Chondrocyte‐intrinsic Smad3 represses Runx2‐inducible matrix metalloproteinase 13 expression to maintain articular cartilage and prevent osteoarthritis

Objective

To identify mechanisms by which Smad3 maintains articular cartilage and prevents osteoarthritis.

Methods

A combination of in vivo and in vitro approaches was used to test the hypothesis that Smad3 represses Runx2‐inducible gene expression to prevent articular cartilage degeneration. Col2‐Cre;Smad3^fl/fl mice allowed study of the chondrocyte‐intrinsic role of Smad3 independently of its role in the perichondrium or other tissues. Primary articular cartilage chondrocytes from Smad3^fl/fl mice and ATDC5 chondroprogenitor cells were used to evaluate Smad3 and Runx2 regulation of matrix metalloproteinase 13 (MMP‐13) messenger RNA (mRNA) and protein expression.

Results

Chondrocyte‐specific reduction of Smad3 caused progressive articular cartilage degeneration due to imbalanced cartilage matrix synthesis and degradation. In addition to reduced type II collagen mRNA expression, articular cartilage from Col2‐Cre;Smad3^fl/fl mice was severely deficient in type II collagen and aggrecan protein due to excessive MMP‐13–mediated proteolysis of these key cartilage matrix constituents. Normally, transforming growth factor β (TGFβ) signals through Smad3 to confer a rapid and dynamic repression of Runx2‐inducible MMP‐13 expression. However, we found that in the absence of Smad3, TGFβ signals through p38 and Runx2 to induce MMP‐13 expression.

Conclusion

Our findings elucidate a mechanism by which Smad3 mutations in humans and mice cause cartilage degeneration and osteoarthritis. Specifically, Smad3 maintains the balance between cartilage matrix synthesis and degradation by inducing type II collagen expression and repressing Runx2‐inducible MMP‐13 expression. Selective activation of TGFβ signaling through Smad3, rather than p38, may help to restore the balance between matrix synthesis and proteolysis that is lost in osteoarthritis.

     

Pathogenesis of osteoarthritis‐like changes in the joints of mice deficient in type IX collagen

Objective

To examine the pathogenetic mechanisms of osteoarthritis (OA)–like changes in Col9a1^−/− mice, which are deficient in type IX collagen.

Methods

Knee joints and temporomandibular joints (TMJs) from Col9a1^−/− mice and their wild‐type (Col9a1^+/+) littermates were examined by light microscopy. Immunohistochemical staining was performed to examine the expression of matrix metalloproteinase 3 (MMP‐3) and MMP‐13, degraded type II collagen, and the discoidin domain receptor 2 (DDR‐2) in knee joints. Cartilage mechanics were also evaluated for compressive properties by microindentation testing of the tibial plateau and for tensile properties by osmotic loading of the femoral condyle.

Results

Histologic analysis showed age‐dependent OA‐like changes in the knee and TMJs of Col9a1^−/− mice starting at the age of 3 months. At the age of 6 months, enhanced proteoglycan degradation was observed in the articular cartilage of the knee and TMJs of the mutant mice. The expression of MMP‐13 and DDR‐2 protein and the amount of degraded type II collagen were higher in the knee joints of Col9a1^−/− mice than in their wild‐type littermates at the age of 6 months. Changes in cartilage mechanics were observed in the femoral and tibial plateaus of Col9a1^−/− mice at 6 months, including a decrease in the compressive modulus and uniaxial modulus. At 3 and 6 months of age, tibial cartilage in Col9a1^−/− mice was found to be more permeable to fluid flow, with an associated compromise in the fluid pressurization mechanism of load support. All of these changes occurred only at medial sites.

Conclusion

Lack of type IX collagen in Col9a1^−/− mice results in age‐dependent OA‐like changes in the knee joints and TMJs.

