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... Show moreDysregulation 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. Show less
Timmermans, R.G.M.; Blom, A.B.; Bloks, N.G.C.; Nelissen, R.G.H.H.; Linden, E.H.M.J. van der; Kraan, P.M. van der; ... ; Bosch, M.H.J. van den 2022
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)... Show moreObjectives: 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 rho >=|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 rho >=|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. Show less
Ascone, G.; Ceglie, I. di; Walgreen, B.; Sloetjes, A.W.; Lindhout, E.; Bot, I.; ... ; Lent, P.L.E.M. van 2020
Rheumatoid arthritis (RA) is a chronic inflammatory disease, characterized by severe joint inflammation and bone destruction as the result of increased numbers and activity of osteoclasts. RA is... Show moreRheumatoid arthritis (RA) is a chronic inflammatory disease, characterized by severe joint inflammation and bone destruction as the result of increased numbers and activity of osteoclasts. RA is often associated with metabolic syndrome, whereby elevated levels of LDL are oxidized into oxLDL, which might affect osteoclastogenesis. In this study, we induced antigen-induced arthritis (AIA) in Apoe(-/-) mice, which spontaneously develop high LDL levels, to investigate the effects of high LDL/oxLDL levels on osteoclast differentiation and bone destruction. Whereas basal levels of bone resorption were comparable between naive WT and Apoe(-/-) mice, induction of AIA resulted in a significant reduction of bone destruction in Apoe(-/-) mice as compared to WT controls. In line with that, the TRAP(+) area on the cortical bone was significantly decreased. The absence of Apoe did affect neither the numbers of CD11b(+) Ly6C(high) and CD11b(-)/Ly6C(high)osteoclast precursors (OCPs) in the BM of naive mice nor their in vitro osteoclastogenic potential as indicated by comparable mRNA expression of osteoclast markers. Addition of oxLDL, but not LDL, to pre-osteoclasts from day 3 and mature osteoclasts from day 6 of osteoclastogenesis strongly reduced the number of TRAP(+) osteoclasts and their resorptive capacity. This coincided with a decreased expression of various osteoclast markers. Interestingly, oxLDL significantly lowered the expression of osteoclast-associated receptor (Oscar) and the DNAX adaptor protein-12 encoding gene Tyrobp, which regulate the immunoreceptor tyrosine-based activation motif (ITAM) co-stimulation pathway that is strongly involved in osteoclastogenesis. Collectively, our findings suggest that under inflammatory conditions in the joint, high LDL levels lessen bone destruction during AIA, probably by formation of oxLDL that inhibits osteoclast formation and activity through modulation of the ITAM-signaling. Show less
Ascone, G.; Di Ceglie, I.; Walgreen, B.; Sloetjes, A.W.; Lindhout, E.; Bot, I.; ... ; Lent, P.L.E.M. van 2019
Rheumatoid arthritis (RA) is a chronic inflammatory disease, characterized by severe joint inflammation andbone destruction as the result of increased numbers and activity of osteoclasts. RA is... Show moreRheumatoid arthritis (RA) is a chronic inflammatory disease, characterized by severe joint inflammation andbone destruction as the result of increased numbers and activity of osteoclasts. RA is often associated withmetabolic syndrome, whereby elevated levels of LDL are oxidized into oxLDL, which might affect osteoclastogenesis.In this study, we induced antigen-induced arthritis (AIA) in Apoe−/− mice, which spontaneously develophigh LDL levels, to investigate the effects of high LDL/oxLDL levels on osteoclast differentiation and bonedestruction. Whereas basal levels of bone resorption were comparable between naive WT and Apoe−/− mice,induction of AIA resulted in a significant reduction of bone destruction in Apoe−/− mice as compared to WTcontrols. In line with that, the TRAP+ area on the cortical bone was significantly decreased. The absence of Apoedid affect neither the numbers of CD11b+Ly6Chigh and CD11b−/Ly6Chigh osteoclast precursors (OCPs) in the BMof naïve mice nor their in vitro osteoclastogenic potential as indicated by comparable mRNA expression ofosteoclast markers. Addition of oxLDL, but not LDL, to pre-osteoclasts from day 3 and mature osteoclasts fromday 6 of osteoclastogenesis strongly reduced the number of TRAP+ osteoclasts and their resorptive capacity. Thiscoincided with a decreased expression of various osteoclast markers. Interestingly, oxLDL significantly loweredthe expression of osteoclast-associated receptor (Oscar) and the DNAX adaptor protein-12 encoding gene Tyrobp,which regulate the immunoreceptor tyrosine-based activation motif (ITAM) co-stimulation pathway that isstrongly involved in osteoclastogenesis. Collectively, our findings suggest that under inflammatory conditions inthe joint, high LDL levels lessen bone destruction during AIA, probably by formation of oxLDL that inhibitsosteoclast formation and activity through modulation of the ITAM-signaling. Show less
Bosch, M.H.J. van den; Ramos, Y.F.M.; Hollander, W. den; Bomer, N.; Nelissen, R.G.H.H.; Bovee, J.V.M.G.; ... ; Meulenbelt, I. 2019
