SirT1 enhances survival of human osteoarthritic chondrocytes by repressing protein tyrosine phosphatase 1B and activating the insulin‐like growth factor receptor pathway

Objective

The protein deacetylase SirT1 inhibits apoptosis in a variety of cell systems by distinct mechanisms, yet its role in chondrocyte death has not been explored. We undertook the present study to assess the role of SirT1 in the survival of osteoarthritic (OA) chondrocytes in humans.

Methods

SirT1, protein tyrosine phosphatase 1B (PTP1B), and PTP1B mutant expression plasmids as well as SirT1 small interfering RNA (siRNA) and PTP1B siRNA were transfected into primary human chondrocytes. Levels of apoptosis were determined using flow cytometry, and activation of components of the insulin‐like growth factor receptor (IGFR)/Akt pathway was assessed using immunoblotting. OA and normal knee cartilage samples were subjected to immunohistochemical analysis.

Results

Expression of SirT1 in chondrocytes led to increased chondrocyte survival in either the presence or the absence of tumor necrosis factor α/actinomycin D, while a reduction of SirT1 by siRNA led to increased chondrocyte apoptosis. Expression of SirT1 in chondrocytes led to activation of IGFR and the downstream kinases phosphatidylinositol 3‐kinase, phosphoinosite‐dependent protein kinase 1, mTOR, and Akt, which in turn phosphorylated MDM2, inhibited p53, and blocked apoptosis. Activation of IGFR occurs at least in part via SirT1‐mediated repression of PTP1B. Expression of PTP1B in chondrocytes increased apoptosis and reduced IGFR phosphorylation, while down‐regulation of PTP1B by siRNA significantly decreased apoptosis. Examination of cartilage from normal donors and OA patients revealed that PTP1B levels are elevated in OA cartilage in which SirT1 levels are decreased.

Conclusion

For the first time, it has been demonstrated that SirT1 is a mediator of human chondrocyte survival via down‐regulation of PTP1B, a potent proapoptotic protein that is elevated in OA cartilage.

     

Role of interleukin‐8 in PiT‐1 expression and CXCR1‐mediated inorganic phosphate uptake in chondrocytes

Objective

The proinflammatory chemokine interleukin‐8 (IL‐8) induces chondrocyte hypertrophy. Moreover, chondrocyte hypertrophy develops in situ in osteoarthritic (OA) articular cartilage and promotes dysregulated matrix repair and calcification. Growth plate chondrocyte hypertrophy is associated with expression of the type III sodium‐dependent inorganic phosphate (Pi) cotransporter phosphate transporter/retrovirus receptor 1 (PiT‐1). This study was undertaken to test the hypothesis that IL‐8 promotes chondrocyte hypertrophy by modulating chondrocyte PiT‐1 expression and sodium‐dependent Pi uptake, and to assess differential roles in this activity.

Methods

The selective IL‐8 receptor CXCR1 and the promiscuous chemokine receptor CXCR2 were used. Human knee OA cartilage, cultured normal bovine knee chondrocytes, and immortalized human articular chondrocytic CH‐8 cells were transfected with CXCR1/CXCR2 chimeric receptors in which the 40–amino acid C‐terminal cytosolic tail domains were swapped and site mutants of a CXCR1‐specific region were generated.

Results

Up‐regulated PiT‐1 expression was detected in OA cartilage. IL‐8, but not IL‐1 or the CXCR2 ligand growth‐related oncogene α, induced PiT‐1 expression and increased sodium‐dependent Pi uptake by >40% in chondrocytes. The sodium/phosphate cotransport inhibitor phosphonoformic acid blocked IL‐8–induced chondrocyte hypertrophic differentiation. Signaling mediated by kinase Pyk‐2 was essential for IL‐8 induction of PitT‐1 expression and Pi uptake. Signaling through the TSYT^346–349 region of the CXCR1 cytosolic tail, a region divergent from the CXCR2 cytosolic tail, was essential for IL‐8 to induce Pi uptake.

Conclusion

Our results link low‐grade IL‐8–mediated cartilaginous inflammation in OA to altered chondrocyte differentiation and disease progression through PiT‐1 expression and sodium‐dependent Pi uptake mediated by CXCR1 signaling.