     

Developmental and osteoarthritic changes in Col6a1‐knockout mice: Biomechanics of type VI collagen in the cartilage pericellular matrix

Objective

Chondrocytes, the sole cell type in articular cartilage, maintain the extracellular matrix (ECM) through a homeostatic balance of anabolic and catabolic activities that are influenced by genetic factors, soluble mediators, and biophysical factors such as mechanical stress. Chondrocytes are encapsulated by a narrow tissue region termed the “pericellular matrix” (PCM), which in normal cartilage is defined by the exclusive presence of type VI collagen. Because the PCM completely surrounds each cell, it has been hypothesized that it serves as a filter or transducer for biochemical and/or biomechanical signals from the cartilage ECM. The present study was undertaken to investigate whether lack of type VI collagen may affect the development and biomechanical function of the PCM and alter the mechanical environment of chondrocytes during joint loading.

Methods

Col6a1^−/− mice, which lack type VI collagen in their organs, were generated for use in these studies. At ages 1, 3, 6, and 11 months, bone mineral density (BMD) was measured, and osteoarthritic (OA) and developmental changes in the femoral head were evaluated histomorphometrically. Mechanical properties of articular cartilage from the hip joints of 1‐month‐old Col6a1^−/−, Col6a1^+/−, and Col6a1^+/+ mice were assessed using an electromechanical test system, and mechanical properties of the PCM were measured using the micropipette aspiration technique.

Results

In Col6a1^−/− and Col6a1^+/− mice the PCM was structurally intact, but exhibited significantly reduced mechanical properties as compared with wild‐type controls. With age, Col6a1^−/− mice showed accelerated development of OA joint degeneration, as well as other musculoskeletal abnormalities such as delayed secondary ossification and reduced BMD.

Conclusion

These findings suggest that type VI collagen has an important role in regulating the physiology of the synovial joint and provide indirect evidence that alterations in the mechanical environment of chondrocytes, due to either loss of PCM properties or Col6a1^−/−‐derived joint laxity, can lead to progression of OA.

     

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.

     

Osteoarthritis‐like changes and decreased mechanical function of articular cartilage in the joints of mice with the chondrodysplasia gene (cho)

Objective

To investigate whether heterozygosity for a loss‐of‐function mutation in the gene encoding the α1 chain of type XI collagen (Col11a1) in mice (chondrodysplasia, cho) causes osteoarthritis (OA), and to understand the biochemical and biomechanical effects of this mutation on articular cartilage in knee and temporomandibular (TM) joints.

Methods

Articular cartilage from the knee and TM joints of mice heterozygous for cho (cho/+) and their wild‐type littermates (+/+) was examined. The morphologic properties of cartilage were evaluated, and collagen fibrils were examined by transmission electron microscopy. Immunohistochemical staining was performed to examine the protein expression levels of matrix metalloproteinase 3 (MMP‐3) and MMP‐13 in knee joints. In 6‐month‐old animals, fixed‐charge density was determined using a semiquantitative histochemical method, and tensile stiffness was determined using an osmotic loading technique.

Results

The diameter of collagen fibrils in articular cartilage of knee joints from heterozygous cho/+ mice was increased relative to that in control cartilage, and histologic analysis showed OA‐like degenerative changes in knee and TM joints, starting at age 3 months. The changes became more severe with aging. At 3 months, protein expression for MMP‐3 was increased in knee joints from cho/+ mice. At 6 months, protein expression for MMP‐13 was higher in knee joints from cho/+ mice than in joints from their wild‐type littermates, and negative fixed‐charge density was significantly decreased. Moreover, tensile stiffness in articular cartilage of knee joints from cho/+ mice was moderately reduced and was inversely correlated with the increase in articular cartilage degeneration.

Conclusion

Heterozygosity for a loss‐of‐function mutation in Col11a1 results in the development of OA in the knee and TM joints of cho/+ mice. Morphologic and biochemical evidence of OA appears to precede significant mechanical changes, suggesting that the cho mutation leads to OA through a mechanism that does not initially involve mechanical factors.

     

Attenuation of osteoarthritis progression by reduction of discoidin domain receptor 2 in mice

Objective

To investigate whether the reduction of discoidin domain receptor 2 (DDR‐2), a cell membrane tyrosine kinase receptor for native type II collagen, attenuates the progression of articular cartilage degeneration in mouse models of osteoarthritis (OA).

