Chondrocyte Catabolism Research Articles

Parthenolide inhibits tumor necrosis factor-alpha induced catabolism of aggrecan in chondrocytes in osteoarthritis: in vitro experiment with cultured human chondrocytes

To investigate the effect of parthenolide (PAR) on the tumor necrosis factor (TNF)-alpha induced aggrecan catabolism of chondrocytes in osteoarthritis (OA). Human chondrocytes were obtained from the condyles of femur of OA patients undergoing knee joint replacement during operation, cultured, and randomly divided into 4 groups: control group, TNF group (cultured in medium containing 10 ng/ml TNF-alpha) , PAR group (cultured in medium containing 10 micromol/L PAR), and PAR + TNF group (cultured in medium containing 10 micromol/L PAR and 10 ng/ml TNF-alpha). Eight days later 1, 9-dimethylmethylene blue spectrophotometric assay was used to measure the content of glycosaminoglycan (GAG) , marker of aggrecan catabolism, in the culture fluid and the cells. Using anti-aggrecan monoclonal antibodies Mab 5D4 and 3B3, ELISA was employed to detect the contents of 5D4 and 3B3, aggrecan catabolic fragments. RT-PCR was used to detect the mRNA expression of the aggrecan, ADAMTS-4 and ADAMTS-5, both aggrecanases, tissue inhibitor of metalloprotease (TIMP)-1, mitogen-activated protein kinase (MAPK)-1 and 18S, a marker. The GAG percentage in the culture fluid of the TNF-alpha was 57.1% +/- 2.0%, significantly higher than those of the control and TNF-alpha + PAR groups (P = 0.001 and 0.02). The 5D4 fragment level of the TNF-alpha group was 509 ng/ml +/- 32 ng/ml, significantly higher than that of the control group (166 ng/ml +/- 15 ng/ml, t = 11.60, P =0.007), and the level of 5D4 fragment of the PAR + TNF-alpha group was 333 ng/ml +/- 15 ng/ml, significantly lower than that of the TNF-alpha group (t = 7.93, P = 0.016). There was not significant difference in the 3B3 fragment level among the 4 groups (F = 1.316, P = 0.335). The aggrecan mRNA expression level of the TNF-alpha group was significantly lower than those of the other 3 groups (F = 133.7, P = 0.000), the mRNA expression levels of ADAMTS-5 and MAPK-1 of the TNF-alpha group were significantly higher than those of the other 3 groups (F = 209. 7, 117.1; P =0. 000), the ADAMTS-5 mRNA expression level of the PAR group was significantly lower than that of the control group (t = 11.1, P= 0.008) , and there was not significant differences in the mRNA expression levels of ADAMTS-4 and TIMP-1 among the 4 groups (F = 1.87, 0.73; P > 0.05) . PAR inhibits TNF-alpha induced catabolism of aggrecan in the chondrocytes of OA and reduces the mRNA expression of ADAMTS-5 and MAPK-1. PAR may be useful in the treatment of OA and other inflammatory joint diseases.

Cytokines as therapeutic targets for osteoarthritis.

Osteoarthritis (OA) is a debilitating, progressive disease of diarthrodial joints associated with the aging process. With the exception of anti-inflammatory corticosteroids and nonsteroidal anti-inflammatory drugs which inhibit cyclo-oxygenase-2, the enzyme responsible for prostaglandin biosynthesis in inflammation, no specific therapy based on fundamental intracellular pathways of chondrocytes and synoviocytes exists for the medical management of OA. At the molecular level, OA is characterized by an imbalance between chondrocyte anabolism and catabolism. Disruption of chondrocyte homeostasis primarily affects the cartilage extracellular matrix (ECM), which is responsible for the biomechanical properties of the tissue. Recent evidence has implicated cytokines, among which interleukin (IL)-1, tumor necrosis factor-alpha, IL-6, and IL-17 seem most involved in the OA process of cartilage destruction. The primary role of these cytokines is to modulate the expression of matrix metalloproteinases and cartilage ECM proteins. Cartilage repair that could restore the functional integrity of the joint is also impaired because chondrocytes in OA cartilage appear unable to respond to insulin-like growth factor-1 or respond abnormally to transforming growth factor-beta. As these growth factors also modulate cytokine expression, they may prove useful in designing strategies for suppressing 'chondrocyte activation'. Although cytokines and growth factors provide a potential therapeutic target for OA, it will be necessary to elucidate the fundamental mechanisms that cytokines employ to cause chondrocyte and synoviocyte dysfunction before 'anti-cytokine' therapy can be employed in the medical management of the disease.

