Protective effect of atorvastatin in cultured osteoarthritic chondrocytes

The aim of our study was to evaluate the in vitro effect of an HMG‐CoA reductase inhibitor, atorvastatin, on the expression of significant anabolic and catabolic genes in human osteoarthritic chondrocytes and to explore the metabolic pathways involved in this process. Human articular osteoarthritic chondrocytes were cultured in the presence and absence of atorvastatin (10 and 50 µmol/L) for 24 h. Metalloproteinase 13 (MMP‐13), collagen type II (COL2A1), and aggrecan (AGC) mRNA expression levels were evaluated by real‐time PCR, and protein expression levels by Western blot analysis. IL‐1β levels in culture medium was analyzed with ELISA. The effect of the treatment with the mevalonate isoprenoid derivatives farnesol and geranylgeraniol, or the cholesterol precursor squalene, was evaluated in the atorvastatin osteoarthritic chondrocyte cultures. Incubation of osteoarthritic chondrocyte cultures with atorvastatin produced a significant dose‐dependent reduction in IL‐1β production. Atorvastatin supplementation in cultures produced a decrease in MMP‐13 mRNA and protein expression levels, which was reversed by the addition of farnesol. Regarding AGC and COL2A1 mRNA expression, a significant increase was observed only in chondrocytes cultures treated with 50 µmol/L atorvastatin. Our findings suggest that atorvastatin may have potential chondroprotective effects mostly by reducing cartilage degradation. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:110–115, 2010     

Cartilage-derived morphogenetic protein-1 and -2 are endogenously expressed in healthy and osteoarthritic human articular chondrocytes and stimulate matrix synthesis

OBJECTIVE: We investigated whether chondrocytes derived from osteoarthritic cartilage may lose their responsiveness to cartilage-derived morphogenetic protein-1, -2 (CDMP-1, -2) and osteogenic protein-1 (OP-1) compared with healthy cells, thus leading to an impaired maintenance of matrix integrity. DESIGN: Chondrocytes were isolated from articular cartilage from patients with and without osteoarthritic lesions. Cells were grown as monolayer cultures for 7 days in a chemically defined serum-free basal medium (BM) in the presence of recombinant CDMP-1, -2, and OP-1. Glycosaminoglycan synthesis was measured by [35S]Sulfate incorporation into newly synthesized macromolecules. Cell proliferation was investigated by [3H]Thymidine incorporation. The endogenous gene expression of CDMPs/OP-1 and their respective type I and type II receptors was examined using RT-PCR. The presence of CDMP proteins in tissue and cultured cells was detected by Western immunoblots. RESULTS: mRNAs coding for CDMPs and their respective receptors are endogenously expressed not only in healthy, but also in osteoarthritic cartilage. CDMP proteins are present in both normal and osteoarthritic articular cartilage and cultured chondrocytes. CDMP-1, CDMP-2 and OP-1 markedly increased glycosaminoglycan synthesis in both healthy (P< 0.01) and osteoarthritic (P< 0.05) human articular chondrocytes. A comparison of the glycosaminoglycan biosynthetic activity between healthy and osteoarthritic samples revealed no detectable difference, neither in stimulated nor in unstimulated cultures. [(3)H]Thymidine incorporation showed that CDMPs/OP-1 did not affect cell proliferation in vitro. CONCLUSION: CDMPs and OP-1 exert their anabolic effects on both healthy and osteoarthritic chondrocytes indicating no loss in responsiveness to these growth factors in OA. The endogenous expression of CDMPs/OP-1 and their receptors suggest an important role in cartilage homeostasis.     

Intermittent compressive strain may reduce aggrecanase expression in cartilage: a study of chondrocytes in agarose gel

We tested the hypothesis that intermittent compressive strain reduces the catabolic actions of interleukin-1 beta on chondrocyte metabolism at the gene expression level. We investigated the effects of intermittent compressive strain on mRNA expression in bovine chondrocytes cultured in agarose gel supplemented with or without interleukin-1 beta. Fifteen percent compressive strain amplitude was applied to agarose-chondrocyte constructs at a frequency of 1 Hz. In the absence of interleukin-1 beta, the strain caused an increase in the mRNA levels of anabolic factors like aggrecan and Type II collagen, compared with the levels of anabolic factors in unstrained constructs. These results suggest that the strain may stimulate matrix production in normal cartilage. Interleukin-1 beta is a powerful catabolic agent; in unstrained agarose-chondrocyte constructs, interleukin-1 beta caused a decrease in the mRNA levels of anabolic factors. However, interleukin-1 beta also caused an increase in the mRNA of catabolic factors like aggrecanase-1, aggrecanase-2, and matrix metalloproteinase-3. In the presence of interleukin-1 beta, the strain reduced the mRNA levels of aggrecanase-1 and aggrecanase-2. These results suggest that intermittent compressive strain may protect cartilage by suppressing the expression of aggrecanase-1 and aggrecanase-2, which are thought to be the major matrix-degrading enzymes responsible for cleaving aggrecan.     

