Matriptase is a novel initiator of cartilage matrix degradation in osteoarthritis

Objective

Increasing evidence implicates serine proteinases in pathologic tissue turnover. The aim of this study was to assess the role of the transmembrane serine proteinase matriptase in cartilage destruction in osteoarthritis (OA).

Methods

Serine proteinase gene expression in femoral head cartilage obtained from either patients with hip OA or patients with fracture to the neck of the femur (NOF) was assessed using a low‐density array. The effect of matriptase on collagen breakdown was determined in cartilage degradation models, while the effect on matrix metalloproteinase (MMP) expression was analyzed by real‐time polymerase chain reaction. ProMMP processing was determined using sodium dodecyl sulfate–polyacrylamide gel electrophoresis/N‐terminal sequencing, while its ability to activate proteinase‐activated receptor 2 (PAR‐2) was determined using a synovial perfusion assay in mice.

Results

Matriptase gene expression was significantly elevated in OA cartilage compared with NOF cartilage, and matriptase was immunolocalized to OA chondrocytes. We showed that matriptase activated proMMP‐1 and processed proMMP‐3 to its fully active form. Exogenous matriptase significantly enhanced cytokine‐stimulated cartilage collagenolysis, while matriptase alone caused significant collagenolysis from OA cartilage, which was metalloproteinase‐dependent. Matriptase also induced MMP‐1, MMP‐3, and MMP‐13 gene expression. Synovial perfusion data confirmed that matriptase activates PAR‐2, and we demonstrated that matriptase‐dependent enhancement of collagenolysis from OA cartilage is blocked by PAR‐2 inhibition.

Conclusion

Elevated matriptase expression in OA and the ability of matriptase to activate selective proMMPs as well as induce collagenase expression make this serine proteinase a key initiator and inducer of cartilage destruction in OA. We propose that the indirect effects of matriptase are mediated by PAR‐2, and a more detailed understanding of these mechanisms may highlight important new therapeutic targets for OA treatment.

     

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.

     

Bone morphogenetic protein and transforming growth factor β inhibitory Smads 6 and 7 are expressed in human adult normal and osteoarthritic cartilage in vivo and are differentially regulated in vitro by interleukin‐1β

Objective

Bone morphogenetic protein (BMP) and transforming growth factor β (TGFβ) are potent anabolic factors in adult articular chondrocytes. In this study, we investigated whether intracellular inhibitors of BMP and TGFβ signaling, inhibitory Smad6 (I‐Smad6) and I‐Smad7, are expressed in articular chondrocytes in normal and osteoarthritic (OA) cartilage, and whether their expression shows a correlation with the anabolic activity of OA chondrocytes in vivo and after interleukin‐1β (IL‐1β) stimulation in vitro.

Methods

RNA isolated directly from normal and OA human knee cartilage as well as from cultured articular chondrocytes was analyzed by (quantitative) polymerase chain reaction technology. Immunolocalization of the I‐Smads was performed on tissue sections and compared with the anabolic cellular activity as documented by in situ hybridization experiments for aggrecan and type II collagen.

Results

Both Smad6 and Smad7 were expressed in all samples of normal and OA cartilage. Immunostaining (including confocal microscopy) confirmed the presence of Smad6 and Smad7 in the majority of normal and degenerated articular chondrocytes; localization was mostly cytoplasmic. No correlation between expression of the main anabolic genes and expression of the I‐Smads was found. In cultured articular chondrocytes, stimulation with IL‐1β showed up‐regulation of Smad7, whereas Smad6 was down‐regulated.

Conclusion

Both Smad6 and Smad7 are expressed in adult human articular chondrocytes. The primarily cytoplasmic localization suggests permanent activation of the I‐Smads in articular cartilage in vivo. No evidence was found that up‐regulation or down‐regulation of I‐Smads in OA cartilage correlates directly with the anabolic (or catabolic) activity of articular chondrocytes. The regulation in chondrocytes of Smad6 and Smad7 expression by IL‐1β suggests a potentially important role of IL‐1β signaling in chondrocytes, via indirect influencing of the BMP/TGFβ signaling cascade.

