Takahiro Iino

Deficiency of tenascin-C delays articular cartilage repair in mice

OBJECTIVE In human articular cartilage, tenascin-C (TN-C) expression decreases during maturation of chondrocytes, and almost disappears in adults; however, it reappears in damaged cartilage. To examine the effects of TN-C on cartilage degeneration and repair, we compared articular cartilage degeneration between wild-type (WT) and tenascin-C knockout mouse (TNKO) mice using a spontaneous osteoarthritis (OA) in aged joints and surgical OA model. In addition, we made full-thickness cartilage defects and compared the cartilage repair process between the two groups. METHODS The surgical procedure to create degenerative OA model was performed by transecting the anterior cruciate ligament and medial collateral ligament. Full-thickness defects were created in the center of the femoral trochlea to evaluate cartilage repair. Sections of cartilage were stained with hematoxylin and eosin or safranin-O, and immunostaining for TN-C. The degrees of degeneration and repair were graded. RESULTS In the WT surgical OA model, the articular cartilage was almost normal at 2 weeks, but safranin-O decreased staining at 4 weeks. In TNKO mice, safranin-O decreased staining at 2 weeks, and cartilage was injured intensely at 4 weeks. In the cartilage repair model, TN-C was expressed after 1 week, was strongly expressed in the upper layer of regenerated tissue after 3 weeks, and disappeared after 6 weeks. The defects were restored until 6 weeks in WT mice; however, defects in TNKO mice were filled with fibrous tissue with no cartilage-like tissue. CONCLUSIONS This study revealed that cartilage repair in TNKO mice was significantly slower than that in WT mice and that the deficiency of TN-C progressed during cartilage degeneration.

Thrombin-Cleaved Osteopontin in Synovial Fluid of Subjects with Rheumatoid Arthritis

Objective. Osteopontin (OPN) is an extracellular matrix glycoprotein that has been recognized as a potential inflammatory cytokine. The function of OPN is modulated by protease digestion, and a thrombin-cleaved form of OPN is involved in the pathogenesis of various inflammatory disorders. We examined thrombin-cleaved OPN products in synovial fluid from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Methods. Synovial fluid samples were obtained from knees of 20 patients with RA and 111 patients with OA. Thrombin-cleaved OPN product was determined using Western blotting. Levels of thrombin- cleaved and full-length OPN in synovial fluid were determined by ELISA. Synovia were analyzed by immunohistochemistry using an antibody specific to the thrombin-cleaved form. Results. Immunoblotting showed the presence of thrombin-cleaved OPN in synovial fluid from patients with RA and OA. ELISA results showed no difference between concentrations of full-length OPN in the synovial fluid of RA and OA patients; however, thrombin-cleaved OPN concentrations in RA synovial fluid samples were roughly 30-fold higher compared with OA samples (p < 0.001). Synovial fluid concentrations of thrombin-cleaved OPN in RA did not correlate with C-reactive protein levels. Immunohistochemistry of the synovium showed stronger reactivity in RA than in OA samples. Conclusion. Local generation of thrombin-cleaved OPN was increased in RA joints. Thrombin-cleaved OPN may be a useful biochemical marker of RA.

Distribution and role of tenascin-C in human osteoarthritic cartilage

BackgroundTenascin-C (TN-C) is expressed in the cartilage of osteoarthritis (OA). We examined whether TN-C was involved in cartilage repair of the diseased joints. Human articular cartilage samples were obtained from patients with OA and those with normal joints.MethodsImmunohistochemistry testing of TN-C, chondroitin sulfate (CS), and proliferating cell nuclear antigen (PCNA) was performed. Chondrocytes were isolated from human cartilage and cultured. After treatment with TN-C, chondrocyte proliferation s was analyzed by bromodeoxyuridine (BrdU) incorporation assay using an enzyme-linked immunosorbent assay kit. Glycosaminoglycan content was determined by dimethylmethylene blue (DMMB) assay. The mRNA expression of aggrecan was also analyzed, by quantitative real-time polymerase chain reaction (PCR).ResultsIn osteoarthritic cartilage, increased TN-C staining was observed with the degeneration of articular cartilage in comparison with normal cartilage. TN-C staining was shown in the cartilage surface overlying CS-positive areas. In addition, the expression of PCNA in the positive areas for TN-C was significantly higher than that in the negative areas. Treatment of human articular chondrocytes with 10 μg/ml TN-C accelerated chondrocyte proliferation, increased the proteoglycan amount in culture, and increased the expression of aggrecan mRNA.ConclusionsOur findings indicate that the distribution of TN-C is related to CS production and chondrocyte proliferation in osteoarthritic cartilage and that TN-C has effects on DNA synthesis, proteoglycan content, and aggrecan mRNA expression in vitro. TN-C may be responsible for repair in human osteoarthritic cartilage.

