Giovanna Nalesso

University of Surrey

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Francesco Dell’Accio

Queen Mary University of London

Costantino Pitzalis

Queen Mary University of London

Jessica Bertrand

University Hospital Magdeburg

J. Sherwood

University Hospital Münster

Thomas Pap

University Hospital Münster

B.L. Thomas

Queen Mary University of London

S. Eldridge

Queen Mary University of London

Anne‐Sophie Thorup

William Harvey Research Institute

Frank Echtermeyer

Medizinische Hochschule Hannover

Lars Godmann

University Hospital Münster

Cooperative Institutions

Queen Mary University of London

William Harvey Research Institute

University Hospital Münster

University of Münster

University of Surrey

Universidade de São Paulo

University College London

University of Oxford

GlaxoSmithKline (United Kingdom)

University of Padua

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A8.13 Syndecan-4 Function is Essential for Matrix Remodelling Under Inflammatory Conditions, But Dispensable During Embryogenesis

Annals of the Rheumatic Diseases (2013)

Jessica Bertrand Richard Stange Heriburg Hidding Frank Echtermeyer Giovanna Nalesso

Objective

The heparan sulphate proteoglycan syndecan-4 (Sdc4) has been associated strongly with osteoarthritis, a disease that mimics key aspects of early cartilage remodelling during endochondral ossification, but its role in embryonic and adult bone formation remains unclear. Therefore, we used Sdc4 ^-/- mice to analyse the distribution and functional role of Scd4 in endochondral ossification of mouse embryos and in adult fracture repair, which recapitulates endochondral ossification, but like osteoarthritis, involves an inflammatory component.

Methods

Sdc4 promoter activity was analysed in Sdc4 ^-/- /LacZ knock-in animals using β-galactosidase stainings. E16.5 embyros were used for histological (alcian blue/alizarin red) and immunohistological (PCNA, Col10a1, ADAMTS-4, BC-3, Sdc2) staining and the calcified bone area was quantified using whole mount staining of these embryos. Histological (Masson-Goldner, alcian blue) and immunohistological (Col10a1, Sdc2, PCNA) staining at day 7, 14 and 28 fracture calli were performed. These experiments were repeated with anti-TNF treatment during fracture healing. Callus size and cartilage area were quantified using image J Chondrocytes were isolated from neonatal knee joints and embyronal cartilage. Proliferation was investigated using MTT assay. Gene expression analysis for Sdc-2, Sdc-4 with and without stimulation using TNFα and WNT3a was performed using quantitative RT-PCR.

Results

In Sdc4 ^-/- /LacZ knock-in animals, Sdc4 promoter activity was detectable in all stages of chondrocyte differentiation during embryogenesis. Sdc4 deficiency inhibited chondrocyte proliferation both in vivo and in vitro, but this did not lead to a growth phenotype at birth. In contrast to embryogenesis, fracture healing in adult mice was markedly delayed in Sdc4 ^-/- animals and accompanied by increased callus formation. Analysing the discrepancy between the mild embryonic and the severe adult phenotype, we found a compensatory up-regulation of Sdc2 in the developing cartilage of Sdc4 ^-/- mice that was absent in adult tissue. Stimulation of chondrocytes with Wnt3a in vitro, led to an increased expression of Sdc2, while stimulation with TNFα resulted in an up-regulation of Sdc4 but a decreased expression of Sdc2. In consequence treatment with a blocking anti-TNF antibody during fracture healing abolished the difference in callus size between wildtype and sdc4 ^-/- mice.

Conclusions

We conclude that Sdc4 is functionally involved in endochondral ossification and that the loss of Sdc4 impairs adult fracture healing due to the inhibition of compensatory mechanisms under inflammatory conditions.