     

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.

     

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.

     

ADAM15 exerts an antiapoptotic effect on osteoarthritic chondrocytes via up‐regulation of the X‐linked inhibitor of apoptosis

Objective

To investigate the capacity of ADAM15, a disintegrin metalloproteinase that is up‐regulated in osteoarthritic (OA) cartilage, to protect chondrocytes against apoptosis induced by growth factor deprivation and genotoxic stress.

Methods

Caspase 3/7 activity was determined in primary OA and ADAM15‐transfected T/C28a4 chondrocytes upon exposure to the DNA‐damaging agent camptothecin or serum withdrawal. Camptothecin‐induced cytotoxicity was determined by measuring cellular ATP content. (Anti‐)apoptotic proteins were analyzed by immunoblotting, and levels of messenger RNA (mRNA) for X‐linked inhibitor of apoptosis (XIAP) were determined using real‐time polymerase chain reaction. RNA interference was applied for down‐regulation of ADAM15 and XIAP expression. Immunohistochemistry analysis of normal and OA cartilage samples was performed using XIAP‐ and ADAM15‐specific antibodies.

Results

ADAM15‐transfected chondrocytes cultured on a collagen matrix displayed significantly reduced caspase 3/7 activity upon serum or intermittent matrix withdrawal, compared with vector‐transfected control cells. Apoptosis induction by camptothecin exposure also led to significantly elevated caspase 3/7 activity and reduced cell viability of the vector‐transfected compared with ADAM15‐transfected chondrocytes. Increased levels of activated caspase 3 and cleaved poly(ADP‐ribose) polymerase were detected in the vector controls. XIAP, an inhibitor of activated caspase 3, was significantly up‐regulated (∼3‐fold) at the protein and mRNA levels in ADAM15‐transfected chondrocytes upon camptothecin treatment. Specific down‐regulation of either ADAM15 or XIAP in OA chondrocytes led to significant sensitization to camptothecin‐induced caspase 3/7 activity. Immunohistochemical analysis revealed low to moderate XIAP expression in normal specimens and markedly increased XIAP staining, colocalizing with ADAM15, in OA cartilage.

Conclusion

ADAM15 conveys antiapoptotic properties to OA chondrocytes that might sustain their potential to better resist the influence of death‐inducing stimuli under pathophysiologic conditions.

     

Matrix metalloproteinases and aggrecanases cleave aggrecan in different zones of normal cartilage but colocalize in the development of osteoarthritic lesions in STR/ort mice

Objective

To map aggrecan cleavage by matrix metalloproteinases (MMPs) and aggrecanases in normal murine tibial articular cartilage (CBA strain) and in the development of spontaneous osteoarthritis (OA) in the STR/ort mouse and to assess the influence of sex hormone status on these conditions in gonadectomized STR/ort mice.

Methods

The distributions of neoepitopes of aggrecan generated by MMP (VDIPEN) and aggrecanase (NITEGE) cleavage were investigated by immunohistochemistry.

Results

VDIPEN neoepitope was detected mainly in the pericellular matrix of deep‐zone chondrocytes in normal tibial cartilage from STR/ort and CBA mice. In early OA, VDIPEN immunostaining also localized to the pericellular matrix of chondrocytes at the site of the lesion. With increasing severity of OA lesions, VDIPEN immunostaining was also detected in the interterritorial matrix, close to the site of the lesion. In contrast, NITEGE mapped most strongly to the pericellular matrix of upper‐zone chondrocytes in normal tibial cartilage. As with VDIPEN, NITEGE was strongly expressed in the pericellular matrix at the site of early OA lesions. With advancing OA, NITEGE colocalized with VDIPEN in both the pericellular and interterritorial matrices of chondrocytes adjacent to OA lesions and in those of the deep zones. Hormone status did not appear to influence the development of OA or the distribution of aggrecan neoepitopes in STR/ort mice.