     

Accelerated,aging‐dependent development of osteoarthritis in α1 integrin–deficient mice

Objective

Cell–matrix interactions regulate chondrocyte differentiation and survival. The α1β1 integrin is a major collagen receptor that is expressed on chondrocytes. Mice with targeted inactivation of the integrin α1 gene (α1‐KO mice) provide a model that can be used to address the role of cell–matrix interactions in cartilage homeostasis and osteoarthritis (OA) pathogenesis.

Methods

Knee joints from α1‐KO and wild‐type (WT) BALB/c mice were harvested at ages 4–15 months. Knee joint sections were examined for inflammation, cartilage degradation, and loss of glycosaminoglycans (by Safranin O staining). Immunohistochemistry was performed to detect the distribution of α1 integrin, matrix metalloproteinases (MMPs), and chondrocyte apoptosis.

Results

In WT mice, the α1 integrin subunit was detected in hypertrophic chondrocytes in the growth plate and in a subpopulation of cells in the deep zone of articular cartilage. There was a marked increase in α1‐positive chondrocytes in the superficial and upper mid‐zones in OA‐affected areas in joints from old WT mice. The α1‐KO mice showed more severe cartilage degradation, glycosaminoglycan depletion, and synovial hyperplasia as compared with the WT mice. MMP‐2 and MMP‐3 expression was increased in the OA‐affected areas. In cartilage from α1‐KO mice, the cellularity was reduced and the frequency of apoptotic cells was increased. These results suggest that the α1 integrin subunit is involved in the early remodeling process in OA cartilage.

Conclusion

Deficiency in the α1 integrin subunit is associated with an earlier deregulation of cartilage homeostasis and an accelerated, aging‐dependent development of OA.

     

Osteoarthritic chondrocyte–secreted morphogens induce chondrogenic differentiation of human mesenchymal stem cells

Objective

The potential of stem cells to repair compromised cartilage tissue, such as in osteoarthritis (OA), depends strongly on how transplanted cells respond to factors secreted from the residing OA chondrocytes. This study was undertaken to determine the effect of morphogenetic signals from OA chondrocytes on chondrogenic differentiation of human mesenchymal stem cells (MSCs).

Methods

The effect of OA chondrocyte–secreted morphogens on chondrogenic differentiation of human MSCs was evaluated using a coculture system involving both primary and passaged OA chondrocytes. The findings were compared against findings for human MSCs cultured in OA chondrocyte–conditioned medium. Gene expression analysis, biochemical assays, and immunofluorescence staining were used to characterize the chondrogenic differentiation of human MSCs. Mass spectrometry analysis was used to identify the soluble factors. Numerical analysis was carried out to model the concentration profile of soluble factors within the human MSC–laden hydrogels.

Results

The human MSCs cocultured with primary OA chondrocytes underwent chondrogenic differentiation even in the absence of growth factors; however, the same effect could not be mimicked using OA chondrocyte–conditioned medium or expanded cells. Additionally, the cocultured environment down‐regulated hypertrophic differentiation of human MSCs. Mass spectrometry analysis demonstrated cell–cell communication and chondrocyte phenotype–dependent effects on cell‐secreted morphogens.

Conclusion

The experimental findings, along with the results of the numerical analysis, suggest a crucial role of soluble morphogens and their local concentrations in the differentiation pattern of human MSCs in a 3‐dimensional environment. The concept of using a small number of chondrocytes to promote chondrogenic differentiation of human MSCs while preventing their hypertrophic differentiation could be of great importance in formulating effective stem cell–based cartilage repair.

     

Chondrocyte AMP‐activated protein kinase activity suppresses matrix degradation responses to proinflammatory cytokines interleukin‐1β and tumor necrosis factor α

Objective

Interleukin‐1β (IL‐1β) and tumor necrosis factor α (TNFα) stimulate chondrocyte matrix catabolic responses, thereby compromising cartilage homeostasis in osteoarthritis (OA). AMP‐activated protein kinase (AMPK), which regulates energy homeostasis and cellular metabolism, also exerts antiinflammatory effects in multiple tissues. This study was undertaken to test the hypothesis that AMPK activity limits chondrocyte matrix catabolic responses to IL‐1β and TNFα.