Prevalence and consequences of musculoskeletal symptoms in symphony orchestra musicians vary by gender: a cross-sectional study

Background

Tenascin-C (TN-C) is an extracellular matrix glycoprotein that is involved in tissue injury and repair processes. We analyzed TN-C expression in normal and osteoarthritic (OA) human cartilage, and evaluated its capacity to induce inflammatory and catabolic mediators in chondrocytes in vitro. The effect of TN-C on proteoglycan loss from articular cartilage in culture was also assessed.

Methods

TN-C in culture media, cartilage extracts, and synovial fluid of human and animal joints was quantified using a sandwich ELISA and/or analyzed by Western immunoblotting. mRNA expression of TN-C and aggrecanases were analyzed by Taqman assays. Human and bovine primary chondrocytes and/or explant culture systems were utilized to study TN-C induced inflammatory or catabolic mediators and proteoglycan loss. Total proteoglycan and aggrecanase -generated ARG-aggrecan fragments were quantified in human and rat synovial fluids by ELISA.

Results

TN-C protein and mRNA expression were significantly upregulated in OA cartilage with a concomitant elevation of TN-C levels in the synovial fluid of OA patients. IL-1 enhanced TN-C expression in articular cartilage. Addition of TN-C induced IL-6, PGE₂, and nitrate release and upregulated ADAMTS4 mRNA in cultured primary human and bovine chondrocytes. TN-C treatment resulted in an increased loss of proteoglycan from cartilage explants in culture. A correlation was observed between TN-C and aggrecanase generated ARG-aggrecan fragment levels in the synovial fluid of human OA joints and in the lavage of rat joints that underwent surgical induction of OA.

Conclusions

TN-C expression in the knee cartilage and TN-C levels measured in the synovial fluid are significantly enhanced in OA patients. Our findings suggest that the elevated levels of TN-C could induce inflammatory mediators and promote matrix degradation in OA joints.     

Reactive nitrogen and oxygen species in interleukin-1-mediated DNA damage associated with osteoarthritis

OBJECTIVE: Osteoarthritis (OA) is associated with increased levels of reactive nitrogen and oxygen species and pro-inflammatory cytokines, such as interleukin-1 (IL-1). Nitric oxide (NO) can mediate a number of the catabolic effects of IL-1 in articular cartilage. The aims of this study were to determine if OA cartilage shows evidence of DNA damage, and if IL-1 could induce DNA damage in non-OA cartilage by increasing NO or superoxide. METHODS: Articular chondrocytes were isolated from porcine femoral condyles and embedded in 1.2% alginate. The effects of 24h incubation with IL-1, the nitric oxide synthase 2 (NOS2)-selective inhibitor, the free radical scavenger superoxide dismutase (SOD), the NO donor NOC18, or the combined NO and peroxynitrite donor SIN-1 on DNA damage were tested, using the "comet" assay. NO production was measured using the Griess assay. The type of oxidative damage present was assessed using a modified comet assay. RESULTS: OA cartilage had significantly more DNA damage than non-OA cartilage (P<0.001). IL-1 caused an increase in DNA damage (P<0.01), which was associated with increased NO production (P<0.01). Both oxidative DNA strand breaks and base modifications of purines and pyrimidines were observed. IL-1-induced DNA damage was inhibited by an NOS2 inhibitor or by SOD (P<0.01). Furthermore, NOC18 or SIN-1 caused DNA damage (P<0.001). CONCLUSION: Our work shows chondrocytes in osteoarthritic cartilage exhibit DNA damage, and that IL-1 induces DNA damage and reactive oxygen and nitrogen species in non-OA chondrocytes in alginate.     