Conclusion

MMP‐ and aggrecanase‐generated neoepitopes map predominantly to different regions in normal murine tibial cartilage. However, both groups of enzymes generate increased amounts of neoepitopes in pericellular and interterritorial matrix adjacent to histopathologic lesions of OA. Aggrecan degradation and the development of OA appear to be independent of sex hormone status in this model.

     

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.

     

Mitochondrial dysfunction in osteoarthritis is associated with down‐regulation of superoxide dismutase 2

Objective

Superoxide dismutase 2 (SOD2) is down‐ regulated in osteoarthritis (OA). This study was undertaken to investigate the functional effects of this down‐regulation in the context of oxidative damage and mitochondrial dysfunction.

Methods

Lipid peroxidation in articular cartilage from OA patients and from lesion‐free control subjects with femoral neck fracture was assessed by measuring malondialdehyde levels using the thiobarbituric acid reactive substances assay. Long‐range polymerase chain reaction amplification and a mitochondrial DNA (mtDNA) strand break assay were used to investigate the presence of somatic large‐scale mtDNA rearrangements in cartilage. Microscale oxygraphy was used to explore possible changes in mitochondrial respiratory activity between OA and control chondrocytes. RNA interference was used to determine the effects of SOD2 depletion on lipid peroxidation, mtDNA damage, and mitochondrial respiration.

Results

OA cartilage had higher levels of lipid peroxidation compared to control cartilage, and lipid peroxidation was similarly elevated in SOD2‐depleted chondrocytes. SOD2 depletion led to a significant increase in mtDNA strand breaks in chondrocytes, but there was no notable difference in the level of strand breaks between OA and control chondrocytes. Furthermore, only very low levels of somatic, large‐scale mtDNA rearrangements were identified in OA cartilage. OA chondrocytes showed less spare respiratory capacity (SRC) and higher proton leak compared to control chondrocytes. SOD2‐depleted chondrocytes also showed less SRC and higher proton leak.

Conclusion

This is the first study to analyze the effects of SOD2 depletion in human articular chondrocytes in terms of changes to oxidation and mitochondrial function. The findings indicate that SOD2 depletion in chondrocytes leads to oxidative damage and mitochondrial dysfunction, suggesting that SOD2 down‐regulation is a potential contributor to the pathogenesis of OA.

     

Inhibition of interleukin‐1β‐stimulated production of matrix metalloproteinases by hyaluronan via CD44 in human articular cartilage

Objective

To investigate the mechanism of the inhibitory action of hyaluronan (HA) on interleukin‐1β (IL‐1β)‐stimulated production of matrix metalloproteinases (MMPs) in human articular cartilage.

Methods

IL‐1β was added to normal and osteoarthritic (OA) human articular cartilage in explant culture to stimulate MMP production. Articular cartilage was incubated or preincubated with a clinically used form of 800‐kd HA to assess its effect on IL‐1β‐induced MMPs. Levels of secreted MMPs 1, 3, and 13 in conditioned media were detected by immunoblotting; intracellular MMP synthesis in chondrocytes was evaluated by immunofluorescence microscopy. Penetration of HA into cartilage tissue and its binding to CD44 were analyzed by fluorescence microscopy using fluoresceinated HA. Blocking experiments with anti‐CD44 antibody were performed to investigate the mechanism of action of HA.

Results

Treatment and pretreatment with 800‐kd HA at 1 mg/ml resulted in significant suppression of IL‐1β‐stimulated production of MMPs 1, 3, and 13 in normal and OA cartilage explant culture. Fluorescence histocytochemistry revealed that HA penetrated cartilage tissue and localized in the pericellular matrix around chondrocytes. HA‐binding blocking experiments using anti‐CD44 antibody demonstrated that the association of HA with chondrocytes was mediated by CD44. Preincubation with anti‐CD44 antibody, which suppressed IL‐1β‐stimulated MMPs, reversed the inhibitory effect of HA on MMP production that was induced by IL‐1β in normal and OA cartilage.