Methods

Expression of AMPK subunits was examined, and AMPKα activity was ascertained by the phosphorylation status of AMPKα Thr¹⁷² in human knee articular chondrocytes and cartilage by Western blotting and immunohistochemistry, respectively. Procatabolic responses to IL‐1β and TNFα, such as release of glycosaminoglycan, nitric oxide, and matrix metalloproteinases 3 and 13 were determined by dimethylmethylene blue assay, Griess reaction, and Western blotting, respectively, in cartilage explants and chondrocytes with and without knockdown of AMPKα by small interfering RNA.

Results

Normal human knee articular chondrocytes expressed AMPKα1, α2, β1, β2, and γ1 subunits. AMPK activity was constitutively present in normal articular chondrocytes and cartilage, but decreased in OA articular chondrocytes and cartilage and in normal chondrocytes treated with IL‐1β and TNFα. Knockdown of AMPKα resulted in enhanced catabolic responses to IL‐1β and TNFα in chondrocytes. Moreover, AMPK activators suppressed cartilage/chondrocyte procatabolic responses to IL‐1β and TNFα and the capacity of TNFα and CXCL8 (IL‐8) to induce type X collagen expression.

Conclusion

Our findings indicate that AMPK activity is reduced in OA cartilage and in chondrocytes following treatment with IL‐1β or TNFα. AMPK activators attenuate dephosphorylation of AMPKα and procatabolic responses in chondrocytes induced by these cytokines. These observations suggest that maintenance of AMPK activity supports cartilage homeostasis by protecting cartilage matrix from inflammation‐induced degradation.

     

αvβ5 Integrin promotes dedifferentiation of monolayer‐cultured articular chondrocytes

Objective

When cultured in monolayers, articular chondrocytes undergo an obvious phenotypic change. Although the involvement of integrins has been suggested, the exact mechanisms of the change have not been determined. This study was undertaken to clarify the mechanisms underlying the loss of chondrocyte phenotype early after plating.

Methods

Primary cultured human articular chondrocytes were used for the experiments. Involvement of respective integrins in the phenotypic change was investigated in RNA interference (RNAi) experiments. A signaling pathway involved in the change was identified in experiments using specific inhibitors and adenoviruses encoding mutated genes involved in the pathway. Adenoviruses carrying mutated GTPases were used to determine the involvement of small GTPases in the process.

Results

In monolayer‐cultured chondrocytes, suppression of αv or β5 integrin expression by RNAi inhibited morphologic changes in the cells and increased (or prevented a reduction in) the expression of various cartilage matrix genes. Consistent results were obtained in experiments using a blocking antibody and a synthetic inhibitor of αvβ5 integrin. The decrease in cartilage matrix gene expression in chondrocytes after plating was mediated by ERK signaling, which was promoted primarily by αvβ5 integrin. In articular chondrocytes, the affinity of αvβ5 integrin for ligands was regulated by the small GTPase R‐Ras. R‐Ras was gradually activated in monolayer‐cultured chondrocytes after plating, which caused a gradual decline in cartilage matrix gene expression through enhanced αvβ5 integrin activation and the subsequent increase in ERK signaling.

Conclusion

Our findings indicate that αvβ5 integrin may be involved in the change that occurs in monolayer‐cultured chondrocytes after plating.

     

Regulation of plasminogen activator inhibitor 1 expression in human osteoarthritic chondrocytes by fluid shear stress: Role of protein kinase Cα

Objective

To test a fluid flow system for the investigation of the influence of shear stress on expression of plasminogen activator inhibitor 1 (PAI‐1) in human osteoarthritic (OA) articular chondrocytes (from lesional and nonlesional sites) and human SW‐1353 chondrocytes.

Methods

Human SW‐1353 chondrocytes and OA and normal human articular chondrocytes were cultured on type II collagen–coated glass plates under static conditions or placed in a flow chamber to form a closed fluid‐circulation system for exposure to different levels of shear stress (2–20 dyn/cm²). Real‐time polymerase chain reaction was used to analyze PAI‐1 gene expression, and protein kinase C (PKC) inhibitors and small interfering RNA were used to investigate the mechanism of shear stress–induced signal transduction in SW‐1353 and OA (lesional and nonlesional) articular chondrocytes.