Connective tissue growth factor mRNA expression pattern in cartilages is associated with their type I collagen expression

Connective tissue growth factor (CTGF) has been identified as a secretory protein encoded by an immediate early gene and is a member of the CCN family. In vitro CTGF directly regulates the proliferation and differentiation of chondrocytes; however, a previous study showed that it was localized only in the hypertrophic chondrocytes in the costal cartilages of E 18 mouse embryos. We described the expression of CTGF mRNA and protein in chondrocytes of different types of cartilages, including femoral growth plate cartilage, costal cartilage, femoral articular cartilage, mandibular condylar cartilage, and cartilage formed during the healing of mandibular ramus fractures revealed by in situ hybridization and immunohistochemistry. To characterize the CTGF-expressing cells, we also analyzed the distribution of the type I, type II, and type X collagen mRNA expression. Among these different types of cartilages we found distinct patterns of CTGF mRNA and protein expression. Growth plate cartilage and the costal cartilage showed localization of CTGF mRNA and protein in the hypertrophic chondrocytes that expressed type X collagen mRNA with less expression in proliferating chondrocytes that expressed type II collagen mRNA, whereas it was also expressed in the proliferating chondrocytes that expressed type I collagen mRNA in the condylar cartilage, the articular cartilage, and the cartilage appearing during fracture healing. In contrast, the growth plate cartilages or the costal cartilages were negative for type I collagen and showed sparse expression of CTGF mRNA in the proliferating chondrocytes. We found for the first time that CTGF mRNA could be differentially expressed in five different types of cartilage associated with those expressing type I collagen. Moreover, the spatial distribution of CTGF mRNA in the cartilages with type I collagen mRNA suggested its roles in the early differentiation, as well as in the proliferation and the terminal differentiation, of those cartilages.     

Glucosamine decreases expression of anabolic and catabolic genes in human osteoarthritic cartilage explants

OBJECTIVE: To investigate the effect of glucosamine (GlcN) in a human osteoarthritic explant model on expression of genes involved in anabolic and catabolic activities of chondrocytes. METHODS: Human osteoarthritic explants, obtained during knee arthroplasty surgery, were pre-cultured (3 days) and treated with glucosamine-hydrochloride (GlcN-HCl) or glucosamine-3-sulphate (GlcN-S) at 0.5mM and 5mM (4 days). RNA was isolated from the explants and real time RT-PCR was performed. Additionally, total matrix metalloproteinase (MMP) activity was measured in culture medium. RESULTS: Addition of 5mM GlcN led to significant down-regulation of aggrecan (2.65-7.73-fold) and collagen type II (7.75-22.17-fold) gene expression, indicating inhibited anabolic activity. Considering catabolic activities, 5mM GlcN significantly down-regulated aggrecanase-1 and MMP3 and 5mM GlcN-S additionally down-regulated aggrecanase-2 and tissue inhibitor of MMP gene expression significantly. Gene expression was not significantly altered by 0.5mM GlcN. Total MMP activity in culture medium was only significantly reduced after addition of 5mM GlcN-HCl. CONCLUSION: The effects of GlcN on gene expression in a human osteoarthritic explant model suggest that enzymatic breakdown of the extra-cellular matrix might be reduced by the addition of 5mM GlcN. Additionally, restoration of already damaged cartilage is not to be expected, because gene expression of anabolic genes is also down-regulated. We suggest that chondroprotective properties of GlcN in vivo may be based on inhibiting further degradation due to catabolic activities, rather than on the ability to rebuild cartilage.     

Susceptibility to physiological concentrations of IL-1beta varies in cartilage at different anatomical locations on human osteoarthritic knee joints

OBJECTIVES: To determine whether cartilage biopsies from specific regions of osteoarthritic knee joints differ in susceptibility to the degradative effects of the amounts of interleukin-1 beta (IL-1 beta; 1-10 pg/ml) found in osteoarthritic joints. To establish whether biopsies are sensitive to the effects of either IL-1 beta or TNFalpha or both catabolic cytokines. METHODS: Cartilage from specified regions of 22 osteoarthritic knee joints was examined. Biopsies were incubated for 14 days without or with IL-1 beta or TNFalpha at physiological concentrations and GAG release into supernatants assessed. RESULTS: Variation was observed in susceptibility to the effects of 1-10 pg/ml IL-1 beta in biopsies from different sites within the same joints and the same site in different joints. The number of regions responding to the cytokine increased significantly (P< 0.0063, Chi square test) with concentration: only 10% (2/21) of all regions tested were susceptible to the effects of 1 pg/ml IL-1 beta, whereas 45% (9/20) were susceptible to the effects of 5 pg/ml and 56% (10/18) to the effects of 10 pg/ml IL-1 beta. Significantly fewer regions (4%, 2/47) responded to both IL-1 beta and TNFalpha (P< 0.047, Chi square test); biopsies from some patients responded to neither cytokine. CONCLUSIONS: IL-1 beta, at the low concentrations detected in joints, can degrade cartilage from susceptible locations. Susceptibility of some cartilage biopsies to the effects of either IL-1 beta and TNFalpha, but not both, suggests the signalling receptors for the two major catabolic cytokines are not usually expressed concurrently. The fact that some biopsies respond to neither cytokine suggests that in some patients the local concentration of inhibitors may be high or that other catabolic stimuli predominate. These results could have important implications for pharmacological intervention strategies.     

