Articular cartilage is a connective tissue consisting of a specialized extracellular matrix (ECM) that dominates the bulk of its wet and dry weight. Type II collagen and aggrecan are the main ECM proteins in cartilage. However, little attention has been paid to less abundant molecular components, especially minor collagens, including type IV, VI, IX, X, XI, XII, XIII, and XIV, etc. Although accounting for only a small fraction of the mature matrix, these minor collagens not only play essential structural roles in the mechanical properties, organization, and shape of articular cartilage, but also fulfil specific biological functions. Genetic studies of these minor collagens have revealed that they are associated with multiple connective tissue diseases, especially degenerative joint disease. The progressive destruction of cartilage involves the degradation of matrix constituents including these minor collagens. The generation and release of fragmented molecules could generate novel biochemical markers with the capacity to monitor disease progression, facilitate drug development and add to the existing toolbox for in vitro studies, preclinical research and clinical trials.
A T-cell permissive tumor microenvironment, characterized by the presence of activated T cells and low fibrotic activity is crucial for response to immune checkpoint inhibitors (ICIs). Granzyme B has been shown to promote T-cell migration through the basement membrane by the degradation of type IV collagen. In this study, we evaluated the biomarker potential of measuring granzyme B-mediated degradation of type IV collagen (C4G) in combination with a fibroblast activation biomarker (PRO-C3) non-invasively for identifying metastatic melanoma patients responding to the ICI ipilimumab. A monoclonal antibody was generated against C4G and used to develop a competitive electro-chemiluminescence immunoassay. C4G and PRO-C3 were measured in pretreatment serum from metastatic melanoma patients (n = 54). The C4G assay was found specific for a granzyme B-generated neo-epitope on type IV collagen. The objective response rate (ORR) was 2.6-fold higher (18% vs. 7%) in patients with high C4G levels (>25th percentile) vs. low levels (≤25th percentile). Likewise, high C4G levels at baseline were associated with longer overall survival (OS) (log-rank, p = 0.040, and hazard ratio (HR) = 0.48, 95%CI: 0.24–0.98, p = 0.045). Combining high C4G with low PRO-C3 correlated with improved OS with a median OS of 796 days vs. 273 days (p = 0.0003) and an HR of 0.30 (95%CI: 0.15–0.60, p = 0.0006). In conclusion, these results suggest that high granzyme B degraded type IV collagen (C4G) combined with low PRO-C3 quantified non-invasively has the potential to identify the responders to ICI therapy.
Background: Osteoarthritis (OA) is a progressive, chronic disease characterized by articular cartilage destruction. The pro-inflammatory cytokine IL-17 levels have been reported elevated in serum and synovial fluid of OA patients and correlated with increased cartilage defects and bone remodeling. The aim of this study was to characterize an IL-17-mediated articular cartilage degradation ex-vivo model and to investigate IL-17 effect on cartilage extracellular matrix protein turnover. Methods: Full-depth bovine femoral condyle articular cartilage explants were cultured in serum-free medium for three weeks in the absence, or presence of cytokines: IL-17A (100 ng/ml or 25 ng/ml), or 10 ng OSM combined with 20 ng/ml TNFα (O + T). RNA isolation and PCR analysis were performed on tissue lysates to confirm IL-17 receptor expression. GAG and ECM-turnover biomarker release into conditioned media was assessed with dimethyl methylene blue and ELISA assays, respectively. Gelatin zymography was used for matrix metalloproteinase (MMP) 2 and MMP9 activity assessment in conditioned media, and shotgun LC-MS/MS for identification and label-free quantification of proteins and protein fragments in conditioned media. Western blotting was used to validate MS results. Results: IL-17RA mRNA was expressed in bovine full-depth articular cartilage and the treatment with IL-17A did not interfere with metabolic activity of the model. IL-17A induced cartilage breakdown; conditioned media GAG levels were 3.6-fold-elevated compared to untreated. IL-17A [100 ng/ml] induced ADAMTS-mediated aggrecan degradation fragment release (14-fold increase compared to untreated) and MMP-mediated type II collagen fragment release (6-fold-change compared to untreated). MS data analysis revealed 16 differentially expressed proteins in IL-17A conditioned media compared to untreated, and CHI3L1 upregulation in conditioned media in response to IL-17 was confirmed by Western blotting. Conclusions: We showed that IL-17A has cartilage modulating potential. It induces collagen and aggrecan degradation indicating an upregulation of MMPs. This was confirmed by zymography and mass spectrometry data. We also showed that the expression of other cytokines is induced by IL-17A, which provide further insight to the pathways that are active in response to IL-17A. This exploratory study confirms that IL-17A may play a role in cartilage pathology and that the applied model may be a good tool to further investigate it.
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