Endochondral ossification

Syndecan 1

10.1136/annrheumdis-2013-203222.13

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WNT signalling in joint repair and homeostasis

Giovanna Nalesso

Homeostasis

Signalling

Source

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Faculty Opinions recommendation of Roles of β-catenin signaling in phenotypic expression and proliferation of articular cartilage superficial zone cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature (2012)

Francesco Dell’Accio Giovanna Nalesso

Expression (computer science)

10.3410/f.716947877.792352801

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WNT3A‐loaded exosomes enable cartilage repair

Journal of Extracellular Vesicles (2021)

B.L. Thomas S. Eldridge Babak Nosrati Mario Alvarez Anne‐Sophie Thorup

Cartilage defects repair poorly. Recent genetic studies suggest that WNT3a may contribute to cartilage regeneration, however the dense, avascular cartilage extracellular matrix limits its penetration and signalling to chondrocytes. Extracellular vesicles actively penetrate intact cartilage. This study investigates the effect of delivering WNT3a into large cartilage defects in vivo using exosomes as a delivery vehicle. Exosomes were purified by ultracentrifugation from conditioned medium of either L-cells overexpressing WNT3a or control un-transduced L-cells, and characterized by electron microscopy, nanoparticle tracking analysis and marker profiling. WNT3a loaded on exosomes was quantified by western blotting and functionally characterized in vitro using the SUPER8TOPFlash reporter assay and other established readouts including proliferation and proteoglycan content. In vivo pathway activation was assessed using TCF/Lef:H2B-GFP reporter mice. Wnt3a loaded exosomes were injected into the knees of mice, in which large osteochondral defects were surgically generated. The degree of repair was histologically scored after 8 weeks. WNT3a was successfully loaded on exosomes and resulted in activation of WNT signalling in vitro. In vivo, recombinant WNT3a failed to activate WNT signalling in cartilage, whereas a single administration of WNT3a loaded exosomes activated canonical WNT signalling for at least one week, and eight weeks later, improved the repair of osteochondral defects. WNT3a assembled on exosomes, is efficiently delivered into cartilage and contributes to the healing of osteochondral defects.

WNT3A

Exosome

10.1002/jev2.12088

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Hydroquinone, a cigarette smoke compound, affects cartilage homeostasis through activation of the aryl hydrocarbon receptor pathway

bioRxiv (Cold Spring Harbor Laboratory) (2022)

Cíntia Scucuglia Heluany Anna De Palma Nicholas James Day Sandra Helena Poliselli Farsky Giovanna Nalesso

Abstract Exposure to cigarette smoke has a proven detrimental impact on different aspects of human health. Increasing evidences link smoking to degeneration of joint tissues. However, the toxic mechanisms elicited by the different components of cigarette smoke have not been fully elucidated yet. We have previously shown that exposure to hydroquinone (HQ), a pro-oxidant chemical present in cigarette smoke, can promote joint tissue degradation in murine models of rheumatoid arthritis through the activation of the aryl hydrocarbon receptor (AhR) pathway. Osteoarthritis (OA) is a chronic debilitating articular disease characterized by progressive degradation of the articular cartilage, whose onset and progression have also been associated with smoking. In this work we aimed to investigate the effect of HQ exposure on articular chondrocytes and how it affects cartilage homeostasis. Cell viability, gene expression, oxidative stress and inflammatory parameters were quantified in primary articular chondrocytes exposed to HQ in presence or absence of IL-1β pre-stimulation. HQ stimulation downregulated phenotypic markers genes such as SOX-9 and Col2a1, whereas upregulated the expression of the catabolic enzymes MMP-3 and ADAMTS5. HQ also promoted oxidative stress and reduced proteoglycan content. HQ exacerbated the pro-inflammatory effects mediated by the IL-1β co-stimulation. Finally, we showed that HQ-degenerative effects were mediated by the activation of AhR. Together, our findings address the harmful effects of HQ in the articular cartilage health, providing novel evidence surrounding the toxic mechanisms of environmental pollutants underlying the onset of articular diseases.