Results

There was a significant reduction in PAI‐1 expression in OA chondrocytes obtained from lesional sites compared with those obtained from nonlesional sites. In SW‐1353 chondrocytes subjected to 2 hours of shear flow, moderate shear stresses (5 and 10 dyn/cm²) generated significant PAI‐1 expression, which was regulated through PKCα phosphorylation and Sp‐1 activation. These levels of shear stress also increased PAI‐1 expression in articular chondrocytes from nonlesional sites and from normal healthy cartilage through the activation of PKCα and Sp‐1 signal transduction, but no effect of these levels of fluid shear stress was observed on OA chondrocytes from lesional sites.

Conclusion

OA chondrocytes from lesional sites and those from nonlesional sites of human cartilage have differential responses to shear stress with regard to PAI‐1 gene expression, and therefore diverse functional consequences can be observed.

     

One of two chondrocyte‐expressed isoforms of cartilage intermediate‐layer protein functions as an insulin‐like growth factor 1 antagonist

Objective

Aging and osteoarthritic (OA) cartilage commonly demonstrate enhanced expression of the large, transforming growth factor β (TGFβ)–inducible glycoprotein cartilage intermediate‐layer protein (CILP) as well as enhanced extracellular inorganic pyrophosphate (PPi) that promotes the deposition of calcium pyrophosphate dihydrate crystals. In normal chondrocytes, TGFβ induces elevated chondrocyte extracellular PPi. Insulin‐like growth factor 1 (IGF‐1) normally blocks this response and reduces extracellular PPi. However, chondrocyte resistance to IGF‐1 is observed in OA and aging. Because CILP was reported to chromatographically fractionate with PPi‐generating nucleotide pyrophosphatase phosphodiesterase (NPP) activity, it has been broadly assumed that CILP itself has NPP activity. Our objective was to directly define CILP functions and their relationship to IGF‐1 in chondrocytes.

Methods

Using primary cultures of articular chondrocytes from the knee, we defined the function of the previously described CILP (CILP‐1) and of a recently described 50.6% identical protein that we designated the CILP‐2 isoform.

Results

Both CILP isoforms were constitutively expressed by primary cultured articular chondrocytes, but only CILP‐1 expression was detectable in cultured knee meniscal cartilage cells. Neither CILP isoform had intrinsic NPP activity. But CILP‐1 blocked the ability of IGF‐1 to decrease extracellular PPi, an activity specific for the CILP‐1 N‐terminal domain. The CILP‐1 N‐terminal domain also suppressed IGF‐1–induced (but not TGFβ‐induced) proliferation and sulfated proteoglycan synthesis, and it inhibited ligand‐induced IGF‐1 receptor autophosphorylation.

Conclusion

Two CILP isoforms are differentially expressed by chondrocytes. Neither CILP isoform exhibits PPi‐generating NPP activity. But, increased expression of CILP‐1, via N‐terminal domain–mediated inhibitory effects of CILP‐1 on chondrocyte IGF‐1 responsiveness, could impair chondrocyte growth and matrix repair and indirectly promote PPi supersaturation in aging and OA cartilage.

     

Inhibition of CD95 apoptotic signaling by interferon‐γ in human osteoarthritic chondrocytes is associated with increased expression of FLICE inhibitory protein

Objective

Cartilage homeostasis dysregulation during osteoarthritis (OA) has been linked to an increased rate of apoptosis of chondrocytes, the only cell type resident in the cartilage. In addition, the CD95–CD95 ligand (the Fas system) has emerged as one of the major pathways of cell death in the cartilage. We undertook the present study to investigate the role of interferon‐γ (IFNγ) in the regulation of the Fas system by analyzing the modulation of intracellular signaling molecules (FLICE inhibitory protein [FLIP] and caspases 3 and 8) in primary cultures of human OA chondrocytes.

Methods

CD95‐induced apoptotic death of human OA chondrocytes was analyzed in the presence or absence of IFNγ using cell death immunoassay for apoptosis, real‐time polymerase chain reaction for FLIP and caspase 8 expression, Western blotting for FLIP, and proteolytic activity for caspases 3 and 8.