Effects of transforming growth factor-beta on aggrecanase production and proteoglycan degradation by human chondrocytes in vitro

OBJECTIVE: Aggrecan is degraded by Aggrecanases (ADAMTS-4 and -5) and MMPs, which cleave its core protein at different sites. Transforming growth factor (TGF)beta is known to stimulate matrix formation in cartilage, and ADAMTS-4 production in synoviocytes. The aim of this in-vitro study was to examine the effects of TGFbeta on aggrecanase production in human cartilage. DESIGN: Expression of ADAMTS-4 and -5 in chondrocyte cultures from normal or osteoarthritic cartilage was studied at mRNA level by RT-PCR. Aggrecanase activity was examined by western blot of aggrecanase-generated neoepitope NITEGE, and by measure of proteoglycan degradation in cartilage explants. RESULTS: TGFbeta strongly increased mRNA levels of ADAMTS-4, while ADAMTS-5 was expressed in a constitutive way in chondrocytes from normal and osteoathritic cartilage. TGFbeta also increased NITEGE levels and proteoglycan degradation. Addition of an aggrecanase inhibitor blocked the increase of NITEGE, and partially inhibited proteoglycan degradation. CONCLUSIONS: TGFbeta stimulates ADAMTS-4 expression and aggrecan degradation in cartilage. This catabolic action seems to be partially mediated by aggrecanases. It is, therefore, proposed that the role of TGFbeta in cartilage matrix turnover is not limited to anabolic and anti-catabolic actions, but also extends to selective degradation of matrix components such as aggrecan.     

Mitochondrial activity is modulated by TNFalpha and IL-1beta in normal human chondrocyte cells

OBJECTIVE: Pro-inflammatory cytokines play an important role in osteoarthritis (OA). In osteoarthritic cartilage, chondrocytes exhibit an alteration in mitochondrial activity. This study analyzes the effect of tumor necrosis factor-alpha (TNFalpha) and interleukin-1beta (IL-1beta) on the mitochondrial activity of normal human chondrocytes. MATERIALS AND METHODS: Mitochondrial function was evaluated by analyzing the activities of respiratory chain enzyme complexes and citrate synthase, as well as by mitochondrial membrane potential (Deltapsim) and adenosine triphosphate (ATP) synthesis. Bcl-2 family mRNA expression and protein synthesis were analyzed by RNase protection assay (RPA) and Western-blot, respectively. Cell viability was analyzed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and apoptosis by 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) stain. Glycosaminoglycans were quantified in supernatant by a dimethyl-methylene blue binding assay. RESULTS: Compared to basal cells, stimulation with TNFalpha (10 ng/ml) and IL-1beta (5 ng/ml) for 48 h significantly decreased the activity of complex I (TNFalpha=35% and IL-1beta=35%) and the production of ATP (TNFalpha=18% and IL-1beta=19%). Both TNFalpha and IL-1beta caused a definitive time-dependent decrease in the red/green fluorescence ratio in chondrocytes, indicating depolarization of the mitochondria. Both cytokines induced mRNA expression and protein synthesis of the Bcl-2 family. Rotenone, an inhibitor of complex I, caused a significant reduction of the red/green ratio, but it did not reduce the viability of the chondrocytes. Rotenone also increased Bcl-2 mRNA expression and protein synthesis. Finally, rotenone as well as TNFalpha and IL-1beta, reduced the content of proteoglycans in the extracellular matrix of normal cartilage. CONCLUSION: These results show that both TNFalpha and IL-1beta regulate mitochondrial function in human articular chondrocytes. Furthermore, the inhibition of complex I by both cytokines could play a key role in cartilage degradation induced by TNFalpha and IL-1beta. These data could be important for understanding of the OA pathogenesis.     

Collagen degradation products modulate matrix metalloproteinase expression in cultured articular chondrocytes.