Conclusion

This study demonstrates that HA effectively inhibits IL‐1β‐stimulated production of MMP‐1, MMP‐3, and MMP‐13, which supports the clinical use of HA in the treatment of OA. The action of HA on IL‐1β may involve direct interaction between HA and CD44 on chondrocytes.

     

Apoptotic cell death is not a widespread phenomenon in normal aging and osteoarthritic human articular knee cartilage: A study of proliferation,programmed cell death (apoptosis), and viability of chondrocytes in normal and osteoarthritic human knee cartilage

Objective

Chondrocytes are crucial for adequate matrix balance and function. Cell proliferation and, recently, extensive apoptotic cell death have been reported in osteoarthritic (OA) cartilage. Apoptotic cell death would be an obvious central factor in the initiation and progression of OA, since there is no potential for replacing articular chondrocytes in the adult. Therefore, we studied the occurrence of apoptotic cell disintegration and cell proliferation in OA and normal articular cartilage obtained from the knees of adult donors of all ages.

Methods

Following immunostaining for cellular proteins as well as staining for nuclear DNA, we performed triple‐channel confocal laser scanning microscopy on thick cartilage slices to evaluate lacunar emptying and cell viability. Cell proliferation and apoptotic cell death were evaluated morphologically, by immunodetection of the proliferation‐associated Ki‐67 antigen, and by the TUNEL reaction.

Results

With the exception of the calcified layer, we were not able to detect any major (apoptotic or nonapoptotic) cell disintegration in normal young or aged articular knee cartilage. Single apoptotic cells were detected in OA articular knee cartilage. A significant increase in lacunar emptying was observed in late‐stage specimens with higher Mankin scores compared with age‐matched normal control cartilage specimens, but not in low‐grade lesions. A significant (but lesser) increase in empty lacunae was also observed with age in normal cartilage. Cell proliferation was rarely detected in OA cartilage samples and was not detected at all in normal cartilage samples.

Conclusion

Our results confirm the findings of previous studies showing that cell proliferation occurs in OA cartilage. They also show that, contrary to previous suggestions, apoptotic cell death is not a widespread phenomenon in aging or OA cartilage.

     

Association between the abnormal expression of matrix‐degrading enzymes by human osteoarthritic chondrocytes and demethylation of specific CpG sites in the promoter regions

Objective

To investigate whether the abnormal expression of matrix metalloproteinases (MMPs) 3, 9, and 13 and ADAMTS‐4 by human osteoarthritic (OA) chondrocytes is associated with epigenetic “unsilencing.”

Methods

Cartilage was obtained from the femoral heads of 16 patients with OA and 10 control patients with femoral neck fracture. Chondrocytes with abnormal enzyme expression were immunolocalized. DNA was extracted, and the methylation status of the promoter regions of MMPs 3, 9, and 13 and ADAMTS‐4 was analyzed with methylation‐sensitive restriction enzymes, followed by polymerase chain reaction amplification.

Results

Very few chondrocytes from control cartilage expressed the degrading enzymes, whereas all clonal chondrocytes from late‐stage OA cartilage were immunopositive. The overall percentage of nonmethylated sites was increased in OA patients (48.6%) compared with controls (20.1%): 20% versus 4% for MMP‐13, 81% versus 47% for MMP‐9, 57% versus 30% for MMP‐3, and 48% versus 0% for ADAMTS‐4. Not all CpG sites were equally susceptible to loss of methylation. Some sites were uniformly methylated, whereas in others, methylation was generally absent. For each enzyme, there was 1 specific CpG site where the demethylation in OA patients was significantly higher than that in controls: at −110 for MMP‐13, −36 for MMP‐9, −635 for MMP‐3, and −753 for ADAMTS‐4.