Results

CD95‐induced apoptotic death of human OA chondrocytes was strongly counteracted by IFNγ treatment, although the surface expression of CD95 was slightly up‐regulated by this cytokine. The messenger RNA (mRNA) expression of FLIP and caspase 8, mediators involved in CD95 signaling, revealed that FLIP expression in human OA chondrocytes was significantly up‐regulated (2‐fold increase) by IFNγ treatment. Moreover, the FLIP:caspase 8 mRNA ratio increased significantly. FLIP up‐regulation by IFNγ was confirmed at the protein level. Caspase 8 and caspase 3 proteolytic activities, both induced in these cells by stimulation with anti‐CD95, were also significantly down‐modulated by IFNγ.

Conclusion

These findings suggest that IFNγ impairs CD95‐mediated signaling and apoptotic death in human chondrocytes. Its mechanism of action involves down‐regulation of caspase 8 and caspase 3 activities and increased expression of the antiapoptotic protein FLIP, suggesting an interesting mechanism for the inhibition of chondrocyte apoptosis.

     

MicroRNA‐140 is expressed in differentiated human articular chondrocytes and modulates interleukin‐1 responses

Objective

MicroRNA (miRNA) are a class of noncoding small RNAs that act as negative regulators of gene expression. MiRNA exhibit tissue‐specific expression patterns, and changes in their expression may contribute to pathogenesis. The objectives of this study were to identify miRNA expressed in articular chondrocytes, to determine changes in osteoarthritic (OA) cartilage, and to address the function of miRNA‐140 (miR‐140).

Methods

To identify miRNA specifically expressed in chondrocytes, we performed gene expression profiling using miRNA microarrays and quantitative polymerase chain reaction with human articular chondrocytes compared with human mesenchymal stem cells (MSCs). The expression pattern of miR‐140 was monitored during chondrogenic differentiation of human MSCs in pellet cultures and in human articular cartilage from normal and OA knee joints. We tested the effects of interleukin‐1β (IL‐1β) on miR‐140 expression. Double‐stranded miR‐140 (ds–miR‐140) was transfected into chondrocytes to analyze changes in the expression of genes associated with OA.

Results

Microarray analysis showed that miR‐140 had the largest difference in expression between chondrocytes and MSCs. During chondrogenesis, miR‐140 expression in MSC cultures increased in parallel with the expression of SOX9 and COL2A1. Normal human articular cartilage expressed miR‐140, and this expression was significantly reduced in OA tissue. In vitro treatment of chondrocytes with IL‐1β suppressed miR‐140 expression. Transfection of chondrocytes with ds–miR‐140 down‐regulated IL‐1β–induced ADAMTS5 expression and rescued the IL‐1β–dependent repression of AGGRECAN gene expression.

Conclusion

This study shows that miR‐140 has a chondrocyte differentiation–related expression pattern. The reduction in miR‐140 expression in OA cartilage and in response to IL‐1β may contribute to the abnormal gene expression pattern characteristic of OA.

     

Semaphorin 3A is expressed in human osteoarthritic cartilage and antagonizes vascular endothelial growth factor 165–promoted chondrocyte migration: An implication for chondrocyte cloning

Objective

Vascular endothelial growth factor 165 (VEGF₁₆₅) and its receptors, including neuropilin 1 (NRP‐1), are overexpressed in human osteoarthritic (OA) articular cartilage, although their functional roles in the cartilage are not fully understood. An axon‐guidance molecule, semaphorin 3A (Sema3A), which binds to NRP‐1, acts as an antagonist of VEGF signaling in endothelial cells. The aim of this study was to examine the expression of Sema3A and the functions of the VEGF₁₆₅/Sema3A/NRP‐1 axis in OA cartilage.

Methods

The expression of Sema3A in OA and normal cartilage samples was examined by real‐time polymerase chain reaction and immunohistochemical analyses. Functional analyses of VEGF₁₆₅ and Sema3A were carried out using OA chondrocytes in culture. The migration activity of chondrocytes was examined in a monolayer wound assay. The effects of Sema3A on VEGF₁₆₅‐induced up‐regulation of matrix metalloproteinases (MMPs) and intracellular signaling were also studied in cultured chondrocytes.

Results

Sema3A expression was significantly elevated in OA cartilage as compared to normal cartilage. Sema3A immunoreactivity directly correlated with the Mankin score and with chondrocyte cloning. VEGF₁₆₅ promoted the migration of chondrocytes, and this activity was suppressed by VEGF receptor 2 tyrosine kinase inhibitors. Sema3A antagonized the chondrocyte migration promoted by VEGF₁₆₅, and the activity was blocked by a selective inhibitor of, or small interfering RNA for, Sema3A. VEGF₁₆₅‐induced overexpression of MMPs and phosphorylation of ERK and focal adhesion kinase in chondrocytes were inhibited by Sema3A.