Continuous cyclic load reduces proteoglycan release from articular cartilage

Objective: to study the effect of a continuous cyclic mechanical load on the release of newly synthesized proteoglycans (PGs) from mature bovine articular cartilage.Methods: Viable cartilage explants were continuously loaded with 1 MPa cyclic stress at 1 Hz frequency for 24 h, and the release of labeled (35SO4) PGs measured before, during and after application of the compressive load. To separate the effect of active chondrocyte catabolism from that of passive PG release, PG release in live explants, with and without protease inhibitors to inhibit PG breakdown, was compared to PG release in explants whose chondrocytes were killed prior to loading.Results: In live explants, a continuous cyclic load significantly reduced PG release by as much as 50% compared to unloaded explants. In killed explants which were unloaded, the PG release increased five to 10 times, while a cyclic load reduced PG release to that found in viable, loaded explants. Twenty-four hours after load removal PG release in all loaded explants returned (increased) to that of the unloaded explants.Conclusions: These results indicate that PG release from the cartilage matrix is inhibited by continuous cyclic mechanical loading, independent of cellular metabolism, and suggest that a primary mechanism for reducing PG release is by decreasing the interstitial porosity through which the PGs can escape.

Increased Expression of CD44 in Bovine Articular Chondrocytes by Catabolic Cellular Mediators

Bovine articular chondrocytes cultured in alginate beads were used to study the effect of catabolic cellular mediators on CD44 expression. Treatment with either the 29-kDa fragment of fibronectin or interleukin-1 alpha results in a time- and dose-dependent inhibition of proteoglycan synthesis as well as a stimulation in the expression of CD44 mRNA level as determined by semi-quantitative polymerase chain reaction following reverse transcription. No noticeable effect at 6 h was observed. By 24 h, the major CD44 product (CD44H) from fibronectin fragment-treated cultures showed an 8-fold increase; CD44H from interleukin-1 alpha-treated cultures showed a 6-fold increase as compared to control cultures. In addition, a minor band, determined to be an isoform of CD44, was also shown to be up-regulated by both mediators. Stimulation of CD44 mRNA via interleukin-1 was also evident by in situ hybridization studies of bovine as well as human articular cartilage in organ culture. The increased in CD44 mRNA is matched by an increase at the protein level as determined by Western blot analysis. The Western blot reveals a doublet protein band at 80-90 kDa that corresponds to the molecular mass of CD44H. Cultures incubated with fibronectin fragments for 24 h had an 8.0-fold increase in CD44, while a 6.6-fold was observed for interleukin-1 alpha. Fluorescein-conjugated hyaluronan binding and internalization studies indicate that the increase in CD44 protein, induced by interleukin-1 alpha, closely correlates with an increase in functional hyaluronan receptors present at the chondrocyte cell surface. Taken together these results indicate that conditions that up-regulate chondrocyte catabolism also up-regulate the expression of CD44, a cell surface hyaluronan receptor involved in hyaluronan endocytosis.

Catabolic effects of muramyl dipeptide on rabbit chondrocytes

Muramyl dipeptide, an essential structure for the diverse biologic activities of bacterial cell wall peptidoglycan, inhibited the synthesis of glycosaminoglycan/proteoglycan in cultured rabbit costal chondrocytes in a dose-dependent manner. Muramyl dipeptide, as well as lipopolysaccharide and interleukin-1 alpha, also enhanced the release of 35S-sulfate-prelabeled glycosaminoglycan/proteoglycan from the cell layer, which seems to reflect, at least partially, the increasing degradation of glycosaminoglycan/proteoglycan. Five synthetic analogs of muramyl dipeptide known to be adjuvant active or adjuvant inactive were tested for their potential to inhibit synthesis of glycosaminoglycan/proteoglycan and to enhance the release of glycosaminoglycan/proteoglycan in chondrocytes. The structural dependence of these synthetic analogs on chondrocytes was found to parallel that of immunoadjuvant activity. These results suggest that muramyl dipeptide is a potent mediator of catabolism in chondrocytes.