Methods

Double‐heterozygous (type XI collagen–deficient [Col11a1^+/−] and Ddr2‐deficient [Ddr2^+/−]) mutant mice were generated. Knee joints of Ddr2^+/− mice were subjected to microsurgical destabilization of the medial meniscus. Conditions of the articular cartilage from the knee joints of the double‐heterozygous mutant and surgically treated mice were examined by histology, evaluated using a modified Mankin scoring system, and characterized by immunohistochemistry.

Results

The rate of progressive degeneration in knee joints was dramatically reduced in the double‐heterozygous mutant mice compared with that in the type XI collagen–deficient mice. The progression in the double‐heterozygous mutant mice was delayed by ∼6 months. Following surgical destabilization of the medial meniscus, the progressive degeneration toward OA was dramatically delayed in the Ddr2^+/− mice compared with that in their wild‐type littermates. The articular cartilage damage present in the knee joints of the mice was directly correlated with the expression profiles of DDR‐2 and matrix metalloproteinase 13.

Conclusion

Reduction of DDR‐2 expression attenuates the articular cartilage degeneration of knee joints induced either by type XI collagen deficiency or by surgical destabilization of the medial meniscus.

     

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.

     

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.

     

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.

     

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.

     

Increased collagen and aggrecan degradation with age in the joints of Timp3−/− mice

Objective

To investigate the in vivo effect of an imbalance between metalloproteinases and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs), in mouse articular cartilage.

Methods

Hind joints of Timp3^−/− and wild‐type mice were examined by routine staining and by immunohistochemical analysis using antibodies specific for type X collagen and for the neoepitopes produced on proteolytic cleavage of aggrecan (… VDIPEN and … NVTEGE) and type II collagen. The neoepitope generated on cleavage of type II collagen by collagenases was quantitated in sera by enzyme‐linked immunosorbent assay.

Results

Articular cartilage from Timp3‐knockout animals (ages ≥6 months) showed reduced Safranin O staining and an increase in …VDIPEN content compared with cartilage from heterozygous and wild‐type animals. There was also a slight increase in … NVTEGE content in articular cartilage and menisci of Timp3^−/− animals. Chondrocytes showed strong pericellular staining for type II collagen cleavage neoepitopes, particularly in the superficial layer, in knockout mice. Also, there was more type X collagen expression in the superficial zone of articular cartilage, especially around clusters of proliferating chondrocytes, in the knockout mice. More type II collagen cleavage product was found in the serum of Timp3^−/− mice compared with wild‐type animals. This increase was significant in 15‐month‐old animals.

Conclusion

These results indicate that TIMP‐3 deficiency results in mild cartilage degradation similar to changes seen in patients with osteoarthritis, suggesting that an imbalance between metalloproteinases and TIMP‐3 may play a pathophysiologic role in the development of this disease.

     

Gene deletion of either interleukin‐1β, interleukin‐1β–converting enzyme,inducible nitric oxide synthase,or stromelysin 1 accelerates the development of knee osteoarthritis in mice after surgical transection of the medial collateral ligament and partial medial meniscectomy

Objective

To investigate the development of osteoarthritis (OA) after transection of the medial collateral ligament and partial medial meniscectomy in mice in which genes encoding either interleukin‐1β (IL‐1β), IL‐1β–converting enzyme (ICE), stromelysin 1, or inducible nitric oxide synthase (iNOS) were deleted.

Methods

Sectioning of the medial collateral ligament and partial medial meniscectomy were performed on right knee joints of wild‐type and knockout mice. Left joints served as unoperated controls. Serial histologic sections were obtained from throughout the whole joint of both knees 4 days or 1, 2, 3, or 4 weeks after surgery. Sections were graded for OA lesions on a scale of 0–6 and were assessed for breakdown of tibial cartilage matrix proteoglycan (aggrecan) and type II collagen by matrix metalloproteinases (MMPs) and aggrecanases with immunohistochemistry studies using anti‐VDIPEN, anti‐NITEGE, and Col2‐3/4C_short neoepitope antibodies. Proteoglycan depletion was assessed by Alcian blue staining and chondrocyte cell death, with the TUNEL technique.