Conclusion

Our findings provide the first evidence that Sema3A is overexpressed, with a direct correlation with cloning, in OA cartilage and that it suppresses the VEGF₁₆₅‐promoted migration of chondrocytes. Our findings also suggest that Sema3A plays a role in chondrocyte cloning through inhibition of cell migration in OA cartilage.

     

Catabolic stress induces features of chondrocyte senescence through overexpression of caveolin 1: Possible involvement of caveolin 1–induced down‐regulation of articular chondrocytes in the pathogenesis of osteoarthritis

Objective

Articular chondrocyte senescence is responsible, at least in part, for the increased incidence of osteoarthritis (OA) with increased age. Recently, it was suggested that caveolin 1, a 21–24‐kd membrane protein, participates in premature cellular senescence. Caveolin 1 is the principal structural component of caveolae, vesicular invaginations of the plasma membrane. This study was undertaken to investigate whether the catabolic factors oxidative stress and interleukin‐1β (IL‐1β) induce features of premature senescence of articular chondrocytes through up‐regulation of caveolin 1 expression.

Methods

Caveolin 1 expression was investigated in human OA cartilage by real‐time polymerase chain reaction and in rat OA cartilage by immunohistologic analysis. We studied whether IL‐1β and H₂O₂ induce caveolin 1 expression in OA chondrocytes and analyzed the relationship between cellular senescent phenotypes and caveolin 1 expression in human chondrocytes.

Results

In human and rat OA articular cartilage, caveolin 1 positivity was associated with cartilage degeneration. Both IL‐1β and H₂O₂ up‐regulated caveolin 1 messenger RNA and protein levels, and both treatments induced marked expression of senescent phenotypes: altered cellular morphology, cell growth arrest, telomere erosion, and specific senescence‐associated β‐galactosidase activity. Caveolin 1 overexpression induced p38 MAPK activation and impaired the ability of chondrocytes to produce type II collagen and aggrecan. In contrast, down‐regulation of caveolin 1 with antisense oligonucleotide significantly inhibited the features of chondrocyte senescence induced by catabolic factors. Caveolin 1 induction and stresses with both IL‐1β and H₂O₂ up‐regulated p53 and p21 and down‐regulated phosphorylated retinoblastoma (Rb), suggesting that the p53/p21/Rb phosphorylation pathway, as well as prolonged p38 MAPK activation, may mediate the features of chondrocyte senescence induced by stress.

Conclusion

Our findings suggest that IL‐1β and oxidative stress induce features of premature senescence in OA chondrocytes, mediated, at least in part, by stress‐induced caveolin 1 expression. This indicates that caveolin 1 plays a role in the pathogenesis of OA via promotion of chondrocyte down‐regulation.

     

Involvement of the Wnt signaling pathway in experimental and human osteoarthritis: Prominent role of Wnt‐induced signaling protein 1

Objective

Wnt signaling pathway proteins are involved in embryonic development of cartilage and bone, and, interestingly, developmental processes appear to be recapitulated in osteoarthritic (OA) cartilage. The present study was undertaken to characterize the expression pattern of Wnt and Fz genes during experimental OA and to determine the function of selected genes in experimental and human OA.

Methods

Longitudinal expression analysis was performed in 2 models of OA. Levels of messenger RNA for genes from the Wnt/β‐catenin pathway were determined in synovium and cartilage, and the results were validated using immunohistochemistry. Effects of selected genes were assessed in vitro using recombinant protein, and in vivo by adenoviral overexpression.

Results

Wnt‐induced signaling protein 1 (WISP‐1) expression was strongly increased in the synovium and cartilage of mice with experimental OA. Wnt‐16 and Wnt‐2B were also markedly up‐regulated during the course of disease. Interestingly, increased WISP‐1 expression was also found in human OA cartilage and synovium. Stimulation of macrophages and chondrocytes with recombinant WISP‐1 resulted in interleukin‐1–independent induction of several matrix metalloproteinases (MMPs) and aggrecanase. Adenoviral overexpression of WISP‐1 in murine knee joints induced MMP and aggrecanase expression and resulted in cartilage damage.