Conclusion

This study provides the first evidence that altered synthesis of cartilage‐degrading enzymes by late‐stage OA chondrocytes may have resulted from epigenetic changes in the methylation status of CpG sites in the promoter regions of these enzymes. These changes, which are clonally transmitted to daughter cells, may contribute to the development of OA.

     

Detection of nitrotyrosine in aging and osteoarthritic cartilage: Correlation of oxidative damage with the presence of interleukin‐1β and with chondrocyte resistance to insulin‐like growth factor 1

Objective

To determine whether oxidative damage to cartilage proteins can be detected in aging and osteoarthritic (OA) cartilage, and to correlate the results with the local production of interleukin‐1β (IL‐1β) and the responsiveness of isolated chondrocytes to stimulation with insulin‐like growth factor 1 (IGF‐1).

Methods

The presence of nitrotyrosine was used as a measure of oxidative damage. Histologic sections of knee articular cartilage, obtained from young adult and old adult cynomolgus monkeys, which develop age‐related, naturally occurring OA, were evaluated. Each cartilage section was graded histologically on a scale of 0–7 for the presence of OA‐like changes, and serial sections were immunostained using antibodies to nitrotyrosine and IL‐1β. Chondrocytes isolated and cultured from cartilage adjacent to the sections used for immunostaining were tested for their response to IGF‐1 stimulation by measuring sulfate incorporation in alginate cultures. For comparison with the monkey tissues, cartilage sections from human tissue donors and from tissue removed at the time of OA‐related joint replacement surgery were also immunostained for nitrotyrosine and IL‐1β.

Results

The presence of nitrotyrosine was associated with aging and with the development of OA in cartilage samples from both monkeys and humans. All sections that were highly positive for IL‐1β also showed staining for nitrotyrosine. However, in a few sections from older adult monkeys and humans, nitrotyrosine was present but IL‐1β was absent, suggesting that some age‐related oxidative damage is independent of IL‐1β. In chondrocytes that were isolated from monkey cartilage positive for nitrotyrosine or IL‐1β, the response to stimulation with IGF‐1 was significantly reduced. In some samples from older adult monkeys, IGF‐1 resistance was seen in cells isolated from tissue that did not stain for nitrotyrosine or IL‐1β.

Conclusion

Oxidative damage due to the concomitant overproduction of nitric oxide and other reactive oxygen species is present in both aging and OA cartilage. This damage can contribute to the resistance of chondrocytes to IGF‐1 stimulation, but it is unlikely to be the sole cause of IGF‐1 resistance in these chondrocytes.

     

Quantitative imaging of murine osteoarthritic cartilage by phase‐contrast micro–computed tomography

Objective

The mouse is an optimal model organism in which gene–environment interactions can be used to study the pathogenesis of osteoarthritis (OA). The gold standard for arthritis research in mice is based on histopathology and immunohistochemistry, which are labor‐intensive, prone to sampling bias and technical variability, and limited in throughput. The aim of this study was to develop a new technique that assesses mouse cartilage by integrating quantitative volumetric imaging techniques.

Methods

A novel mouse model of OA was generated by cruciate ligament transection (CLT) and evaluated by histopathology and immunohistochemistry. Knee joint specimens were then imaged using a new technique that combines high‐resolution micro–computed tomography (micro‐CT) and phase‐contrast optics followed by quantitative analyses. A comparative analysis was also performed in a previously established mouse model of OA generated by destabilization of the medial meniscus (DMM).

Results

Phase‐contrast micro‐CT achieved cellular resolution of chondrocytes and quantitative assessment of parameters such as articular cartilage volume and surface area. In mouse models of OA generated by either CLT or DMM, we showed that phase‐contrast micro‐CT distinguished control and OA cartilage by providing quantitative measures with high reproducibility and minimal variability. Features of OA at the cellular or tissue level could also be observed in images generated by phase‐contrast micro‐CT.

