WNT signalling in joint repair and homeostasis

PhDDisruption of the Wnt canonical signalling leads to the development of osteoarthritis both in human and in mice but the underlying molecular mechanisms are poorly understood. Both forced activation and blockade of Wnt/β catenin signalling lead to cartilage breakdown. This study attempts to unravel the mechanisms leading to such paradox and is based on the hypothesis that WNT-3A triggers multiple signalling pathways simultaneously, with distinct outcomes. WNT-3A-stimulation induced activation of Wnt/β-catenin pathway in articular chondrocytes and promoted proliferation and loss of chondrocytes phenotype markers, such as COL2A1, Aggrecan and SOX9 mRNA. However, whereas the inhibition of the Wnt/β-catenin pathway by DKK1 rescued the proliferative effect of WNT-3A, it did not rescue the loss of chondrocyte phenotype but, paradoxically, it further enhanced it. Therefore I tested the possibility that WNT-3A-induced chondrocyte de-differentiation could be mediated by other WNT pathways independent of β-catenin. Indeed, in AHAC WNT-3A induced intracellular calcium mobilization and phosphorylation and nuclear localization of CaMKII in a G-protein dependent manner, suggesting the activation of the Wnt/CaMKII pathway. Inhibition of the Wnt/CaMKII pathway rescued the loss of the phenotypic markers SOX9 and COL2A1 induced by WNT-3A, indicating that this pathway drives WNT-3A-induced chondrocyte de-differentiation. Finally, my data show that the Wnt/β-catenin and the Wnt/CaMKII pathways are mutually inhibitory, explaining why both exogenous WNT-3A and its inhibitor DKK1 lead to chondrocyte de-differentiation: the first through direct activation of CaMKII, and the second indirectly by removal of the inhibition of CaMKII exerted by the β-catenin pathway. My results show for the first time that a single WNT ligand can simultaneously activate at least two different pathways in the same cells with different outcomes. These findings highlight the possibility to therapeutically target individual outcomes of Wnt signalling, for instance to prevent chondrocyte de-differentiation without affecting crucial anabolic processes such as cell proliferation

Agrin mediates chondrocyte homeostasis and requires both LRP4 and alpha-dystroglycan to enhance cartilage formation in vitro and in vivo

Objectives Osteoarthritis (OA) is a leading cause of disability for which there is no cure. The identification of molecules supporting cartilage homeostasis and regeneration is therefore a major pursuit in musculoskeletal medicine. Agrin is a heparan sulfate proteoglycan which, through binding to low-density lipoprotein receptor-related protein 4 (LRP4), is required for neuromuscular synapse formation. In other tissues, it connects the cytoskeleton to the basement membrane through binding to α-dystroglycan. Prompted by an unexpected expression pattern, we investigated the role and receptor usage of agrin in cartilage. Methods Agrin expression pattern was investigated in human osteoarthritic cartilage and following destabilisation of the medial meniscus in mice. Extracellular matrix (ECM) formation and chondrocyte differentiation was studied in gain and loss of function experiments in vitro in three-dimensional cultures and gain of function in vivo, using an ectopic cartilage formation assay in nude mice. Receptor usage was investigated by disrupting LRP4 and α-dystroglycan by siRNA and blocking antibodies respectively. Results Agrin was detected in normal cartilage but was progressively lost in OA. In vitro, agrin knockdown resulted in reduced glycosaminoglycan content, downregulation of the cartilage transcription factor SOX9 and other cartilage-specific ECM molecules. Conversely, exogenous agrin supported cartilage differentiation in vitro and ectopic cartilage formation in vivo. In the context of cartilage differentiation, agrin used an unusual receptor repertoire requiring both LRP4 and α-dystroglycan. Conclusions We have discovered that agrin strongly promotes chondrocyte differentiation and cartilage formation in vivo. Our results identify agrin as a novel potent anabolic growth factor with strong therapeutic potential in cartilage regeneration

ROR2 blockade as a therapy for osteoarthritis

Osteoarthritis is characterized by the loss of the articular cartilage, bone remodeling, pain, and disability. No pharmacological intervention can currently halt progression of osteoarthritis. Here, we show that blocking receptor tyrosine kinase–like orphan receptor 2 (ROR2) improves cartilage integrity and pain in osteoarthritis models by inhibiting yes-associated protein (YAP) signaling. ROR2 was up-regulated in the cartilage in response to inflammatory cytokines and mechanical stress. The main ligand for ROR2, WNT5A, and the targets YAP and connective tissue growth factor were up-regulated in osteoarthritis in humans. In vitro, ROR2 overexpression inhibited chondrocytic differentiation. Conversely, ROR2 blockade triggered chondrogenic differentiation of C3H10T1/2 cells and suppressed the expression of the cartilage-degrading enzymes a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)–4 and ADAMTS-5. The chondrogenic effect of ROR2 blockade in the cartilage was independent of WNT signaling and was mediated by down-regulation of YAP signaling. ROR2 signaling induced G protein and Rho-dependent nuclear accumulation of YAP, and YAP inhibition was required but not sufficient for ROR2 blockade–induced chondrogenesis. ROR2 silencing protected mice from instability-induced osteoarthritis with improved structural outcomes, sustained pain relief, and without apparent side effects or organ toxicity. Last, ROR2 silencing in human articular chondrocytes transplanted in nude mice led to the formation of cartilage organoids with more and better differentiated extracellular matrix, suggesting that the anabolic effect of ROR2 blockade is conserved in humans. Thus, ROR2 blockade is efficacious and well tolerated in preclinical animal models of osteoarthritis

WNT-3A modulates articular chondrocyte phenotype by activating both canonical and noncanonical pathways

Peer reviewedPublisher PD

Prolonged higher dose methylprednisolone vs. conventional dexamethasone in COVID-19 pneumonia: a randomised controlled trial (MEDEAS)

Dysregulated systemic inflammation is the primary driver of mortality in severe COVID-19 pneumonia. Current guidelines favor a 7-10-day course of any glucocorticoid equivalent to dexamethasone 6 mg·day-1. A comparative RCT with a higher dose and a longer duration of intervention was lacking

Extracellular genomic biomarkers of osteoarthritis

Introduction: Osteoarthritis (OA), a chronic, debilitating and degenerative disease of the joints, is the most common form of arthritis. The seriousness of this prevalent and chronic disease is often overlooked. Disease modifying OA drug development is hindered by the lack of soluble biomarkers to detect OA early. The objective of OA biomarker research is to identify early OA prior to the appearance of radiographic signs and the development of pain. Areas covered: This review has focused on extracellular genomic material that could serve as biomarkers of OA. Recent studies have examined the expression of extracellular genomic material such as miRNA, lncRNA, snoRNA, mRNA and cell-free DNA, which are aberrantly expressed in the body fluids of OA patients. Changes in genomic content of peripheral blood mononuclear cells in OA could also function as biomarkers of OA. Expert commentary: There is an unmet need for soluble biomarkers for detecting and then monitoring OA disease progression. Extracellular genomic material research may also reveal more about the underlying pathophysiology of OA. Minimally-invasive liquid biopsies such as synovial fluid and blood sampling of genomic material may be more sensitive over radiography in the detection, diagnosis and monitoring of OA in the future.<br /

EARLY DOWN-REGULATIONOF ANGIOTENSIN II RECEPTORS GENE EXPRESSION IN CULTURED HUMAN MESANGIAL CELLS BY ANGIOTENSIN II

Internat. Forum on Angiotensin II Receptor Antagonism, Montecarlo, Jenuary 27-30, 1999, page 4