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Radboud University, 01 september 2020, Promotor : Kraan, P.M. van der Co-promotores : Caam, A.P.M. van, Blaney Davidson, E.N., Contains fulltext : 220315.pdf (publisher's version ) (Open Access)

Contains fulltext : 201050.pdf (publisher's version ) (Closed access)

Item does not contain fulltext, IMPACT STATEMENT: Catabolic factors present in a damaged joint inhibit chondrogenic differentiation of mesenchymal stem cells, thereby reducing the chance for successful cartilage formation. By improving stem cell-based cartilage repair with interleukin-37 (IL37), we might be able to inhibit the worsening progression of focal cartilage defects and prevent further development of joint diseases such as osteoarthritis. This will avoid chronic pain and impaired joint mobility for patients and reduce costs for society.

Contains fulltext : 173999.pdf (publisher's version ) (Closed access), Objective.: A crucial feature of OA is cartilage degradation. This process is mediated by pro-inflammatory cytokines, among other factors, via induction of matrix-degrading enzymes. Interleukin 37 (IL37) is an anti-inflammatory cytokine and is efficient in blocking the production of pro-inflammatory cytokines during innate immune responses. We hypothesize that IL37 is therapeutic in treating the inflammatory cytokine cascade in human OA chondrocytes and can act as a counter-regulatory cytokine to reduce cartilage degradation in OA. Methods.: Human OA cartilage was obtained from patients undergoing total knee or hip arthroplasty. Immunohistochemistry was applied to study IL37 protein expression in cartilage biopsies from OA patients. Induction of IL37 expression by IL1beta, OA synovium-conditioned medium and TNFalpha was investigated in human OA chondrocytes. Adenoviral overexpression of IL37 followed by IL1beta stimulation was performed to investigate the anti-inflammatory potential of IL37. Results.: IL37 expression was detected in cartilage biopsies of OA patients and induced by IL1beta. After IL1beta stimulation, increased IL1beta, IL6 and IL8 expression was observed in OA chondrocytes. Elevated IL37 levels diminished the IL1beta-induced IL1beta , IL6 and IL8 gene levels and IL1beta and IL8 protein levels. In addition to the reduction in pro-inflammatory cytokine expression, IL37 reduced MMP1 , MMP3 , MMP13 and disintegrin and metalloproteinase with thrombospondin motifs 5 gene levels and MMP3 and MMP13 protein levels. Conclusion.: IL37 is induced by IL1beta, and IL37 itself reduced IL1beta, IL6 and IL8 production, indicating that IL37 is able to induce a counter-regulatory anti-inflammatory feedback loop in chondrocytes. In addition, IL37 dampens catabolic enzyme expression. This supports IL37 as a potential therapeutic target in OA.

Item does not contain fulltext, OBJECTIVE: In osteoarthritic cartilage, expression of the receptor ALK1 correlates with markers of deleterious chondrocyte hypertrophy. Recently, bone morphogenetic protein 9 (BMP9) was identified as a high affinity ligand for ALK1. Therefore, we studied if BMP9 signaling results in expression of hypertrophy markers in chondrocytes. Furthermore, because transforming growth factorss1 (TGFbeta1) is a well known anti-hypertrophic factor, the interaction between BMP9 and TGFbeta1 signaling was also studied. DESIGN: Primary chondrocytes were isolated from bovine cartilage and stimulated with BMP9 and/or TGFbeta1 to measure intracellular signaling via pSmads with the use of Western blot. Expression of Smad-responsive genes or hypertrophy-marker genes was measured using qPCR. To confirm observations on TGFbeta/Smad3 responsive genes, a Smad3-dependent CAGA12-luc transcriptional reporter assay was performed in the chondrocyte G6 cell line. RESULTS: In primary chondrocytes, BMP9 potently induced phosphorylation of Smad1/5 and Smad2 to a lesser extent. BMP9-induced Smad1/5 phosphorylation was rapidly (2 h) reflected in gene expression, whereas Smad2 phosphorylation was not. Remarkably, BMP9 and TGFbeta1 dose-dependently synergized on Smad2 phosphorylation, and showed an additive effect on expression of Smad3-dependent genes like bSerpine1 after 24 h. The activation of the TGFbeta/Smad3 signaling cascade was confirmed using the CAGA12-luc transcriptional reporter. BMP9 selectively induced bAlpl and bColX expression, which are considered early markers of cellular hypertrophy, but this was potently antagonized by addition of a low dose of TGFbeta1. CONCLUSIONS: This study shows that in vitro in chondrocytes, BMP9 potently induces pSmad1/5 and a chondrocyte hypertrophy-like state, which is potently blocked by TGFbeta1. This observation underlines the importance of TGFbeta1 in maintenance of chondrocyte phenotype.