Results

All knockout mice showed accelerated development of OA lesions in the medial tibial cartilage after surgery, compared with wild‐type mice. ICE‐, iNOS‐, and particularly IL‐1β–knockout mice developed OA lesions in the lateral cartilage of unoperated limbs. Development of focal histopathologic lesions was accompanied by increased levels of MMP‐, aggrecanase‐, and collagenase‐generated cleavage neoepitopes in areas around lesions, while nonlesional areas showed no change in immunostaining. Extensive cell death was also detected by TUNEL staining in focal areas around lesions.

Conclusion

We postulate that deletion of each of these genes, which encode molecules capable of producing degenerative changes in cartilage, leads to changes in the homeostatic controls regulating the balance between anabolism and catabolism, favoring accelerated cartilage degeneration. These observations suggest that these genes may play important regulatory roles in maintaining normal homeostasis in articular cartilage matrix turnover.

     

In vivo bone‐specific EphB4 overexpression in mice protects both subchondral bone and cartilage during osteoarthritis

Objective

In vitro activation of the receptor EphB4 positively affects human osteoarthritis (OA) articular cell metabolism. However, the specific in vivo role of this ephrin receptor in OA remains unknown. We investigated in mice the in vivo effect of bone‐specific EphB4 overexpression on OA pathophysiology.

Methods

Morphometric, morphologic, and radiologic evaluations were performed on postnatal day 5 (P5) mice and on 10‐week‐old mice. Knee OA was induced surgically by destabilization of the medial meniscus (DMM) in 10‐week‐old male EphB4 homozygous transgenic (EphB4‐Tg) and wild‐type (WT) mice. Medial compartment evaluations of cartilage were performed using histology and immunohistochemistry, and evaluations of subchondral bone using histomorphometry, osteoclast staining, and micro–computed tomography.

Results

There was no obvious phenotype difference in skeletal development between EphB4‐Tg mice and WT mice at P5 or at 10 weeks. At 8 and 12 weeks post‐DMM, the EphB4‐Tg mice demonstrated significantly less cartilage alteration in the medial tibial plateau and the femoral condyle than did the WT mice. This was associated with a significant reduction of aggrecan and type II collagen degradation products, type X collagen, and collagen fibril disorganization in the operated EphB4‐Tg mice. The medial tibial subchondral bone demonstrated a significant reduction in sclerosis, bone volume, trabecular thickness, and number of tartrate‐resistant acid phosphatase–positive osteoclasts at both times assessed post‐DMM in the EphB4‐Tg mice than in the WT mice.

Conclusion

This is the first in vivo evidence that bone‐specific EphB4 overexpression exerts a protective effect on OA joint structural changes. The findings of this study stress the in vivo importance of subchondral bone biology in cartilage integrity.

     

Sirtuin 1 enzymatic activity is required for cartilage homeostasis in vivo in a mouse model

Objective

We and others previously demonstrated that sirtuin 1 (SIRT‐1) regulates apoptosis and cartilage‐specific gene expression in human chondrocytes and mouse models. This study was undertaken to determine if SIRT‐1 enzymatic activity plays a protective role in cartilage homeostasis in vivo, by investigating mice with SIRT‐1 mutations to characterize their cartilage.

Methods

Articular cartilage was harvested from the paws and knees of 5‐ and 6‐month‐old wild‐type (WT) mice and mice homozygous for SIRT‐1^tm2.1Mcby (SIRT‐1^y/y), an allele carrying a point mutation that encodes a SIRT‐1 protein with no enzymatic activity (y/y mice). Mice ages 2 days old and 6–7 days old were also examined. Mouse joint cartilage was processed for histologic examination or biochemical analyses of chondrocyte cultures.

Results

We found that articular cartilage tissue sections from y/y mice of up to 6 months of age contained reduced levels of type II collagen, aggrecan, and glycosaminoglycan compared to sections from WT mice. In contrast, protein levels of matrix metalloproteinase 8 (MMP‐8), MMP‐9, and MMP‐13 were elevated in the cartilage of y/y mice. In addition, chondrocyte apoptosis was elevated in SIRT‐1 mutant mice as compared to their WT littermates. Consistent with these observations, protein tyrosine phosphatase 1b was elevated in the y/y mice.

