Effect of Laminin-A4 inhibition on cluster formation of human osteoarthritic chondrocytes (original) (raw)
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Laminin Chain Expression by Chick Chondrocytes and Mouse Cartilaginous Tissuesin Vivoandin Vitro
Experimental Cell Research, 1997
protein . The resident cells, chondrocytes, are re-We have observed that laminins are expressed in sponsible for the production of these matrix molecules the chondrocytes of chick embryo sternum, mouse as determined by in vitro and in vivo studies . limb bud, and adult mouse knee joint by the methods During the formation of cartilage in development, there of in situ hybridization, immunohistochemistry, are changes in the amount and types of these compo-Western blotting, and immunoprecipitation. From in nents .
Histology and histopathology
Morphological and functional changes of chondrocytes are typical in OA cartilage. In this work, we have described noteworthy changes in intermediate filaments cytoskeleton evidenced by transmission electron microscopy. Alterations in the distribution as well as in the content of vimentin, actin, and tubulin have been described by specific fluorescence labelling of each cytoskeletal component and confocal analysis. Normal vs OA cartilages showed a reduction in the percentage of labelled chondrocytes of 37.1% for vimentin, 4.7% for actin, and 20.1% for tubulin. Statistical analysis of fluorescence intensities (mean % +/- SEM) between normal and OA rat cartilage revealed a highly significant difference in vimentin, a significant difference in tubulin, and a non-significant difference in actin. Moreover, by western blot, altered electrophoretic patterns were observed mainly for vimentin and tubulin in OA cartilage in comparison with normal cartilage. These results allow us to suggest th...
Calcified Tissue International, 2000
Osteocalcin (OC), which is a marker of the mature osteoblasts, can also be found in posthypertrophic chondrocytes of the epiphyseal growth plate, but not in chondrocytes of the resting zone or in adult cartilage. In human osteoarthritis (OA), chondrocytes can differentiate to a hypertrophic phenotype characterized by type X collagen. The protein-and mRNA-expression pattern of OC was systematically analyzed in decalcified cartilage and bone sections and nondecalcified cartilage sections of human osteoarthritic knee joints with different stages of OA to investigate the differentiation of chondrocytes in OA. In severe OA, we found an enhanced expression of the OC mRNA in the subchondral bone plate, demonstrating an increased osteoblast activity. Interestingly, the OC protein and OC mRNA were also detected in osteoarthritic chondrocytes, whereas in chondrocytes of normal adult cartilage, both the protein staining and the specific mRNA signal were negative. The OC mRNA signal increased with the severity of OA and chondrocytes from the deep cartilage layer, and proliferating chondrocytes from clusters showed the strongest signal for OC mRNA. In this late stage of OA, chondrocytes also stained for alkaline phosphatase and type X collagen. Our results clearly show that the expression of OC in chondrocytes correlates with chondrocyte hypertrophy in OA. Although the factors including this phenotypic shift in OA are still unknown, it can be assumed that the altered microenvironment around osteoarthritic chondrocytes and systemic mediators could be potential inducers of this differentiation.
Regulation of MMP3 by laminin alpha 4 in human osteoarthritic cartilage
Scandinavian Journal of Rheumatology, 2011
The degradation of cartilage is characterized by a loss of extracellular matrix (ECM) and the appearance of clusters of hypertrophic chondrocytes (1). The transmembrane proteoglycan syndecan is involved in the activation of aggrecanase-2 (ADAMTS5), which, together with other metalloproteinases, promotes matrix degradation (2). Syndecan-4 is a receptor for a number of ECM constituents and is a key regulator of cartilage degradation in osteoarthritis (OA) (2). Syndecans bind to laminins containing the alpha 4 subunit (3). Laminin alpha 4 chain (LAMA4) was first described as a constituent of basal membranes in blood vessels, the heart, and the kidney. Mutations in the murine gene encoding LAMA4 are associated with endothelial defects, cardiomyopathy, and chronic kidney disease (4, 5). We demonstrate that LAMA4 can be detected by immunohistochemistry in human osteoarthritic cartilage and that it colocalizes with syndecan-4 around hypertrophic chondrocytes. Moreover, treatment of human chondrocyte cultures with a function-inhibiting antibody to LAMA4 leads to a marked decrease in the transcription of the matrix metalloproteinase 3 (MMP3) gene, pointing to a possible regulatory role of laminin in human osteoarthritic cartilage.
2018
Osteoarthritis (OA) is a joint disease involving cartilage degeneration. This study aimed 15 to compare properties of chondrocytes from less-affected (LA-Cartilage) and severely-affected (SA16 Cartilage) of human OA articular cartilage. Based on Dougados classification, OA cartilage was 17 classified into two groups; less-affected (Grade 0-1) and severely-affected (Grade 2-3). Chondrocytes 18 from each group were cultured until passage (P) 4. Growth, migration, stem cell properties and 19 chondrogenic properties under normal and inflammatory conditions, and the formation of in vitro 20 3D cartilage tissues were compared between groups. The growth and migratory properties of LA21 chondrocytes and SA-chondrocytes were similar, except that the migration rate of SA-chondrocytes 22 was significantly higher at P0 compared to LA-chondrocytes. Both LA-chondrocytes and SA23 chondrocytes expressed mesenchymal stem cell markers and tri-lineage differentiation, but the 24 expression of stem cel...
