Masanori Wako

TWEAK/Fn14 interaction regulates RANTES production, BMP-2-induced differentiation, and RANKL expression in mouse osteoblastic MC3T3-E1 cells

Tumour necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK), a member of the TNF family, is a multifunctional cytokine that regulates cell growth, migration, and survival principally through a TWEAK receptor, fibroblast growth factor-inducible 14 (Fn14). However, its physiological roles in bone are largely unknown. We herein report various effects of TWEAK on mouse osteoblastic MC3T3-E1 cells. MC3T3-E1 cells expressed Fn14 and produced RANTES (regulated upon activation, healthy T cell expressed and secreted) upon TWEAK stimulation through PI3K-Akt, but not nuclear factor-κB (NF-κB), pathway. In addition, TWEAK inhibited bone morphogenetic protein (BMP)-2-induced expression of osteoblast differentiation markers such as alkaline phosphatase through mitogen-activated protein kinase (MAPK) Erk pathway. Furthermore, TWEAK upregulated RANKL (receptor activation of NF-κB ligand) expression through MAPK Erk pathway in MC3T3-E1 cells. All these effects of TWEAK on MC3T3-E1 cells were abolished by mouse Fn14-Fc chimera. We also found significant TWEAK mRNA or protein expression in osteoblast – and osteoclast-lineage cell lines or the mouse bone tissue, respectively. Finally, we showed that human osteoblasts expressed Fn14 and induced RANTES and RANKL upon TWEAK stimulation. Collectively, TWEAK/Fn14 interaction regulates RANTES production, BMP-2-induced differentiation, and RANKL expression in MC3T3-E1 cells. TWEAK may thus be a novel cytokine that regulates several aspects of osteoblast function.

TNF-α–induced NF-κB signaling reverses age-related declines in VEGF induction and angiogenic activity in intervertebral disc tissues

We previously demonstrated that VEGF and its receptors were expressed in human herniated discs (HD). TNF‐α induced VEGF, resulting in neovascularization of disc tissues in a model of HD. The goal of the current research was to investigate the precise role of TNF‐α–induced VEGF and the mechanism of angiogenesis in disc tissues. We performed ELISAs, Western blots, and immunohistological examinations to assess the role of TNF‐α–induced VEGF using organ disc cultures with wild type, TNF receptor 1‐null (TNF‐RInull), or TNF receptor 2‐null (TNF‐RIInull) mice. VEGF induction was inhibited when we used TNF‐RInull‐derived disc tissues. NF‐κB pathway inhibitors also strongly suppressed VEGF induction. Thus, TNF‐α induced VEGF expression in disc cells primarily through the NF‐κB pathway. In addition, VEGF immunoreactivity was detected predominantly in annulus fibrosus cells and increased after TNF‐α stimulation. TNF‐α treatment also resulted in CD31 expression on endothelial cells and formation of an anastomosing network. In contrast, angiogenic activity was strongly inhibited in the presence of NF‐κB inhibitors or anti‐VEGF antibody. Our data show angiogenesis activity in disc tissues is regulated by VEGF and the NF‐κB pathway, both of which are induced by TNF‐α. The level of angiogenic activity in disc tissues was closely related to aging. Because neovascularization of HD is indispensable for HD resorption, the prognosis of HD and the rate of the resorption process in patients may vary as a function of the patients age.

High molecular weight hyaluronic acid increases the differentiation potential of the murine chondrocytic ATDC5 cell line.

Osteoarthritis (OA) is a group of common, chronic, and painful inflammatory joint diseases. One important finding in OA patients is a remarkable decrease in the molecular weight of hyaluronic acid (HA) in the synovial fluid of affected joints. Therapeutic HA is available to patients in most parts of the world as a viscosupplementation product for the treatment of OA. Previous clinical reports show that high molecular weight HA (HMWHA) more effectively relieves pain than low molecular weight HA (LMWHA). However, the mechanism behind this finding remains unclear. In this study, we investigated whether a LMWHA (Low‐0.9 MDa) and two types of HMWHA (High‐1.9 MDa and 6 MDa) differentially affected chondroregulatory action. We tested this using ATDC5 cell, a murine chondrocytic cell line widely used in culture systems to study chondrogenic differentiation. We found that HMWHA, especially hylan G‐F 20 (High‐6 MDa), significantly induced aggrecan and proteoglycan accumulation, nodule formation, and mRNA expression of chondrogenic differentiation markers in a time‐ and dose‐dependent manner. In addition, we showed that HMWHA prevented TNF‐α induced inhibition of chondrogenic differentiation, with no effect on cell proliferation or viability. These results reveal that HMWHA significantly promotes chondrogenic differentiation of ATDC5 cells in vitro, and suggest that HMWHA plays a significant chondroregulatory role in vivo.