10.1101/2022.04.25.489372

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microRNA-544a as a new modulator of the Wnt-signalling network in the articular cartilage and osteoarthritis

bioRxiv (Cold Spring Harbor Laboratory) (2024)

Cíntia Scucuglia Heluany Nicholas Day K. Delos Santos Anna De Palma T.E. Swingler

Abstract Objective Determining the effect of microRNA-544a (miR-544a) in articular chondrocytes isolated from patients affected by osteoarthritis (OA) and its role in the modulation of the Wnt signalling. Methods Articular chondrocytes were isolated from patients undergoing joint replacement because of OA. Expression levels of miR-544a were measured by PCR and by in situ hybridization. Putative targets of miR-544a were confirmed by reporter assay and by qPCR in cells stimulated with a miR-544a mimic. The effect of miR-544a on chondrocyte metabolism was monitored by qPCR for phenotypic markers, protein expression levels of aggrecan neoepitopes/MMP-13 and modulation of alcian blue content in micromass cultures, upon stimulation with a miR-544a mimic. The expression levels of MMP-13 and Aggrecan neoepitopes in response to miR-544a stimulation was also measured in co-stimulation with Xav-939 and KN93, which are respectively β-catenin and CaMKII inhibitors. Results Our results suggest that miR-544a enhances the activation of the Wnt-signalling in the articular chondrocytes, by downregulating the expression of components of the Wnt/β-catenin destruction complex. The expression of miR-544a is higher in chondrocytes isolated from damaged areas of the articular cartilage removed from OA patients, and can be upregulated by pro-inflammatory and pro-fibrotic cytokines. miR-544a exerts a pro-catabolic effect of articular chondrocytes, which is rescued both by the inhibition of the Wnt/β-catenin and Wnt/CaMKII signalling pathways. Conclusion our results point to miR-544a as a new, important modulator of the Wnt signalling network within the articular cartilage suggesting a key role for microRNAs in regulating how the multiple branches of the network and their interaction modulate cartilage homeostasis.

Aggrecan

10.1101/2024.11.28.625937

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Syndecan-4 deficient chondrocytes exhibit alterations in activation of WNT signaling pathways leading to higher phenotypic stability

Osteoarthritis and Cartilage (2013)

Jessica Bertrand Richard Stange Giovanna Nalesso J. Sherwood Lars Godmann

Aggrecan

WNT3A

10.1016/j.joca.2013.02.061

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WNT Signalling in Osteoarthritis and Its Pharmacological Targeting

Handbook of experimental pharmacology (2021)

Anna De Palma Giovanna Nalesso

Signalling pathways

Mediator

Signalling

10.1007/164_2021_525

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Citations (13)

Faculty Opinions recommendation of Transcriptional regulation of endochondral ossification by HIF-2alpha during skeletal growth and osteoarthritis development.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature (2010)

Francesco Dell’Accio Giovanna Nalesso

Endochondral ossification

10.3410/f.3516956.3288058

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OP0200 BLOCKING ROR2 IMPROVES CARTILAGE INTEGRITY AND PROVIDES PAIN RELIEF IN OSTEOARTHRITIS

Annals of the Rheumatic Diseases (2021)