Conclusion

Our in vivo findings in this animal model demonstrate that mice with defective SIRT‐1 also have defective cartilage, with elevated rates of cartilage degradation with age. Hence, normal cartilage homeostasis requires enzymatically active SIRT‐1 protein.

     

Stress‐induced cartilage degradation does not depend on the NLRP3 inflammasome in human osteoarthritis and mouse models

Objective

The main feature of osteoarthritis (OA) is degradation and loss of articular cartilage. Interleukin‐1β (IL‐1β) is thought to have a prominent role in shifting the metabolic balance toward degradation. IL‐1β is first synthesized as an inactive precursor that is cleaved to the secreted active form mainly in the “inflammasome,” a complex of initiators (including NLRP3), adaptor molecule ASC, and caspase 1. The aim of this study was to clarify the roles of IL‐1β and the inflammasome in cartilage breakdown.

Methods

We assessed IL‐1β release by cartilage explants from 18 patients with OA. We also evaluated the lipopolysaccharide (LPS)–, IL‐1α–, and tumor necrosis factor α (TNFα)–induced activity of matrix metalloproteinase 3 (MMP‐3), MMP‐9, and MMP‐13 in NLRP3‐knockout mice and wild‐type mice and the inhibition of caspase 1 with Z‐YVAD‐FMK and the blockade of IL‐1β with IL‐1 receptor antagonist (IL‐1Ra). Cartilage explants from NLRP3‐knockout mice and IL‐1R type I (IL‐1RI)–knockout mice were subjected to excessive dynamic compression (0.5 Hz, 1 MPa) to trigger degradation, followed by assessment of load‐induced glycosaminoglycan (GAG) release and MMP enzymatic activity.

Results

Despite the expression of NLRP3, ASC, and caspase 1, OA cartilage was not able to produce active IL‐1β. LPS, IL‐1α, and TNFα dose‐dependently increased MMP‐3, MMP‐9, and MMP‐13 activity in cultured chondrocytes and in NLRP3^−/− chondrocytes, and this effect was not changed by inhibiting caspase 1 or IL‐1β. The load‐induced increase in GAG release and MMP activity was not affected by knockout of NLRP3 or IL‐1RI in cartilage explants.

Conclusion

OA cartilage may be degraded independently of any inflammasome activity, which may explain, at least in part, the lack of effect of IL‐1β inhibitors observed in previous trials.

     

Type II collagen levels correlate with mineralization by articular cartilage vesicles

Objective

Pathologic mineralization is common in osteoarthritic (OA) cartilage and may be mediated by extracellular organelles known as articular cartilage vesicles (ACVs). Paradoxically, ACVs isolated from OA human cartilage mineralize poorly in vitro compared with those isolated from normal porcine cartilage. We recently showed that collagens regulate ACV mineralization. We sought to determine differences between collagens and collagen receptors on human and porcine ACVs as a potential explanation of their different mineralization behaviors.

Methods

ACVs were enzymatically released from old and young human and porcine hyaline articular cartilage. Western blotting was used to determine the presence of types I, II, VI, and X collagen and various collagen receptors on ACVs. Type II collagen was quantified by enzyme‐linked immunosorbent assay. Biomineralization was assessed by measuring the uptake of ⁴⁵Ca by isolated ACVs in agarose gels and by ACVs in situ in freeze‐thawed cartilage.

Results

As previously shown, isolated human ACVs mineralized poorly in response to ATP compared with porcine ACVs, but human and porcine ACVs mineralized similarly in situ in freeze‐thawed cartilage. Type II collagen levels were 100‐fold higher in isolated human ACVs than in porcine ACVs. Type II collagen in human ACVs was of high molecular weight. Transglutaminase‐crosslinked type II collagen showed increased resistance to collagenase, suggesting a possible explanation for residual collagen on human ACVs. Expression of other collagens and collagen receptors was similar on human and porcine ACVs.

Conclusion

Higher levels of type II collagen in human ACV preparations, perhaps mediated by increased transglutaminase crosslinking, may contribute to the decreased mineralization observed in isolated human ACVs in vitro.