Mechanism of signal transduction in tumor necrosis factor-like weak inducer of apoptosis-induced matrix degradation by MMP-3 upregulation in disc tissues.

Study Design. Molecular biologic and immuno-histologic analyses using in vitro murine intervertebral disc tissue culture. Objective. To investigate the role of tumor necrosis factor-like weak inducer of apoptosis (TWEAK) in matrix metalloproteinase 3 (MMP-3) pathway induction, and the effect of TWEAK to induce other cytokines or angiogenesis factors in disc tissues. Summary of Background Data. We previously demonstrated that TWEAK and its receptor Fn14 were expressed in murine disc tissues. TWEAK induced MMP-3 upregulation and aggrecan downregulation in disc tissues. Methods. Enzyme-Linked ImmunoSorbent Assay (ELISA), western blot, and immuno-histologic analyses were used to assess the role of TWEAK-induced MMP-3, using murine disc tissue culture. Results. TWEAK induced disc cells to generate MMP-3 as did TNF-&agr; and IL-1&bgr;. MMP-3 activity was detectable in murine disc cells. MMP-3 induction was markedly inhibited with a c-Jun N-terminal kinase (JNK) inhibitor. Phosphorylation of JNK was also confirmed. Introduction of TWEAK resulted in the degradation of disc matrix in organ disc culture, whereas proteoglycan degradation was markedly abrogated in the presence of an MMP-3 specific inhibitor or a JNK inhibitor. In addition, TWEAK also induced monocyte chemotactic protein (MCP)-1 via the NF-&kgr;B pathway, as phosphorylation of NF-&kgr;B was confirmed by western blotting. Conclusion. TWEAK plays an important role in MMP-3 induction in murine disc cells via JNK that results in degradation of disc matrix. TWEAK also induces MCP-1, which belongs to the chemokine family that recruits inflammatory cells via the NF-&kgr;B pathway.

Efficacy of early surgery and causes of surgical delay in patients with hip fracture.

BACKGROUND Whether early surgery for hip fractures is effective remains controversial. The current Japanese medical system poses some constraints on conducting early surgery. We examined the usefulness of early surgery and factors that delay surgery in patients with hip fractures treated at our hospital. METHODS Among 314 patients aged ≥60 years treated for hip fractures since January 2006, 270 patients (55 men, 215 women; mean age 84.1 years; femoral neck fracture in 111, trochanteric fracture in 159) who underwent surgery were studied. They were divided into an early surgery group (surgery up to 1 day after admission) and a delayed surgery group (later than 1 day). Clinical parameters analyzed included age, gender, pre-injury residence, pre-injury ambulatory ability, admission during public holiday, fracture site, fracture type, blood tests and urinalysis at admission, chest radiography, electrocardiography, number of systemic chronic diseases, dementia, surgical modality, blood transfusion, length of hospital stay, ambulatory ability at discharge, and hospital death. After performing univariate analysis between two groups, the parameters that were identified as significant were further tested by multivariate analysis. RESULTS Among 270 patients treated for hip fracture, 112 patients (41.5%) received early surgery. Multivariate analysis identified admission during public holiday, electrocardiographic abnormalities, femoral head replacement, and length of hospital stay as significant independent factors. CONCLUSION The causes of surgical delay were admission during public holiday, electrocardiographic abnormalities, and femoral head replacement. Although length of hospital stay was shorter in patients with early surgery, there was no difference in outcome.

Thrombin induced by the extrinsic pathway and PAR-1 regulated inflammation at the site of fracture repair