Anne‐Sophie Thorup Danielle Strachan Sara Caxaria B. Poulet B.L. Thomas

Background: Osteoarthritis (OA) is the leading cause of chronic disability worldwide, affecting 12% of the population, and yet we still do not have a disease-modifying treatment. Cartilage breakdown is the hallmark of OA, and patients suffer from pain and loss of joint function/independence, severely affecting quality of life. Therefore, there is a huge unmet clinical need. Receptor tyrosine kinase–like orphan receptor 2 (ROR2) is a non-canonical WNT receptor that regulates the planar cell polarity pathway, controlling limb outgrowth during development. During skeletal development, chondrocytes require ROR2 to undergo hypertrophy throughout the process of endochondral bone formation 1 . Loss of function mutations in humans causes Recessive Robinow Syndrome, leading to limb shortening and brachydactyly 2,3 . Although absent from healthy adult articular cartilage, our initial studies identified high expression levels of ROR2 in chondrocytes from patients with OA, suggesting a role in the disease process Objectives: To test the potential of ROR2 blockade as a disease-modifying treatment for OA. Methods: Human cartilage organoid model in nude mice, menisco-ligament injury (MLI) model of OA in mice, behavioural studies, in vitro studies in cells. Results: ROR2/WNT5A signaling was increased in osteoarthritic cartilage. Blocking ROR2 was sufficient to induce articular chondrogenesis and suppress expression of aggrecanases in a mesenchymal stem cell line, and to support cartilage formation in a human cartilage organoid model in nude mice using primary chondrocytes from patients with OA. In the MLI model of OA, blocking ROR2 in therapeutic regime using atelocollagen-conjugated siRNA resulted in reduced cartilage destruction and in rapid and sustained pain relief. Due to the limited expression pattern of ROR2 in adulthood, no systemic or local toxicity were expected, nor were any observed 4 . With the current technology, ROR2 blockade requires intra-articular (IA) injections of siRNA conjugated to atelocollagen every 5 days. Preliminary efficacy data of potentially longer-acting ROR2 blockers are promising. The mechanism of action of ROR2 blockade was independent of modulation of canonical WNT signaling. ROR2/WNT5A promoted nuclear localization of YAP, which required both Rho and G-proteins. YAP signaling downstream of ROR2 also required Rho, but not G-proteins. YAP and TEAD inhibition was required, but not sufficient, for the chondrogenic effect of blocking ROR2. Therefore, additional, yet unknown mechanisms must be involved downstream of ROR2. Conclusion: ROR2 blockade has potential as a disease-modifying treatment for OA, resulting in cartilage protection and rapid and sustained pain relief in a murine model. This will be crucial for clinical success of any treatment for OA and promote patient compliance. Our current siRNA-atelocollagen based technology requires IA injections too frequently to be acceptable for patients. We are developing ROR2 blockade which can be administered systemically or IA not more often than every 3 months - work funded by FOREUM. References: [1]DeChiara, T. M. et al. Ror2, encoding a receptor-like tyrosine kinase, is required for cartilage and growth plate development. Nat. Genet. 24 , 271–4 (2000). [2]Bokhoven, H. Van, Celli, J. & Kayserili, H. Mutation of the gene encoding the ROR2 tyrosine kinase causes autosomal recessive Robinow syndrome. Nature 25 , 423–426 (2000). [3]Afzal, A., Rajab, A., Fenske, C. & Oldridge, M. Recessive Robinow syndrome, allelic to dominant brachydactyly type B, is caused by mutation of ROR2. Nature 25 , 419–422 (2000). [4]Thorup, A.-S. et al. ROR2 blockade as a therapy for osteoarthritis. Sci. Transl. Med. 12 , eaax3063 (2020). Acknowledgements: We gratefully acknowledge funding support of this work by the Medical College of St Bartholomew’s Hospital Trust, the William Harvey Research Foundation, FOREUM foundation for research in rheumatology (1016807), the MRC (MR/L022893/1, MR/N010973/1, MR/P026362/1, MR/K013076/1), Versus Arthritis (21515, 20886, 21621, 20859), and the DFG Emmy-Noether program (BE4328/5-1). Disclosure of Interests: Anne-Sophie Thorup: None declared, Danielle Strachan: None declared, Sara Caxaria: None declared, Blandine Poulet: None declared, Bethan Thomas: None declared, Suzanne Eldridge: None declared, Giovanna Nalesso: None declared, James Whiteford: None declared, Costantino Pitzalis: None declared, Thomas Aigner: None declared, Roger Corder: None declared, Jessica Bertrand: None declared, Francesco Dell’Accio Consultant of: Samumed and UCB

Chondrogenesis

Endochondral ossification

10.1136/annrheumdis-2021-eular.2358

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