Taku Yatabe

Hyaluronan inhibits expression of ADAMTS4 (aggrecanase-1) in human osteoarthritic chondrocytes

Background: Intra-articular injection of hyaluronan (HA) has been suggested to have a disease-modifying effect in osteoarthritis, but little is known about the possible mechanisms. Objective: To investigate the effects of HA species of different molecular mass, including 800 kDa (HA800) and 2700 kDa (HA2700), on the expression of aggrecanases (ie, ADAMTS species), which play a key role in aggrecan degradation. Methods: The effects of HA species on the expression of ADAMTS1, 4, 5, 8, 9 and 15 in interleukin 1α (IL1α)-stimulated osteoarthritic chondrocytes were studied by reverse transcription PCR and real-time PCR. Expression of ADAMTS4 protein and aggrecanase activity and signal transduction pathways of IL1, CD44 and intracellular adhesion molecule 1 (ICAM1) were examined by immunoblotting. Results: IL1α treatment of chondrocytes induced ADAMTS4, and HA800 and HA2700 significantly decreased IL1α-induced expression of ADAMTS4 mRNA and protein. IL1α-stimulated aggrecanase activity in osteoarthritic chondrocytes was reduced by treatment with HA2700 or transfection of small interfering RNA for ADAMTS4. A similar result was obtained when HA2700 was added to explant cultures of osteoarthritic cartilage. HA2700 neither directly inhibited nor bound to ADAMTS4. Downregulation of ADAMTS4 expression by HA2700 was attenuated by treatment of IL1α-treated chondrocytes with antibodies to CD44 and/or ICAM1. The increased phosphorylation of IL1 receptor-associated kinase-1 and extracellular signal-regulated protein kinase1/2 induced by the IL1α treatment was downregulated by enhanced IRAK-M expression after HA2700 treatment. Conclusion: These data suggest that HA2700 suppresses aggrecan degradation by downregulating IL1α-induced ADAMTS4 expression through the CD44 and ICAM1 signalling pathways in osteoarthritic chondrocytes.

Expression of ADAMTS4 (aggrecanase-1) in human osteoarthritic cartilage.

A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)1, 4, 5, 8, 9 and 15, members of the ADAMTS gene family, have the ability to degrade a major cartilage proteoglycan, aggrecan, at the specific sites, and thus are called ‘aggrecanases’. The expression of these ADAMTS species was examined in human osteoarthritic articular cartilage on reverse transcription–polymerase chain reaction. The results demonstrated the predominant expression of ADAMTS4 in osteoarthritic cartilage, while ADAMTS5 was constitutively expressed in osteoarthritic and normal cartilage. ADAMTS9 was expressed mainly in normal cartilage, whereas no or negligible expression of ADAMTS1, 8 and 15 was observed in either osteoarthritic or normal cartilage. In situ hybridization for ADAMTS4 indicated that chondrocytes in osteoarthritic cartilage expressed the mRNA. Two monoclonal antibodies to ADAMTS4 were developed, and immunolocalized ADAMTS4 to chondrocytes in the proteoglycan‐depleted zones of osteoarthritic cartilage, showing a direct correlation with the Mankin scores. Immunoblotting indicated a major protein band of 58 kDa in the chondrocyte culture media and osteoarthritic cartilage tissue homogenates. These data demonstrate that among the six ADAMTS species, ADAMTS4 is mainly expressed in an active form in osteoarthritic cartilage, and suggest that ADAMTS4 may play an important role in the degradation of aggrecan in human osteoarthritic cartilage.

Repair of full-thickness articular cartilage defects using injectable type II collagen gel embedded with cultured chondrocytes in a rabbit model

BackgroundRecently, tissue-engineered chondrocyte transplantation has been tried to treat full-thickness cartilage defects. We developed an injectable type II collagen gel scaffold by chemically reacting type II collagen with polyethylene glycol crosslinker. This type II collagen was prepared from the nasal septa of cattle. In the present study, chondrocytes embedded in type II collagen gel were injected into rabbit full-thickness cartilage defects without a periosteal graft, and the feasibility for clinical application of the gel was evaluated.MethodsChondrocytes were isolated from 1-kg New Zealand white rabbits. A full-thickness articular cartilage defect (5 mm diameter, 4 mm depth) was created on the patellar groove of the femur of 16 male 3-kg New Zealand white rabbits. A type II collagen solution of mixed chondrocytes at a density of 1 × 107 cells/ml was injected and transplanted into the defect in the right knee. The controls were the defect only in the left knee. At 4, 8, 12, and 24 weeks after operation, four cases from each group were evaluated macroscopically and histologically.ResultsAfter injection into the cartilage defect, the gel bonded to the adjacent cartilage and bone within several minutes. Macroscopic examination revealed that the surface of the transplanted area was smooth and exhibited similar coloration and good integration with the surrounding cartilage at 12 and 24 weeks after transplantation. Histological examination at 8 weeks revealed favorable hyaline cartilage regeneration with good chondrocyte morphology. At 12 and 24 weeks, reparative cartilage remained rich in type II collagen. According to O’Driscoll histological scores, significant differences between the transplanted and control groups were apparent at 12 and 24 weeks. Immunohistochemical staining indicated sufficient type II collagen synthesis in regenerated cartilage 8 weeks after transplantation, and it was maintained until 24 weeks.ConclusionsThese results indicate that type II collagen gel is suitable for injection into cartilage defects without any covering of a graft and offers a useful scaffold during chondrocyte transplantation.

