Crucial role of macrophages in matrix metalloproteinase–mediated cartilage destruction during experimental osteoarthritis : Involvement of matrix metalloproteinase 3

Objective

To explore the involvement of synovial macrophages in early cartilage damage in osteoarthritis (OA), and to identify the role of matrix metalloproteinase 3 (MMP‐3) in the pathology of early and late OA.

Methods

The role of synovial macrophages in MMP‐mediated damage in OA was studied by depleting synovial macrophages prior to elicitation of a collagenase‐induced instability model of OA. The expression of MMP in synovium and cartilage was monitored using TaqMan analysis. In spontaneous and induced OA, cartilage pathology was scored in MMP‐3–knockout mice and control mice, by histologic assessment and VDIPEN staining.

Results

On day 14 following induction of OA, MMP‐mediated neoepitopes were detected in cartilage from mice with mild experimental OA (mean ± SD positively stained surface area 20 ± 3.2%). Remarkably, by depleting synovial macrophages prior to induction of OA, the generation of MMP‐induced neoepitopes was largely prevented (mean ± SD positively stained surface area 5 ± 1%; P< 0.001), indicating an important role for synovial macrophages in the occurrence of MMP‐mediated cartilage damage. We observed a strong decrease in MMP‐3 and MMP‐9 expression in synovial but not cartilage tissue in macrophage‐depleted joints. Among 2‐year‐old mice, spontaneous OA–like changes in the lining layer were significantly decreased in MMP‐3–knockout mice compared with control mice. Even more striking was the 67% reduction in the occurrence of severe cartilage damage in MMP‐3–knockout mice. In addition, MMP‐mediated VDIPEN expression was significantly decreased, indicating reduced MMP‐mediated cartilage breakdown.

Conclusion

The results of this study prove that MMP‐3 is involved in the generation of severe cartilage damage in murine OA. Synovial macrophages are crucial in early MMP activity and appear to mediate MMP production in synovium rather than 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.

     

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.

     

Role of apoptotic and matrix‐degrading genes in articular cartilage and meniscus of mature and aged rabbits during development of osteoarthritis

Objective

To determine expression patterns of apoptotic and matrix‐degrading genes during aging and development of osteoarthritis (OA), using a rabbit model of induced OA.

Methods

Six mature and 6 aged rabbits underwent anterior cruciate ligament transection and were killed 4 and 8 weeks after surgery, respectively, to create early‐grade and advanced‐grade OA. RNA from articular cartilage and menisci was examined for expression of the genes caspase 8, Fas, Fas ligand, p53, aggrecanase, matrix metalloproteinase 1 (MMP‐1), and MMP‐3. A second cohort of animals that had undergone no intervention in the joint was also killed. Parametric data were analyzed with analysis of variance and Student's t‐tests, while nonparametric data were assessed with the Mann‐Whitney U test.

Results

Expression levels of Fas, caspase 8, FasL, and MMP‐1 were significantly higher (>100%) in aged cartilage compared with mature cartilage (P < 0.05). After induction of OA, expression of apoptotic genes in aged rabbits remained high, while significant up‐regulation of Fas and caspase 8 (nearly 150% increase) was observed in mature rabbits (P < 0.05). No significant up‐regulation of these genes was observed in the menisci of aged or mature rabbits prior to or after induction of OA. Development of OA occurred more rapidly in aged cartilage compared with mature cartilage (P < 0.05).

Conclusion

Differential expression of apoptotic and matrix‐degrading genes occurs in aged compared with mature cartilage, both at baseline and during development of OA. This may be responsible for faster degradation of aged cartilage and its predisposition for developing OA.

     

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.

     

Neutrophil gelatinase–associated lipocalin is expressed in osteoarthritis and forms a complex with matrix metalloproteinase 9

Objective

Expression of matrix metalloproteinase 9 (MMP‐9) is up‐regulated in osteoarthritis (OA) and usually presents as multiple bands when synovial fluid (SF) from OA patients is analyzed by zymography. Among these bands is an ∼125–130–kd band for high molecular weight (HMW) gelatinase, which has not been characterized. This study was undertaken to characterize the HMW MMP activity in OA SF.

Methods

MMP activity in OA SF was determined by gelatin zymography. Recombinant MMPs were used to identify MMP activity on the zymogram. Western immunoblotting, immunoprecipitation, and immunodepletion analyses were performed using antibodies specific for human MMP‐9 and human neutrophil gelatinase–associated lipocalin (NGAL). Human cartilage matrix degradation was determined by dimethylmethylene blue assay.

Results

Zymographic analysis showed that the HMW gelatinase in OA SF comigrated with a purified NGAL–MMP‐9 complex. Results of Western immunoblotting showed that the HMW gelatinase was also recognized by antibodies specific for human NGAL or human MMP‐9. These same antibodies also immunoprecipitated the HMW gelatinase activity from OA SF. The NGAL–MMP‐9 complex was reconstituted in vitro in gelatinase buffer. In the presence of NGAL, MMP‐9 activity was stabilized; in the absence of NGAL, rapid loss of MMP‐9 activity occurred. MMP‐9–mediated release of cartilage matrix proteoglycans was significantly higher in the presence of NGAL (P < 0.05).

Conclusion

Our findings demonstrate that the HMW gelatinase activity in OA SF represents a complex of NGAL and MMP‐9. The ability of NGAL to protect MMP‐9 activity is relevant to cartilage matrix degradation in OA and may represent an important mechanism by which NGAL may contribute to the loss of cartilage matrix proteins in OA.

     

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.

     

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.

     

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.