Comparative proteomic analysis of hypertrophic chondrocytes in osteoarthritis
Clinical Proteomics, 2015
Background: Osteoarthritis (OA) is a multi-factorial disease leading progressively to loss of articular cartilage and subsequently to loss of joint function. While hypertrophy of chondrocytes is a physiological process implicated in the longitudinal growth of long bones, hypertrophy-like alterations in chondrocytes play a major role in OA. We performed a quantitative proteomic analysis in osteoarthritic and normal chondrocytes followed by functional analyses to investigate proteome changes and molecular pathways involved in OA pathogenesis. Methods: Chondrocytes were isolated from articular cartilage of ten patients with primary OA undergoing knee replacement surgery and six normal donors undergoing fracture repair surgery without history of joint disease and no OA clinical manifestations. We analyzed the proteome of chondrocytes using high resolution mass spectrometry and quantified it by label-free quantification and western blot analysis. We also used WebGestalt, a web-based enrichment tool for the functional annotation and pathway analysis of the differentially synthesized proteins, using the Wikipathways database. ClueGO, a Cytoscape plug-in, is also used to compare groups of proteins and to visualize the functionally organized Gene Ontology (GO) terms and pathways in the form of dynamical network structures. Results: The proteomic analysis led to the identification of a total of~2400 proteins. 269 of them showed differential synthesis levels between the two groups. Using functional annotation, we found that proteins belonging to pathways associated with regulation of the actin cytoskeleton, EGF/EGFR, TGF-β, MAPK signaling, integrin-mediated cell adhesion, and lipid metabolism were significantly enriched in the OA samples (p ≤10 −5). We also observed that the proteins GSTP1, PLS3, MYOF, HSD17B12, PRDX2, APCS, PLA2G2A SERPINH1/HSP47 and MVP, show distinct synthesis levels, characteristic for OA or control chondrocytes. Conclusion: In this study we compared the quantitative changes in proteins synthesized in osteoarthritic compared to normal chondrocytes. We identified several pathways and proteins to be associated with OA chondrocytes. This study provides evidence for further testing on the molecular mechanism of the disease and also propose proteins as candidate markers of OA chondrocyte phenotype.
PLOS ONE, 2015
The Integrin β1 family is the major receptors of the Extracellular matrix (ECM), and the synthesis and degradation balance of ECM is seriously disrupted during Osteoarthritis (OA). In this scenario, integrins modify their pattern expression and regulate chondrocyte differentiation in the articular cartilage. Members of the Transforming growth factor beta (Tgf-β) Superfamily, such as Growth differentiation factor 5 (Gdf-5) and Bone morphogenetic protein 7 (Bmp-7), play a key role in joint formation and could regulate the integrin expression during chondrocyte differentiation and osteoarthritis progression in an experimental OA rat model. Decrease of α5 integrin expression in articular cartilage was related with chondrocyte dedifferentiation during OA progression, while increase of α1, α2, and α3 integrin expression was related with fibrous areas in articular cartilage during OA. Hypertrophic chondrocytes expressedαV integrin and was increased in the articular cartilage of rats with OA. Integrin expression during chondrocyte differentiation was also analyzed in a micromass culture system of mouse embryo mesenchymal cells, micromass cultures was treated with Gdf-5 or Bmp-7 for 4 and 6 days, respectively. Gdf-5 induced the expression of theα5 sub-unit, while Bmp-7 induced the expression of the αV sub-unit. This suggests a switch in signaling for prehypertrophic chondrocyte differentiation towards hypertrophy, where Gdf-5 could maintain the articular chondrocyte phenotype and Bmp-7 would induce hypertrophy. Decrease of Ihh expression during late stages of OA in rat model suggest that the ossification in OA rat knees and endochondral ossification could be activated by Bmp-7 and αV integrin in absence of Ihh. Thus, chondrocyte phenotype in articular cartilage is similar to prehypetrophic chondrocyte in growth plate, and is preserved due to the presence of Indian hedgehog (Ihh), Gdf-5 and α5 integrin to maintain articular cartilage and prevent hy-pertrophy.
Osteoarthritis (OA) is a joint disease involving cartilage degeneration. This study aimed to compare properties of chondrocytes from less-affected (LA-Cartilage) and severely-affected (SA-Cartilage) of human OA articular cartilage. Based on Dougados classification, OA cartilage was classified into two groups; less-affected (Grade 0–1) and severely-affected (Grade 2–3). Chondrocytes from each group were cultured until passage (P) 4. Growth, migration, stem cell properties and chondrogenic properties under normal and inflammatory conditions, and the formation of in vitro 3D cartilage tissues were compared between groups. The growth and migratory properties of LA-chondrocytes and SA-chondrocytes were similar, except that the migration rate of SA-chondrocytes was significantly higher at P0 compared to LA-chondrocytes. Both LA-chondrocytes and SA-chondrocytes expressed mesenchymal stem cell markers and tri-lineage differentiation, but the expression of stem cell markers decre...