Destruction of collagen within osteoarthritic cartilage depends in part on collagen-degrading matrix metalloproteases (MMP). Degradative fragments of type II collagen (Col II) occur in normal and in osteoarthritic cartilage, and may contribute to regulation of matrix turnover by interfering with normal cell-matrix communication pathways. Therefore, the effects of different types of collagen fragments on mRNA and protein levels of MMP-2, MMP-3, MMP-9, and MMP-13 in cultured bovine articular knee chondrocytes and explants were examined. Primary chondrocytes and explants were incubated with fragments from whole cartilage collagen matrix (Colf) and from purified type II collagen (Col2f), or with a synthetic 29-mer peptide representing the amino-terminal domain of type II collagen (Ntelo). Gelatin zymography revealed increases of proMMP-2, a shift towards active MMP-2 and increases in proMMP-9, depending on the type of fragment. In situ hybridization of cartilage sections displayed MMP-3 mRNA in virtually all cells. Moderate to strong increases in MMP-2, MMP-3, MMP-9, and MMP-13 mRNA levels were detected by quantitative PCR. The results demonstrate stimulating effects of collagen fragments on both mRNA and/or protein from MMP -2, -3, -9, and -13, and suggest a novel mechanism of MMP induction and activation that includes a particular role for N-telo in controlling catabolic pathways of matrix turnover.     

Clusterin expression in adult human normal and osteoarthritic articular cartilage

OBJECTIVE: To characterize the expression pattern of clusterin in adult human normal and osteoarthritic cartilage. METHODS: Clusterin mRNA expression in adult human normal and osteoarthritic cartilage was investigated by analysis of cDNA libraries, TaqMan quantitative RT-PCR, microarray and in situ hybridization. RESULTS: Sequence analysis of ESTs from adult human normal and osteoarthritic cartilage cDNA libraries demonstrated that the abundance of clusterin in these libraries was equivalent to genes which have been more commonly associated with cartilage. To examine tissue distribution, TaqMan Quantitative PCR analysis was performed using RNA from a panel of individual normal tissues. Clusterin was expressed at significant levels in cartilage, brain, liver, and pancreas. The expression of clusterin mRNA was up-regulated in early osteoarthritic vs normal cartilage when analysed by microarray analysis. Using in situ hybridization, chondrocytes of normal cartilage expressed moderate levels of clusterin. Upper mid-zone chondrocytes in cartilage with early stages of osteoarthritic disease expressed high levels of clusterin mRNA. In advanced osteoarthritic cartilage, the overall expression of clusterin was reduced. CONCLUSION: The induction of clusterin has been associated with a variety of disease states where it appears to provide a cytoprotective effect. The increased expression of clusterin mRNA in the early stages of osteoarthritis (OA) may reflect an attempt by the chondrocytes to protect and repair the tissue. In contrast, the decrease in clusterin mRNA in the advanced osteoarthritic cartilage accompanies the final degenerative stages of the disease. An understanding of the expression of clusterin in osteoarthritis may allow consideration of this protein as a marker for cartilage changes in this chronic degenerative condition.     

Expression of bone morphogenetic proteins, receptors, and tissue inhibitors in human fetal, adult, and osteoarthritic articular cartilage.

Coordinate expression of BMPs and their receptors and inhibitors is likely necessary for physiologic BMP regulation and activity. To characterize the expression of such factors in fetal, normal adult, and end-stage osteoarthritic articular cartilage, samples from these sources were analyzed. PCR-amplified sequences (BMPs 1-11), receptors (IA, IB, II), TGF-beta1, TGF-beta2, inhibitors noggin and follistatin, CDMP-1, COMP, and GAPDH from cDNAs generated from extracted total RNA were resolved by gel electrophoresis. Protein levels of BMPs 3, 7, and 8 were also analyzed by SDS-PAGE and Western blotting. RT-PCR revealed that BMPs 1, 2, 4-6, and 11, BMPR-IA and II, noggin, follistatin, CDMP-1, COMP, and GAPDH mRNAs were expressed in similar fashion in both fetal and adult (normal or osteoarthritic) cartilage. BMPs 9 and 10 mRNAs were not expressed in either group. BMPs 7, 8, and BMPR-IB mRNAs were consistently expressed in fetal but not in adult cartilage. BMP-3 mRNA was expressed in fetal and normal adult, but not in osteoarthritic samples. TGF-beta1 was expressed in both adult normal and osteoarthritic, but not fetal, samples. Similarly, Western blotting demonstrated BMPs 7 and 8 to be present in fetal but not in adult samples. BMP-3 protein was present in fetal and adult normal samples, to a lesser extent, but absent in osteoarthritic cartilage.