ADAM‐12 (meltrin α) is involved in chondrocyte proliferation via cleavage of insulin‐like growth factor binding protein 5 in osteoarthritic cartilage

OBJECTIVE ADAMs are a gene family of multifunctional proteins. We undertook this study to determine which ADAM species is up-regulated in osteoarthritic (OA) cartilage and to examine its pathobiologic function. METHODS Expression of the 13 different metalloproteinase-type ADAMs was screened by reverse transcription-polymerase chain reaction (PCR), and expression levels of prototype membrane-anchored ADAM-12 (ADAM-12m) were determined by real-time PCR. ADAM-12m expression in articular cartilage was examined by in situ hybridization, immunohistochemistry, and immunoblotting. Chondrocytes were used for functional analyses of ADAM-12m. RESULTS ADAM-12m was selectively expressed in 87% of OA cartilage, and the expression level was significantly higher in OA cartilage than in normal cartilage. In situ hybridization showed that OA chondrocytes were responsible for the expression. ADAM-12m was immunolocalized on the membranes of OA chondrocytes, and its immunoreactivity correlated directly with the Mankin score and with degrees of chondrocyte cloning and proliferation. Immunoblotting analysis of OA chondrocytes demonstrated an active form of ADAM-12m. ADAM-12m expression in OA chondrocytes was selectively enhanced by transforming growth factor beta (TGFbeta), which also induced chondrocyte proliferation and degradation of insulin-like growth factor binding protein 5 (IGFBP-5). TGFbeta-induced chondrocyte proliferation was inhibited by suppression of IGF-1 signaling. In addition, TGFbeta-induced chondrocyte proliferation, chondrocyte cloning in agarose gel culture, and digestion of IGFBP-5 were inhibited with ADAM inhibitor, anti-ADAM-12 antibody, and small interfering RNA for ADAM-12. CONCLUSION These data suggest a novel function of ADAM-12m in chondrocyte proliferation and cloning in OA cartilage through enhanced bioavailability of IGF-1 from the IGF-1-IGFBP-5 complex by selective IGFBP-5 digestion.

Calcium pentosan polysulfate directly inhibits enzymatic activity of ADAMTS4 (aggrecanase-1) in osteoarthritic chondrocytes

Aggrecanases that include ADAMTS1, 4, 5, 8, 9 and 15 are considered to play key roles in aggrecan degradation in osteoarthritic cartilage. Here we demonstrate that calcium pentosan polysulfate (CaPPS) directly inhibits the aggrecanase activity of ADAMTS4 without affecting the mRNA expression of the ADAMTS species in interleukin‐1α‐stimulated osteoarthritic chondrocytes. Synthetic peptides corresponding to specific regions of the thrombospondin type 1 repeat, cysteine‐rich or spacer domain of ADAMTS4 inhibit the binding to immobilized CaPPS. These data suggest that CaPPS could function as chondroprotective agent for the treatment of osteoarthritis by inhibition of ADAMTS4 through interaction with the C‐terminal ancillary domain.

Flow cytometric technique for the detection of phagocytosed wear particles in patients with total joint arthroplasty

Wear particles from prosthetic implants have been shown to cause inflammatory synovitis and periprosthetic osteolysis. These particle-induced pathologies are manifestations of adverse cellular responses to phagocytosed particles. In this study, phagocytosis of polyethylene particles was analyzed using flow cytometry (FCM), and the clinical utility of FCM in diagnosing particle-induced synovitis was examined. Ultra high molecular weight polyethylene particles exhibited natural autofluorescence at fluorescein isothiocyanate wavelengths when determined by FCM. Using this autofluorescent property of the particles, peripheral blood monocytes (PBMs) phagocytosing the particles could be detected by autofluorescence emission from intracellular particles. This autofluorescence from PBMs increased with particles/cell ratio in a dose-dependent manner. Particle phagocytosis was also detectable in joint fluid cells obtained from the patients with particle-induced synovitis following total joint arthroplasty (TJA). Phenotypic analysis indicated that phagocytes were typically CD14(+)CD16(-) macrophages, with occasional CD14(+)CD16(+) macrophages. Interestingly, decreased autofluorescence intensity of CD14(+) cells was observed after arthroscopic drainage, suggesting that FCM was useful in examining whether the treatment was successful. In summary, these results indicate that FCM analysis offers a simple and useful method of detecting phagocytosis of polyethylene particles and estimating the severity of particle-induced synovitis in post-TJA patients.

RECK is up-regulated and involved in chondrocyte cloning in human osteoarthritic cartilage

Reversion-inducing cysteine-rich protein with Kazal motifs (RECK) is a membrane-anchored matrix metalloproteinase regulator, but its functions in cartilage are not fully understood. The aim of the present study was to examine the expression and functions of RECK in human osteoarthritic (OA) cartilage. Quantitative RT-PCR indicated that the expression level of RECK is significantly higher in OA cartilage than in normal cartilage. By immunohistochemical analysis, RECK was localized to chondrocytes in OA cartilage, and the immunoreactivity directly correlated with the Mankin score and degree of chondrocyte cloning and proliferation. In cultured OA chondrocytes, RECK was expressed on the cell surface by glycosylphosphatidylinositol anchoring. The expression was stimulated by insulin-like growth factor-1 and suppressed by interleukin-1 and tumor necrosis factor-alpha. Down-regulation of RECK by small interfering RNA showed reduced spreading and smaller focal adhesions in the chondrocytes. Chondrocyte migration in a monolayer wounding assay was increased by down-regulation of RECK and inhibited by RECK overexpression in an matrix metalloproteinase activity-dependent manner. On the other hand, chondrocyte proliferation was suppressed by RECK silencing, and this was associated with reduced phosphorylation of focal adhesion kinase and extracellular signal-regulated kinase, whereas the proliferation was enhanced by RECK overexpression. These data are the first to demonstrate that RECK is up-regulated in human OA cartilage and suggest that RECK plays a role in chondrocyte cloning probably through suppression and promotion of chondrocyte migration and proliferation, respectively.