Thrombin (coagulation factor IIa) is a serine protease encoded by the F2 gene. Pro-thrombin (coagulation factor II) is cut to generate thrombin in the coagulation cascade that results in a reduction of blood loss. Procoagulant states that lead to activation of thrombin are common in bone fracture sites. However, its physiological roles and relationship with osteoblasts in bone fractures are largely unknown. We herein report various effects of thrombin on mouse osteoblastic MC3T3-E1 cells. MC3T3-E1 cells expressed proteinase-activated receptor 1 (PAR1), also known as the coagulation factor II receptor. They also produced monocyte chemoattractant protein (MCP-1), tissue factor (TF), MCSF and IL-6 upon thrombin stimulation through the PI3K-Akt and MEK-Erk1/2 pathways. Furthermore, MCP-1 obtained from thrombin-stimulated MC3T3-E1 cells induced migration by macrophage RAW264 cells. All these effects of thrombin on MC3T3-E1 cells were abolished by the selective non-peptide thrombin receptor inhibitor SCH79797. We also found that thrombin, PAR-1, MCP-1, TF as well as phosphorylated AKT and p42/44 were significantly expressed at the fracture site of mouse femoral bone. Collectively, thrombin/PAR-1 interaction regulated MCP-1, TF, MCSF and IL-6 production by MC3T3-E1 cells. Furthermore, MCP-1 induced RAW264 cell migration. Thrombin may thus be a novel cytokine that regulates several aspects of osteoblast function and fracture healing.

Comparison of the prognosis among different age groups in elderly patients with hip fracture.

Background: The outcome of treatment of hip fractures in different age groups in the elderly population is largely unknown. Hence, we stratified elderly patients with hip fracture into age groups and compared the prognosis in various age groups. Materials and Methods: Among 459 patients with hip fracture treated at our hospital from 1997, 430 patients aged 65 years or above at the time of injury were studied. The patients comprised 98 males and 332 females and the ages at injury ranged from 65 to 103 years (mean 83.4 years). There were 167 cases of femoral neck fracture and 263 cases of trochanteric fractures. Surgery was performed in 383 cases, while 47 cases were treated conservatively. The subjects were classified by age into young-old for those aged 65-74 years (group A, n = 55), middle-old for those aged 75-84 years (group B, n = 172), old-old for those aged 85-94 (group C, n = 180), and oldest-old for those aged 95 years or above (group D, n = 23). The functional and survival prognosis at discharge in each group was investigated. Results: Numbers of patients who were ambulatory at discharge among those ambulatory before injury were 43 of 49 (87.8%) in group A, 113 of 152 (74.3%) in group B, 86 of 138 (62.3%) in group C, and 5 of 14 (35.7%) in group D, showing worse recovery of walking ability as age advanced. Among those ambulatory before injury, 42 patients in group A, 139 patients in group B, 130 patients in group C, and 12 patients in group D underwent surgery and of these patients, 38 patients (90.5%) in group A, 109 patients (78.4%) in group B, 83 patients (63.8%) in group C, and 5 patients (41.7%) in group D were ambulatory at discharge. On the other hand, the numbers of patients who were ambulatory at discharge among those receiving conservative treatment were 5 of 7 (71.4%) in group A, 4 of 13 (30.8%) in group B, 3 of 8 (37.5%) in group C, and 0 of 2 (0%) in group D, showing better walking ability in surgical patients than in conservatively treated patients even in the elderly. There were two in-hospital deaths in group B, 11 in group C, and two in group D. Five of the 15 deaths were inoperable cases due to poor performance status at admission. Conclusion: Walking ability at discharge and survival prognosis worsened as age advanced. On the other hand, since surgical cases achieved better walking ability than conservatively treated cases, efforts should be made to achieve better functional prognosis even in the old-olds, including surgery together with early ambulation and rehabilitation.

Twin hook fixation for proximal femoral fractures

Purpose. To report results of twin hook fixation for proximal femoral fractures in comparison to those fixed with the conventional lag screw. Methods. Between August 2005 and July 2006, 2 men and 15 women aged 74 to 94 (mean, 85) years with proximal femoral fractures underwent open reduction and internal fixation using the twin hook system. The tip-apex distance was compared with that in 20 patients treated with the sliding hip screw between August 2004 and July 2005. Results. In the 17 patients, the hook was inserted into the centre of the femoral head. Bone union was achieved and no intra- or post-operative cut-out or device failure was encountered. In patients using the twin hook and sliding hip screw respectively, the mean tip-apex distance was 22.3 mm and 14.6 mm (p<0.001). Conclusion. Using the twin hook system requires more surgical skill than using the sliding hip screw, because failure to insert the pin into the centre of the femoral head risks intra-articular perforation by the hooks.

Arthroscopic washout of the ankle for septic arthritis in a three-month-old boy

There is no report of athroscopic treatment for septic arthritis of the ankle in infants. We report a case of successful management of septic arthritis of the ankle in a three-month-old boy by arthroscopic washout. Arthroscopic washout may be a useful treatment for septic arthritis in young infants when performed early after onset.