Protective effect of edaravone for tourniquet-induced ischemia-reperfusion injury on skeletal muscle in murine hindlimb

BackgroundStudies have shown that ischemia-reperfusion (I/R) produces free radicals leading to lipid peroxidation and damage to skeletal muscle. The purposes of this study were 1) to assess the histological findings of gastrocnemius muscle (GC) and tibialis anterior muscle (TA) in I/R injury model mice, 2) to histologically analyze whether a single pretreatment of edaravone inhibits I/R injury to skeletal muscle in murine models and 3) to evaluate the effect of oxidative stress on these muscles.MethodsC57BL6 mice were divided in two groups, with one group receiving 3 mg/kg intraperitoneal injections of edaravone (I/R + Ed group) and the other group receiving an identical amount of saline (I/R group) 30 minutes before ischemia. Edaravone (3-methy-1-pheny1-2-pyrazolin-5-one) is a potent and novel synthetic scavenger of free radicals. This drug inhibits both nonenzymatic lipid peroxidation and the lipoxygenase pathway, in addition to having potent antioxidant effects against ischemia reperfusion. The duration of the ischemia was 1.5 hours, with reperfusion at either 24 or 72 hours (3 days). Specimens of gastrocnemius (GC) and anterior tibialis (TA) were removed for histological evaluation and biochemical analysis.ResultsThis model of I/R injury was highly reproducible in histologic muscle damage. In the histologic damage score, the mean muscle fibers and inflammatory cell infiltration in the I/R + Ed group were significantly less than the corresponding values of observed in the I/R group. Thus, pretreatment with edaravone was observed to have a protective effect on muscle damage after a period of I/R in mice. In addition, the mean muscle injury score in the I/R + Ed group was also significantly less than the I/R group. In the I/R + Ed group, the mean malondialdehyde (MDA) level was lower than in the I/R group and western-blotting revealed that edaravone pretreatment decreased the level of inducible nitric oxide synthase (iNOS) expression.ConclusionsEdaravone was found to have a protective effect against I/R injury by directly inhibiting lipid peroxidation of the myocyte by free radicals in skeletal muscles and may also reduce the secondary edema and inflammatory infiltration incidence of oxidative stress on tissue.

Prognostic significance of CD155 mRNA expression in soft tissue sarcomas

CD155 was initially identified as a receptor for poliovirus. Several studies have demonstrated that CD155 overexpression in cancer cells is significant in their migration, invasion, proliferation and metastasis. The objective of the present study was to investigate the correlation between CD155 expression and the clinical aggressiveness of soft tissue tumors. The CD155 expression levels in 43 surgically-resected soft tissue tumors were evaluated using the quantitative real-time polymerase chain reaction (PCR). The clinicopathogical factors affecting the expression levels of CD155 mRNA were investigated and the association between the expression levels of CD155 and patient prognosis was identified. The CD155 expression level was not correlated with the patient gender, site of the primary tumor, tumor depth, tumor size or presence of distant metastasis at presentation, but was correlated with patient age (Fisher’s exact test). The local recurrence-free survival rate for patients with a high CD155 expression level was observed to be significantly poorer compared with that of patients with low CD155 expression levels (P=0.0401). Moreover, a multivariate analysis indicated that a high CD155 expression level was an independent adverse prognostic factor for local recurrence-free survival (hazard ratio, 6.369; P=0.0328). The present study therefore suggests that the expression level of CD155 is a useful marker for predicting the local recurrence of soft tissue tumors.

Activated protein C stimulates osteoblast proliferation via endothelial protein C receptor.

INTRODUCTION Bone is continually remodeled by the action of osteoblasts, osteocytes, and osteoclasts. Resting osteoblasts are able to proliferate and differentiate into mature osteoblasts when physiologically required, as after tissue injury. Activated protein C (APC) is a serine protease that functions in anticoagulation, anti-inflammation, anti-apoptosis, cell proliferation, and wound repair. In this study, we examined the effect of APC on osteoblast proliferation and differentiation. MATERIALS AND METHODS We examined the presence of protein C in human fracture hematoma by immunohistochemical staining. We then evaluated the effect of APC, diisopropyl fluorophosphate-inactivated APC (DIP-APC) or protein C zymogen on normal human osteoblast (NHOst) proliferation using tetrazolium salt assay in the presence or absence of aprotinin, hirudin, protein C, antibody against protein C, endothelial protein C receptor (EPCR) or protease-activated receptor (PAR)-1. Finally, activation of p44/42 MAP kinase was evaluated by Western blot analysis. RESULTS Both APC and DIP-APC increased osteoblast proliferation in a dose-dependent manner, while protein C did not. The APC-induced increased proliferation of osteoblast was not affected by aprotinin, hirudin, and anti-protein C antibody which inhibits the protease activity of APC. Treatment with protein C or anti-EPCR antibody which inhibits APC binding to EPCR inhibited APC-mediated osteoblast proliferation, while treatment with anti-PAR-1 antibody did not. APC promoted the phosphorylation of p44/42 MAP kinase within osteoblasts; this effect was inhibited by the anti-EPCR antibody. CONCLUSIONS APC stimulates osteoblast proliferation by activating p44/42 MAP kinase through a mechanism that requires EPCR but not PAR-1 or the proteolytic activity of APC. APC generated at fracture sites may contribute to fracture healing by promoting osteoblast proliferation.

