Soichiro Yamabe

Endoplasmic reticulum stress-induced apoptosis contributes to articular cartilage degeneration via C/EBP homologous protein

OBJECTIVE When endoplasmic reticulum (ER) stress, i.e., the excessive accumulation of unfolded proteins in ER, endangers homeostasis, apoptosis is induced by C/EBP homologous protein (Chop). In osteoarthritis (OA) cartilage, Chop expression and apoptosis increase as degeneration progresses. We investigated the role of Chop in murine chondrocyte apoptosis and in the progression of cartilage degeneration. METHOD We induced experimental OA in Chop-knockout (Chop(-/-)) mice by medial collateral ligament transection and meniscectomy and compared cartilage degeneration, apoptosis, and ER stress in Chop(-/-)- and wild-type (Chop(+/+)) mice. In our in vitro experiments we treated murine Chop(-/-) chondrocytes with the ER stress inducer tunicamycin (TM) and evaluated apoptosis, ER stress, and chondrocyte function. RESULTS In vivo, the degree of ER stress was similar in Chop(-/-)- and Chop(+/+) mice. However, in Chop(-/-) mice apoptosis and cartilage degeneration were lower by 26.4% and 42.4% at 4 weeks, by 26.8% and 44.9% at 8 weeks, and by 26.9% and 32.3% at 12 weeks after surgery than Chop(+/+) mice, respectively. In vitro, the degree of ER stress induction by TM was similar in Chop(-/-)- and Chop(+/+) chondrocytes. On the other hand, apoptosis was 55.3% lower and the suppression of collagen type II and aggrecan mRNA was 21.0% and 23.3% less, and the increase of matrix metalloproteinase-13 mRNA was 20.0% less in Chop(-/-)- than Chop(+/+) chondrocytes. CONCLUSION Our results indicate that Chop plays a direct role in chondrocyte apoptosis and that Chop-mediated apoptosis contributes to the progression of cartilage degeneration in mice.

Enhanced apoptotic and reduced protective response in chondrocytes following endoplasmic reticulum stress in osteoarthritic cartilage

Endoplasmic reticulum (ER) stress has been shown to participate in many disease pathologies. Although recent reports have demonstrated that ER stress in chondrocytes is present in human osteoarthritis (OA), its role in the pathology of cartilage degeneration, such as chondrocyte apoptosis, remains unclear. In the present study, we investigated the expression of phosphorylated PERK (pPERK), ubiquitin (Ub), GRP78, CHOP, phosphorylated JNK (pJNK) and cleaved caspase‐3 (C‐CASP3) and the mRNA splicing of XBP1 (XBP1 splicing) in human OA cartilage by immunohistochemistry and RT‐PCR. Additionally, human chondrocytes were treated with several concentrations of tunicamycin, an ER stress inducer, to assess the impact of ER stress on the mRNA expression of CHOP, XBP1 splicing and apoptosis, as determined by real‐time PCR, RT‐PCR and ELISA analyses respectively. In human OA cartilage, the number of chondrocytes expressing pPERK, Ub, CHOP and pJNK positively correlated with cartilage degeneration and the number of C‐CASP3‐positive chondrocytes. XBP1 splicing and GRP78 expression in severe OA containing the greatest number of C‐CASP3‐positive chondrocytes were similar to the levels in mild OA, however, XBP1 splicing was higher in moderate OA than in mild and severe OA. Tunicamycin dose dependently increased CHOP expression and apoptosis of cultured chondrocytes. Although tunicamycin upregulated XBP1 splicing in cultured chondrocytes, its impact on XBP1 splicing was weakened at higher concentrations. In conclusion, the present results indicate that ER stress may contribute to chondrocyte apoptosis along with OA progression, which was closely associated with an enhanced apoptotic response and a reduced protective response by the cells.

Intracellular accumulation of advanced glycation end products induces apoptosis via endoplasmic reticulum stress in chondrocytes

Mammalian cells attempt to maintain their homeostasis under endoplasmic reticulum (ER) stress. If the stress cannot be alleviated, cells are led to apoptosis through induction of C/EBP homologous protein (CHOP). ER stress is provoked in osteoarthritis chondrocytes, and intracellular accumulation of advanced glycation end products (AGEs) in chondrocytes is a possible cause. To clarify the role of intracellular AGE accumulation in chondrocytes, the present study investigated the effect of intracellular AGE accumulation on ER stress and apoptosis by in vitro and in vivo analysis. Intracellular AGE accumulation induced by AGE precursors caused apoptosis, induced expression of ER stress markers, and led to co‐localization of AGEs with glucose‐regulated protein 78, leading to formation of high‐molecular‐weight complexes in cultured chondrocytes. These reactions were inhibited by an AGE formation inhibitor. CHOP deletion inhibited apoptosis induced by intracellular AGE accumulation. In vivo intracellular AGE accumulation induced by intra‐articular injection of AGE precursors caused ER stress and apoptosis in chondrocytes and led to degradation of articular cartilage. Additionally, intracellular AGE accumulation increased the degree of cartilage degradation in an osteoarthritis model. These data indicate that intracellular accumulation of AGEs induces modification of unfolded protein response‐related protein by AGEs and apoptosis via ER stress in chondrocytes. Moreover, the in vivo study showed that intracellular AGE accumulation in chondrocytes is involved in the occurrence and progression of osteoarthritis through ER stress. Thus, research on mechanisms of apoptosis via ER stress induced by intracellular AGE accumulation in chondrocytes will lead to a new understanding of osteoarthritis pathology.

