differences in mechanically stressed aged human cartilage explants

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-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.Molecular Epidemiolog

Cartilage from human-induced pluripotent stem cells: comparison with neo-cartilage from chondrocytes and bone marrow mesenchymal stromal cells

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 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.Pathogenesis and treatment of chronic pulmonary disease

Inhibiting thyroid activation in aged human explants prevents mechanical induced detrimental signalling by mitigating metabolic processes

Objectives To investigate whether the deiodinase inhibitor iopanoic acid (IOP) has chondroprotective properties, a mechanical stress induced model of human aged explants was used to test both repeated dosing and slow release of IOP. Methods Human osteochondral explants subjected to injurious mechanical stress (65%MS) were treated with IOP or IOP encapsulated in poly lactic-co-glycolic acid-polyethylene glycol nanoparticles (NP-IOP). Changes to cartilage integrity and signalling were determined by Mankin scoring of histology, sulphated glycosaminoglycan (sGAG) release and expression levels of catabolic, anabolic and hypertrophic markers. Subsequently, on a subgroup of samples, RNA sequencing was performed on 65%MS (n = 14) and 65%MS+IOP (n = 7) treated cartilage to identify IOP's mode of action. Results Damage from injurious mechanical stress was confirmed by increased cartilage surface damage in the Mankin score, increased sGAG release, and consistent upregulation of catabolic markers and downregulation of anabolic markers. IOP and, though less effective, NP-IOP treatment, reduced MMP13 and increased COL2A1 expression. In line with this, IOP and NP-IOP reduced cartilage surface damage induced by 65%MS, while only IOP reduced sGAG release from explants subjected to 65%MS. Lastly, differential expression analysis identified 12 genes in IOP's mode of action to be mainly involved in reducing metabolic processes (INSIG1, DHCR7, FADS1 and ACAT2) and proliferation and differentiation (CTGF, BMP5 and FOXM1). Conclusion Treatment with the deiodinase inhibitor IOP reduced detrimental changes of injurious mechanical stress. In addition, we identified that its mode of action was likely on metabolic processes, cell proliferation and differentiation.Molecular Epidemiolog