The cleavage of N-cadherin is essential for chondrocyte differentiation.

The aggregation of chondroprogenitor mesenchymal cells into precartilage condensation represents one of the earliest events in chondrogenesis. N-cadherin is a key cell adhesion molecule implicated in chondrogenic differentiation. Recently, ADAM10-mediated cleavage of N-cadherin has been reported to play an important role in cell adhesion, migration, development and signaling. However, the significance of N-cadherin cleavage in chondrocyte differentiation has not been determined. In the present study, we found that the protein turnover of N-cadherin is accelerated during the early phase of chondrogenic differentiation in ATDC5 cells. Therefore, we generated the subclones of ATDC5 cells overexpressing wild-type N-cadherin, and two types of subclones overexpressing a cleavage-defective N-cadherin mutant, and examined the response of these cells to insulin stimulation. The ATDC5 cells overexpressing cleavage-defective mutants severely prevented the formation of cartilage aggregates, proteoglycan production and the induction of chondrocyte marker gene expression, such as type II collagen, aggrecan and type X collagen. These results suggested that the cleavage of N-cadherin is essential for chondrocyte differentiation.

Effect of tenascin‐C on the repair of full‐thickness osteochondral defects of articular cartilage in rabbits

The purpose of this study was to examine the effect of tenascin‐C (TNC) on the repair of full‐thickness osteochondral defects of articular cartilage in vivo. We used a gellan–gellan–sulfate sponge (Gellan–GS) to maintain a TNC‐rich environment in the cartilage defects. We implanted Gellan‐GS soaked in PBS only (Group 1), Gellan‐GS soaked in 10 µg/ml of TNC (Group 2), and Gellan‐GS soaked in 100 µg/ml of TNC (Group 3) into a full‐thickness osteochondral defect of the patellar groove of rabbits. The defect area was examined grossly and histologically 4–12 weeks after surgery. Sections of synovium were also immunohistochemically investigated. Histologically as well as macroscopically, the defects in Group 2 showed better repair than the other groups at 8 and 12 weeks after surgery. Inflammation of the synovium tended to diminish over time in all groups, and the degree of synovitis was the same for all three groups at each time point. In conclusion, Gellan–GS soaked in TNC can be used as a novel scaffold for the repair of articular cartilage defects. This study also indicates that TNC promotes the repair of full‐thickness osteochondral defects in vivo.

STAT3 inhibitor, cucurbitacin I, is a novel therapeutic agent for osteosarcoma.

The development of clinical agents remains a costly and time-consuming process. Although identification of new uses of existing drugs has been recognized as a more efficient approach for drug discovery than development of novel drugs, little screening of drugs that might be used for a rare malignant tumor such as osteosarcoma (OS) has been performed. In this study, we attempted to identify new molecular targeted agents for OS by employing Screening Committee of Anticancer Drugs (SCADS) kits. To screen compounds for OS treatment, their effect on cell viability of the OS cell lines 143B, MG63, HOS, SAOS-2, and HUO9 were evaluated. Candidate drugs were narrowed down based on a global anti-proliferative effect against these five OS cell lines. After excluding cytotoxic compounds and compounds unsuitable for in vivo administration, cucurbitacin I was extracted. Cucurbitacin I has been found to have cytotoxic and anti-proliferative properties against several tumors through inhibition of signal transducer and activator of transcription 3 (STAT3) activation. Cucurbitacin I dose- and time-dependently inhibited the proliferation of all five OS cell lines. Following cucurbitacin I treatment, STAT3 was inactivated and analysis of Mcl-1, cleaved PARP and caspase-3 indicated apoptosis induction. Expression of cell cycle regulator proteins, such as phospho-cyclin D1, c-Myc and survivin, were suppressed. Finally, cucurbitacin I potently inhibited the tumor growth of human OS 143B cells in nude mice. Our in vitro and in vivo results suggest that STAT3 inhibition by cucurbitacin I will be an effective and new approach for the treatment of OS.

Transfection of nuclear factor-kappaB decoy oligodeoxynucleotide protects against ischemia/reperfusion injury in a rat epigastric flap model.

Nuclear factor‐kappaB (NF‐κB) is considered to play an important role in the response to ischemia/reperfusion (I/R) injury in flap surgery. To inhibit NF‐κB, synthetic double‐stranded oligodeoxynucleotide (ODN) was used as a decoy. The present study aimed to evaluate the suppressive effects of NF‐κB against I/R injury of experimental rat flap model.