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Murine Cartilage (murine + cartilage)
Selected AbstractsMorphometric characterization of murine articular cartilage,Novel application of confocal laser scanning microscopyMICROSCOPY RESEARCH AND TECHNIQUE, Issue 9 2009Kathryn S. Stok Abstract A new technique for characterization of the three-dimensional morphology of murine articular cartilage is proposed. The technique consists of a novel application of confocal laser scanning microscopy (CLSM), where the objective was to develop and validate it for cartilage measurements in murine joints. Murine models are used in arthritis research, because they are well-described for manipulating the disease pathophysiology, facilitating our understanding of the disease, and identifying new targets for therapy. A calibration and reproducibility study was carried out to provide a consistent testing methodology for quantification of murine joints. The proximal tibial condyles from male C57BL/6 mice were scanned using a CLS microscope with an isotropic voxel size of 5.8 ,m. Measurements and analyses were repeated three times on different days, and in a second step the analysis was repeated three times for a single measurement. Calculation of precision errors (coefficient of variation) for cartilage thickness and volume was made. The bias of the system was estimated through comparison with histology. This technique showed good precision, with errors in the repeated analysis ranging from 0.63% (lateral thickness) to 3.48% (medial volume). The repeated analysis alone was robust, with intraclass correlations for the different compartments between 0.918 and 0.991. Measurement bias was corrected by scaling the confocal images to 32% of their width to match histology. CLSM provided a fast and reproducible technique for gathering 3D image data of murine cartilage and will be a valuable tool in understanding the efficacy of arthritis treatments in murine models. Microsc. Res. Tech. 2009. © 2009 Wiley-Liss, Inc. [source] Aurothiomalate inhibits cyclooxygenase 2, matrix metalloproteinase 3, and interleukin-6 expression in chondrocytes by increasing MAPK phosphatase 1 expression and decreasing p38 phosphorylation: MAPK phosphatase 1 as a novel target for antirheumatic drugsARTHRITIS & RHEUMATISM, Issue 6 2010Riina Nieminen Objective Aurothiomalate is a disease-modifying antirheumatic drug that suppresses inflammation and retards cartilage degradation and bone erosion in arthritis. The molecular mechanisms of action of aurothiomalate are not known in detail. MAPK pathways are major signaling pathways in inflammation that regulate the production of many inflammatory and destructive factors in arthritis. The purpose of the present study was to investigate the effects of aurothiomalate on the activity of p38 MAPK and on the expression of MAPK phosphatase 1 (MKP-1), cyclooxygenase 2 (COX-2), matrix metalloproteinase 3 (MMP-3), and interleukin-6 (IL-6) in immortalized murine H4 chondrocytes and in intact human and murine cartilage. Methods Protein expression was examined by Western blotting or by enzyme-linked immunosorbent assay, and messenger RNA (mRNA) expression was examined by real-time reverse transcription,polymerase chain reaction analysis. The mediator role of MKP-1 was investigated by using small interfering RNA (siRNA) methods to down-regulated MKP-1 expression in chondrocytes in culture and by comparing the responses in intact cartilage from MKP-1,deficient and wild-type mice. The effects of aurothiomalate were also confirmed in human rheumatoid cartilage by using tissue samples obtained at the time of total knee replacement surgery. Results Aurothiomalate inhibited IL-1,,induced COX-2 expression and prostaglandin E2 production by destabilizing COX-2 mRNA, as did the p38 MAPK inhibitor SB203580. Interestingly, aurothiomalate also increased the expression of MKP-1 and reduced the IL-1,,induced phosphorylation of p38 MAPK. Knockdown of MKP-1 by siRNA significantly impaired the ability of aurothiomalate to inhibit the phosphorylation of p38 MAPK and the expression of COX-2, MMP-3, and IL-6. Likewise, aurothiomalate reduced COX-2, MMP-3, and IL-6 expression in articular cartilage from patients with rheumatoid arthritis, as well as in articular cartilage from wild-type mice but not from MKP-1,/, mice. Conclusion Our findings indicate a novel mechanism for the antiinflammatory and antierosive actions of aurothiomalate, through increased expression of MKP-1, which leads to reduced activation of p38 MAPK and suppressed expression of COX-2, MMP-3, and IL-6. The results suggest that manipulation of MKP-1 levels is a promising new mechanism to be directed in the search and development of novel antiinflammatory and antierosive compounds that have the good efficacy of gold compounds but not their toxicity. [source] Fibroblast growth factor 2 is an intrinsic chondroprotective agent that suppresses ADAMTS-5 and delays cartilage degradation in murine osteoarthritisARTHRITIS & RHEUMATISM, Issue 7 2009Shi-Lu Chia Objective We have previously identified in articular cartilage an abundant pool of the heparin-binding growth factor, fibroblast growth factor 2 (FGF-2), which is bound to the pericellular matrix heparan sulfate proteoglycan, perlecan. This pool of FGF-2 activates chondrocytes upon tissue loading and is released following mechanical injury. In vitro, FGF-2 suppresses