Immunohistochemical distribution of advanced glycation end products (AGEs) in human osteoarthritic cartilage

Advanced glycation end products (AGEs) may be associated with osteoarthritis (OA), because the accumulation of AGEs in articular cartilage are among the most striking age-related changes. AGEs modify the tissue protein structure and function and stimulate the cellular responses mediated by a specific receptor for AGEs (RAGE). This study investigated the localization of AGEs in degenerated cartilage using newly identified epitope-specific antibodies to determine the linkage between the distribution of AGEs and the development and progression of OA. Osteochondral specimens of the tibial plateau from OA patients were immunostained by specific antibodies against N(ε)-(carboxymethyl)lysine (CML), N(ε)-(carboxyethyl)lysine (CEL), pentosidine, GA-pyridine, and RAGE. The immunohistochemical distribution of these epitopes was evaluated during cartilage degeneration. The immunoreactivity (IR) of AGEs and RAGE was stronger in cells rather than in the extracellular matrix. Higher IR of cellular CML and CEL was observed in both mild and severe OA cartilage in comparison to macroscopically intact cartilage. There was a strong association between GA-pyridine and RAGE in the pattern of increasing IR with the OA grade. These IR patterns of AGEs varying with cartilage degeneration indicate that AGE modified proteins are associated with cartilage degeneration. The coincidental up-regulation of GA-pyridine and RAGE suggests that GA-pyridine is the most significant AGE for cartilage degeneration via the RAGE pathway.

MRI T1ρ and T2 mapping for the assessment of articular cartilage changes in patients with medial knee osteoarthritis after hemicallotasis osteotomy

Objectives The purpose of this study was to clarify the appearance of the reparative tissue on the articular surface and to analyse the properties of the reparative tissue after hemicallotasis osteotomy (HCO) using MRI T1ρ and T2 mapping. Methods Coronal T1ρ and T2 mapping and three-dimensional gradient-echo images were obtained from 20 subjects with medial knee osteoarthritis. We set the regions of interest (ROIs) on the full-thickness cartilage of the medial femoral condyle (MFC) and medial tibial plateau (MTP) of the knee and measured the cartilage thickness (mm) and T1ρ and T2 relaxation times (ms). Statistical analysis of time-dependent changes in the cartilage thickness and the T1ρ and T2 relaxation times was performed using one-way analysis of variance, and Scheffe’s test was employed for post hoc multiple comparison. Results The cartilage-like repair tissue appeared on the cartilage surface of the medial compartment post-operatively, and the cartilage thickness showed a significant increase between the pre-operative and one-year post-operative time points (MFC; p = 0.003, MTP; p < 0.001). The T1ρ values of the cartilage-like repair tissue showed no difference over time, however, the T2 values showed a significant decrease between the pre-operative and one-year post-operative time points (MFC; p = 0.004, MTP; p = 0.040). Conclusion This study clarified that the fibrocartilage-like repair tissue appeared on the articular surface of the medial compartment after HCO as evidenced by MRI T1ρ and T2 mapping. Cite this article: H. Nishioka, E. Nakamura, J. Hirose, N. Okamoto, S. Yamabe, H. Mizuta. MRI T1ρ and T2 mapping for the assessment of articular cartilage changes in patients with medial knee osteoarthritis after hemicallotasis osteotomy. Bone Joint Res 2016;5:294–300. DOI: 10.1302/2046-3758.57.BJR-2016-0057.R1.

Edoxaban Enhances Thromboprophylaxis by Physiotherapy After Total Knee Arthroplasty ― The Randomized Controlled ESCORT-TKA Trial ―

BACKGROUND The pharmacological advantage of combining physiotherapy with anticoagulants for the prevention of venous thromboembolism (VTE) after total knee arthroplasty (TKA) is not fully known. Herein we investigated the potential benefit of this combination therapy in patients undergoing TKA.Methods and Results:The 38 patients were randomly assigned to a physiotherapy group (n=19) or a physiotherapy plus 30 mg/day edoxaban group (n=19). The occurrence of VTE was evaluated, as were serial changes in parameters measured by the Total Thrombus-formation Analysis System, a novel system for quantitatively analyzing thrombus formation using microchips with thrombogenic surfaces (collagen plus tissue factor, atheroma [AR]-chip). Combination therapy significantly reduced the incidence of VTE after TKA compared with monotherapy (P=0.038). The area under the curve (AUC) of thrombus formation for the AR-chip (AR10-AUC30) was significantly lower in the combination group (P=0.001) on Day 7 after TKA than before TKA, but no significant change was observed with monotherapy (P=0.809). In 13 VTE-positive patients, AR10-AUC30was significantly lower in the combination group (n=3) than in the monotherapy group (n=10) on Day 7 (P=0.045). CONCLUSIONS The combination of physiotherapy and edoxaban significantly reduced the incidence of VTE after TKA compared with physiotherapy alone. However, it is possible that VTE occurrence after TKA is not only associated with thrombogenicity, but also rheological factors.