     

Pioglitazone,a peroxisome proliferator–activated receptor γ agonist,reduces the progression of experimental osteoarthritis in guinea pigs

Objective

To evaluate the in vivo therapeutic effect of pioglitazone, a peroxisome proliferator–activated receptor γ (PPARγ) agonist, on the development of lesions in a guinea pig model of osteoarthritis (OA), and to determine the influence of pioglitazone on the synthesis of matrix metalloproteinase 13 (MMP‐13) and interleukin‐1β (IL‐1β) in articular cartilage.

Methods

The OA model was created by partial medial meniscectomy of the right knee joint. The guinea pigs were divided into 4 treatment groups: unoperated animals that received no treatment (normal), operated animals (OA guinea pigs) that received placebo, OA guinea pigs that received oral pioglitazone at 2 mg/kg/day, and OA guinea pigs that received oral pioglitazone at 20 mg/kg/day. The animals began receiving medication 1 day after surgery and were killed 4 weeks later. Macroscopic and histologic analyses were performed on the cartilage. The levels of MMP‐13 and IL‐1β in OA cartilage chondrocytes were evaluated by immunohistochemistry.

Results

OA guinea pigs treated with the highest dosages of pioglitazone showed a significant decrease, compared with the OA placebo group, in the surface area (size) and grade (depth) of cartilage macroscopic lesions on the tibial plateaus. The histologic severity of cartilage lesions was also reduced. A significantly higher percentage of chondrocytes in the middle and deep layers stained positive for MMP‐13 and IL‐1β in cartilage from placebo‐treated OA guinea pigs compared with normal controls. Guinea pigs treated with the highest dosage of pioglitazone demonstrated a significant reduction in the levels of both MMP‐13 and IL‐1β in OA cartilage.

Conclusion

This is the first in vivo study demonstrating that a PPARγ agonist, pioglitazone, could reduce the severity of experimental OA. This effect was associated with a reduction in the levels of MMP‐13 and IL‐1β, which are known to play an important role in the pathophysiology of OA lesions.

     

Pathologic indicators of degradation and inflammation in human osteoarthritic cartilage are abrogated by exposure to n‐3 fatty acids

Objective

To determine if n‐3 polyunsaturated fatty acid (PUFA) supplementation (versus treatment with n‐6 polyunsaturated or other fatty acid supplements) affects the metabolism of osteoarthritic (OA) cartilage.

Methods

The metabolic profile of human OA cartilage was determined at the time of harvest and after 24‐hour exposure to n‐3 PUFAs or other classes of fatty acids, followed by explant culture for 4 days in the presence or absence of interleukin‐1 (IL‐1). Parameters measured were glycosaminoglycan release, aggrecanase and matrix metalloproteinase (MMP) activity, and the levels of expression of messenger RNA (mRNA) for mediators of inflammation, aggrecanases, MMPs, and their natural tissue inhibitors (tissue inhibitors of metalloproteinases [TIMPs]).

Results

Supplementation with n‐3 PUFA (but not other fatty acids) reduced, in a dose‐dependent manner, the endogenous and IL‐1–induced release of proteoglycan metabolites from articular cartilage explants and specifically abolished endogenous aggrecanase and collagenase proteolytic activity. Similarly, expression of mRNA for ADAMTS‐4, MMP‐13, and MMP‐3 (but not TIMP‐1, ‐2, or ‐3) was also specifically abolished with n‐3 PUFA supplementation. In addition, n‐3 PUFA supplementation abolished the expression of mRNA for mediators of inflammation (cyclooxygenase 2, 5‐lipoxygenase, 5‐lipoxygenase–activating protein, tumor necrosis factor α, IL‐1α, and IL‐1β) without affecting the expression of message for several other proteins involved in normal tissue homeostasis.

Conclusion

These studies show that the pathologic indicators manifested in human OA cartilage can be significantly altered by exposure of the cartilage to n‐3 PUFA, but not to other classes of fatty acids.

     

Cartilage degradation biomarkers predict efficacy of a novel,highly selective matrix metalloproteinase 13 inhibitor in a dog model of osteoarthritis: Confirmation by multivariate analysis that modulation of type ii collagen and aggrecan degradation peptides parallels pathologic changes

Objective

To demonstrate that the novel highly selective matrix metalloproteinase 13 (MMP‐13) inhibitor PF152 reduces joint lesions in adult dogs with osteoarthritis (OA) and decreases biomarkers of cartilage degradation.

Methods

The potency and selectivity of PF152 were evaluated in vitro using 16 MMPs, TACE, and ADAMTS‐4 and ADAMTS‐5, as well as ex vivo in human cartilage explants. In vivo effects were evaluated at 3 concentrations in mature beagles with partial medial meniscectomy. Gross and histologic changes in the femorotibial joints were evaluated using various measures of cartilage degeneration. Biomarkers of cartilage turnover were examined in serum, urine, or synovial fluid. Results were analyzed individually and in combination using multivariate analysis.

Results

The potent and selective MMP‐13 inhibitor PF152 decreased human cartilage degradation ex vivo in a dose‐dependent manner. PF152 treatment of dogs with OA reduced cartilage lesions and decreased biomarkers of type II collagen (type II collagen neoepitope) and aggrecan (peptides ending in ARGN or AGEG) degradation. The dose required for significant inhibition varied with the measure used, but multivariate analysis of 6 gross and histologic measures indicated that all doses differed significantly from vehicle but not from each other. Combined analysis of cartilage degradation markers showed similar results.

Conclusion

This highly selective MMP‐13 inhibitor exhibits chondroprotective effects in mature animals. Biomarkers of cartilage degradation, when evaluated in combination, parallel the joint structural changes induced by the MMP‐13 inhibitor. These data support the potential therapeutic value of selective MMP‐13 inhibitors and the use of a set of appropriate biomarkers to predict efficacy in OA clinical trials.