Conclusion

We established an imaging technology that comprehensively assessed and quantified the 2‐dimensional and 3‐dimensional changes of articular cartilage. Application of this technology will facilitate the rapid and high‐throughput assessment of genetic and therapeutic models of OA in mice.

     

ERK‐1/2 and p38 in the regulation of hypertrophic changes of normal articular cartilage chondrocytes induced by osteoarthritic subchondral osteoblasts

Objective

Previous studies have shown the influence of subchondral bone osteoblasts (SBOs) on phenotypical changes of articular cartilage chondrocytes (ACCs) during the development of osteoarthritis (OA). The molecular mechanisms involved during this process remain elusive, in particular, the signal transduction pathways. The aim of this study was to investigate the in vitro effects of OA SBOs on the phenotypical changes in normal ACCs and to unveil the potential involvement of MAPK signaling pathways during this process.

Methods

Normal and arthritic cartilage and bone samples were collected for isolation of ACCs and SBOs. Direct and indirect coculture models were applied to study chondrocyte hypertrophy under the influence of OA SBOs. MAPKs in the regulation of the cell–cell interactions were monitored by phosphorylated antibodies and relevant inhibitors.

Results

OA SBOs led to increased hypertrophic gene expression and matrix calcification in ACCs by means of both direct and indirect cell–cell interactions. In this study, we demonstrated for the first time that OA SBOs suppressed p38 phosphorylation and induced ERK‐1/2 signal phosphorylation in cocultured ACCs. The ERK‐1/2 pathway inhibitor PD98059 significantly attenuated the hypertrophic changes induced by conditioned medium from OA SBOs, and the p38 inhibitor SB203580 resulted in the up‐regulation of hypertrophic genes in ACCs.

Conclusion

The findings of this study suggest that the pathologic interaction of OA SBOs and ACCs is mediated via the activation of ERK‐1/2 phosphorylation and deactivation of p38 phosphorylation, resulting in hypertrophic differentiation of ACCs.

     

Anabolic and catabolic gene expression pattern analysis in normal versus osteoarthritic cartilage using complementary DNA–array technology

Objective

To understand changes in gene expression levels that occur during osteoarthritic (OA) cartilage degeneration, using complementary DNA (cDNA)–array technology.

Methods

Nine normal, 6 early degenerated, and 6 late‐stage OA cartilage samples of human knee joints were analyzed using the Human Cancer 1.2 cDNA array and TaqMan analysis.

Results

In addition to a large variability of expression levels between different patients, significant expression patterns were detectable for many genes. Cartilage types II and VI collagen were strongly expressed in late‐stage specimens, reflecting the high matrix‐remodeling activity of advanced OA cartilage. The increase in fibronectin expression in early degeneration suggests that fibronectin is a crucial regulator of matrix turnover activity of chondrocytes during early disease development. Of the matrix metalloproteinases (MMPs), MMP‐3 appeared to be strongly expressed in normal and early degenerative cartilage and down‐regulated in the late stages of disease. This indicates that other degradation pathways might be more important in late stages of cartilage degeneration, involving other enzymes, such as MMP‐2 and MMP‐11, both of which were up‐regulated in late‐stage disease. MMP‐11 was up‐regulated in OA chondrocytes and, interestingly, also in the early‐stage samples. Neither MMP‐1 nor MMP‐8 was detectable, and MMP‐13 and MMP‐2 were significantly detectable only in late‐stage specimens, suggesting that early stages are characterized more by degradation of other matrix components, such as aggrecan and other noncollagenous molecules, than by degradation of type II collagen fibers.

Conclusion

This investigation allowed us to identify gene expression profiles of the disease process and to get new insights into disease mechanisms, for example, to develop a picture of matrix proteinases that are differentially involved in different phases of the disease process.