interleukin-1,driven aggrecanase activity in human cartilage explants, suggesting a chondroprotective role in vivo. We undertook this study to investigate the in vivo role of FGF-2 in murine cartilage. Methods Basal characteristics of the articular cartilage of Fgf2,/, and Fgf2+/+ mice were determined by histomorphometry, nanoindentation, and quantitative reverse transcriptase,polymerase chain reaction. The articular cartilage was graded histologically in aged mice as well as in mice in which osteoarthritis (OA) had been induced by surgical destabilization of the medial meniscus. RNA was extracted from the joints of Fgf2,/, and Fgf2+/+ mice following surgery and quantitatively assessed for key regulatory molecules. The effect of subcutaneous administration of recombinant FGF-2 on OA progression was assessed in Fgf2,/, mice. Results Fgf2,/, mice were morphologically indistinguishable from wild-type (WT) animals up to age 12 weeks; the cartilage thickness and proteoglycan staining were equivalent, as was the mechanical integrity of the matrix. However, Fgf2,/, mice exhibited accelerated spontaneous and surgically induced OA. Surgically induced OA in Fgf2,/, mice was suppressed to levels in WT mice by subcutaneous administration of recombinant FGF-2. Increased disease in Fgf2,/, mice was associated with increased expression of messenger RNA of Adamts5, the key murine aggrecanase. Conclusion These data identify FGF-2 as a novel endogenous chondroprotective agent in articular cartilage. [source] Regulation of autophagy in human and murine cartilage: Hypoxia-inducible factor 2 suppresses chondrocyte autophagyARTHRITIS & RHEUMATISM, Issue 5 2009Jolene Bohensky Objective We have previously demonstrated that the transcription factor hypoxia-inducible factor 1 (HIF-1) promotes the onset of autophagy in chondrocytes. The overall goal of this study was to test the hypothesis that another HIF family transcription factor, HIF-2, modulates the induction of autophagy by chondrocytes. Methods Expression of HIF-1, HIF-2, and light chain 3 (LC3) in human and murine articular cartilage was visualized by immunohistochemistry. Suppression of HIF-2 was achieved using small interfering RNA technology. Assessments of autophagic flux and lysosomal activity, as well as ultrastructural analysis, were performed in chondrocytes in cell culture. Results HIF-2 was expressed abundantly by cells in human and murine articular cartilage and in the cartilage of mineralizing vertebrae from neonatal mice. Protein levels were reduced in articular cartilage from older mice, in end-plate cartilage from mice, and in chondrocytes from human osteoarthritic (OA) cartilage. HIF-2 was robustly expressed in the prehypertrophic cells of mouse growth cartilage. When HIF-2, was silenced, the generation of reactive oxygen species was found to be elevated, with a concomitant decrease in catalase and superoxide dismutase activity. Suppression of HIF-2 was associated with decreased Akt-1 and mammalian target of rapamycin activities, reduced Bcl-xL expression, and a robust autophagic response, even under nutrient-replete conditions. In these silenced chondrocytes, HIF-1 expression was elevated. Decreased HIF-2 expression was associated with autophagy in OA tissues and aging cartilage samples. The autophagic response of chondrocytes in HIF-2,,knockout mouse growth plate showed an elevated autophagic response throughout the plate. Conclusion Based on these observations, we conclude that HIF-2 is a potent regulator of autophagy in maturing chondrocytes. Our data suggest that this protein acts as a brake on the autophagy-accelerator function of HIF-1. [source] Involvement of the notch pathway in the regulation of matrix metalloproteinase 13 and the dedifferentiation of articular chondrocytes in murine cartilageARTHRITIS & RHEUMATISM, Issue 2 2009Régis Blaise Objective To demonstrate the activation of the Notch signaling pathway during changes in the phenotype of chondrocytes in vitro, and to assess the influence of Notch on the production of chondrocyte markers. Methods Serial monolayer primary cultures of murine articular chondrocytes (MACs), as a model of chondrocyte dedifferentiation, were prepared. MACs were cultured with or without a Notch inhibitor and transfected with different Notch -expressing vectors. The Notch pathway and chondrocyte marker profiles were assessed by quantitative reverse transcription,polymerase chain reaction, immunoblotting, and immunocytochemistry. Results Successive passages of MACs resulted in a loss of type II collagen and aggrecan (chondrocyte differentiation markers), an increase in type I collagen (dedifferentiation marker), an increase in Notch ligands, and augmented target gene activity. The Notch inhibitor decreased the type II collagen protein content but had no effect on Col2a1 messenger RNA, while transfection with the constitutive active forms of the Notch1 receptor led to a decrease in type II collagen in transfected cells. In assays to investigate the mechanism of type II collagen breakdown, matrix metalloproteinase 13 (MMP-13) synthesis was regulated in a Notch-dependent manner, whereas MMP-2 synthesis was unchanged. Conclusion The Notch signaling pathway is associated with decreased type II collagen production during the dedifferentiation of MACs in vitro. This may be correlated with the increase in MMP-13 production linked to activation of Notch. [source] | ||||||||||