Conclusion

This study included a comprehensive characterization of Wnt and Frizzled gene expression in experimental and human OA articular joint tissue. The data demonstrate, for the first time, that WISP‐1 expression is a feature of experimental and human OA and that WISP‐1 regulates chondrocyte and macrophage MMP and aggrecanase expression and is capable of inducing articular cartilage damage in models of OA.

     

Anti‐Fas–induced apoptosis in chondrocytes reduced by hyaluronan: Evidence for CD44 and CD54 (intercellular adhesion molecule 1) involvement

Objective

To investigate the in vitro effect of therapeutic hyaluronan (HA) of 500–730 kd on anti‐Fas–induced apoptosis of chondrocytes from osteoarthritis (OA) patients, and to assess its mechanism of action by analyzing the role of the 2 HA receptors, CD44 and CD54 (intercellular adhesion molecule 1 [ICAM‐1]).

Methods

Chondrocytes isolated from human OA knee cartilage were cultured and the effect of HA on both spontaneous and anti‐Fas–induced apoptosis was evaluated. Apoptosis was analyzed by JAM test (for quantitative analysis of fragmented DNA), cell death detection immunoassay (for quantitative analysis of oligonucleosome), TUNEL assay, and electron microscopy. Blocking experiments with anti‐CD44 and anti‐CD54 alone or in combination were performed to investigate the HA mechanism of action.

Results

Both quantitative tests demonstrated that anti‐Fas significantly induced apoptosis of isolated OA chondrocytes. HA at 1,000 μg/ml significantly reduced the anti‐Fas–induced apoptosis of chondrocytes but did not affect spontaneous chondrocyte apoptosis. These data were also confirmed by TUNEL staining and by electron microscopy morphologic evaluation. The antiapoptotic effects of HA on anti‐FAS–induced chondrocyte apoptosis were significantly decreased by both anti‐CD44 (mean ± SD 57 ± 12% inhibition) and anti–ICAM‐1 (31 ± 22% inhibition). The mixture of the 2 antibodies had an additive effect, since the rate of inhibition increased to 87 ± 13%.

Conclusion

These data demonstrate that 500–730‐kd HA exerts an antiapoptotic effect on anti‐FAS–induced chondrocyte apoptosis by binding its specific receptors (CD44 and ICAM‐1). Furthermore, this HA fraction may be able to slow down chondrocyte apoptosis in OA by regulating the processes of cartilage matrix degradation.

     

Calcification of articular cartilage in human osteoarthritis

Objective

Hypertrophic chondrocyte differentiation is a key step in endochondral ossification that produces basic calcium phosphates (BCPs). Although chondrocyte hypertrophy has been associated with osteoarthritis (OA), chondrocalcinosis has been considered an irregular event and linked mainly to calcium pyrophosphate dihydrate (CPPD) deposition. The aim of this study was to determine the prevalence and composition of calcium crystals in human OA and analyze their relationship to disease severity and markers of chondrocyte hypertrophy.

Methods

One hundred twenty patients with end‐stage OA undergoing total knee replacement were prospectively evaluated. Cartilage calcification was studied by conventional x‐ray radiography, digital‐contact radiography (DCR), field‐emission scanning electron microscopy (FE‐SEM), and synovial fluid analysis. Cartilage calcification findings were correlated with scores of knee function as well as histologic changes and chondrocyte hypertrophy as analyzed in vitro.

Results

DCR revealed mineralization in all cartilage specimens. Its extent correlated significantly with the Hospital for Special Surgery knee score but not with age. FE‐SEM analysis showed that BCPs, rather than CPPD, were the prominent minerals. On histologic analysis, it was observed that mineralization correlated with the expression of type X collagen, a marker of chondrocyte hypertrophy. Moreover, there was a strong correlation between the extent of mineralization in vivo and the ability of chondrocytes to produce BCPs in vitro. The induction of hypertrophy in healthy human chondrocytes resulted in a prominent mineralization of the extracellular matrix.

Conclusion

These results indicate that mineralization of articular cartilage by BCP is an indissociable process of OA and does not characterize a specific subset of the disease, which has important consequences in the development of therapeutic strategies for patients with OA.