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-1ß (IL-1ß). 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-1ß, IL-6, IL-8, IL-10, tumor necrosis factor α, 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-1ß. Furthermore, Wnt inhibitors DKK1 and FRZB decreased in response to OAS-CM or IL-1ß 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.

CCN4/WISP1 Promotes Migration of Human Primary Osteoarthritic Chondrocytes.

OBJECTIVES: Previously, we have shown the involvement of cellular communication network factor 4/Wnt-activated protein Wnt-1-induced signaling protein 1 (CCN4/WISP1) in osteoarthritic (OA) cartilage and its detrimental effects on cartilage. Here, we investigated characteristics of CCN4 in chondrocyte biology by exploring correlations of CCN4 with genes expressed in human OA cartilage with functional follow-up. DESIGN: Spearman correlation analysis was performed for genes correlating with CCN4 using our previously established RNA sequencing dataset of human preserved OA cartilage of the RAAK study, followed by a pathway enrichment analysis for genes with ρ ≥|0.6.| Chondrocyte migration in the absence or presence of CCN4 was determined in a scratch assay, measuring scratch size using a live cell imager for up to 36 h. Changes in expression levels of 12 genes, correlating with CCN4 and involved in migratory processes, were determined with reverse transcription-quantitative polymerase chain reaction (RT-qPCR). RESULTS: Correlation of CCN4 with ρ ≥|0.6| was found for 58 genes in preserved human OA cartilage. Pathway analysis revealed "neural crest cell migration" as most significant enriched pathway, containing among others CORO1C, SEMA3C, and SMO. Addition of CCN4 to primary chondrocytes significantly enhance chondrocyte migration as demonstrated by reduced scratch size over the course of 36 h, but at the timepoints measured no effect was observed on mRNA expression of the 12 genes. CONCLUSION: CCN4 increases cell migration of human primary OA chondrocytes. Since WISP1 expression is known to be increased in OA cartilage, this may serve to direct chondrocytes toward cartilage defects and orchestrate repair.

A human in vitro 3D neo-cartilage model to explore the response of OA risk genes to hyper-physiological mechanical stress.

Objective: Due to the complexity and heterogeneity of osteoarthritis (OA) pathophysiology, studying the interaction between intrinsic molecular changes in chondrocytes after hyper-physiological mechanical stress (MS) and aberrant signalling of OA risk genes remains a challenge. In this study we set out to set up an in vitro 3D neo cartilage pellet model that enables us to explore the responses of OA risk genes to hyper-physiological MS. Design: Human primary chondrocyte neo-cartilage pellets were exposed for 2 days to 2 â€‹× â€‹10 â€‹min of hyper-physiological dynamic MS attained by a 20% strain and a frequency of 5 â€‹Hz. In order to assess cartilage damage, sulphated glycosaminoglycan (sGAG) content in the neo-cartilage was quantified using Alcian blue staining and a dimethyl methylene blue (DMMB) assay, while cleavage of aggrecan was visualized by immunohistochemical staining of aggrecan neo-epitope NITEGE. In addition, changes in expression levels of catabolic, anabolic and hypertrophic genes, and of three OA risk genes; IL11, MGP and TGFA were determined. Results: Hyper-physiological MS induced cartilage damage, as reflected by decreased sGAG content. mRNA levels of aggrecanase ADAMTS5 were increased, while hypertrophic gene RUNX2 was downregulated. MS increased expression of pro-apoptotic marker NOXA. Furthermore, 20% MS led to increased expression of all three OA risk genes IL11, MGP and TGFA. Conclusions: We established a human in vitro model in which hyper-physiological MS induced cartilage damage and catabolic signalling. Next, we demonstrated its usage to study OA risk genes and their response to the mechanical aspects of OA pathophysiology.