Mamoru Igarashi

Low-intensity pulsed ultrasound (LIPUS) increases the articular cartilage type II collagen in a rat osteoarthritis model

In this study, the effect of low‐intensity pulsed ultrasound (LIPUS) on cartilage was evaluated in a rat osteoarthritis (OA) model using serum biomarkers such as CTX‐II (type II collagen degradation) and CPII (type II collagen synthesis) as well as histological criteria (Mankin score and immunohistochemical type II collagen staining). OA was surgically induced in the knee joint of rats by anterior cruciate/medial collateral ligament transection and medial meniscus resection (ACLT + MMx). Animals were divided into three groups: sham‐operated group (Sham), ACLT + MMx group without LIPUS (−LIPUS), and ACLT + MMx group with LIPUS (+LIPUS; 30 mW/cm2, 20 min/day for 28 days). CTX‐II levels were elevated in both −LIPUS and +LIPUS groups compared to that in the Sham group after the operation, but there was no significant difference between +LIPUS and −LIPUS groups, suggesting that LIPUS does not affect the degradation of type II collagen in this model. In contrast, CPII was significantly increased in +LIPUS group compared to −LIPUS and Sham. Moreover, histological damage on the cartilage (Mankin score) was ameliorated by LIPUS, and type II collagen was immunohistochemically increased by LIPUS in the cartilage of an OA model. Of interest, mRNA expression of type II collagen was enhanced by LIPUS in chondrocytes. Together these observations suggest that LIPUS is likely to increase the type II collagen synthesis in articular cartilage, possibly via the activation of chondrocytes and induction of type II collagen mRNA expression, thereby exhibiting chondroprotective action in a rat OA model.

Bone Marrow Adipocytes Facilitate Fatty Acid Oxidation Activating AMPK and a Transcriptional Network Supporting Survival of Acute Monocytic Leukemia Cells

Leukemia cells in the bone marrow must meet the biochemical demands of increased cell proliferation and also survive by continually adapting to fluctuations in nutrient and oxygen availability. Thus, targeting metabolic abnormalities in leukemia cells located in the bone marrow is a novel therapeutic approach. In this study, we investigated the metabolic role of bone marrow adipocytes in supporting the growth of leukemic blasts. Prevention of nutrient starvation-induced apoptosis of leukemic cells by bone marrow adipocytes, as well as the metabolic and molecular mechanisms involved in this process, was investigated using various analytic techniques. In acute monocytic leukemia (AMoL) cells, the prevention of spontaneous apoptosis by bone marrow adipocytes was associated with an increase in fatty acid β-oxidation (FAO) along with the upregulation of PPARγ, FABP4, CD36, and BCL2 genes. In AMoL cells, bone marrow adipocyte coculture increased adiponectin receptor gene expression and its downstream target stress response kinase AMPK, p38 MAPK with autophagy activation, and upregulated antiapoptotic chaperone HSPs. Inhibition of FAO disrupted metabolic homeostasis, increased reactive oxygen species production, and induced the integrated stress response mediator ATF4 and apoptosis in AMoL cells cocultured with bone marrow adipocytes. Our results suggest that bone marrow adipocytes support AMoL cell survival by regulating their metabolic energy balance and that the disruption of FAO in bone marrow adipocytes may be an alternative, novel therapeutic strategy for AMoL therapy. Cancer Res; 77(6); 1453-64. ©2017 AACR.

Biological Activities of Glucosamine and Its Related Substances

Glucosamine (GlcN) has been widely used to treat osteoarthritis (OA) in humans. We revealed that among GlcN-derivatives (GlcN and N-acetyl-d-glucosamine) and uronic acids (d-glucuronic acid and d-galacturonic acid), only GlcN induces the production of hyaluronic acid (HA) by synovial cells and chondrocytes, and the production level is much higher (>10-fold) in synovial cells compared with chondrocytes. Moreover, GlcN increases the expression of HA-synthesizing enzymes (HAS) in synovial cells and chondrocytes. These observations indicate that GlcN likely exhibits the chondroprotective action on OA by modulating the expression of HAS and inducing the production of HA (a major component of glycosaminoglycans contained in the synovial fluid) especially by synovial cells. The pathological change of subchondral bone is implicated in the initiation and progression of cartilage damage in OA. Thus, we further determined the effect of GlcN on the bone metabolism (osteoblastic cell differentiation). The results indicated that GlcN increases the mineralization of mature osteoblasts and the expression of middle and late stage markers (osteopontin and osteocalcin, respectively) during osteoblastic differentiation, and reduces the expression of receptor activator of NF-κB ligand (RANKL), a differentiation and activation factor for osteoclasts. These observations likely suggest that GlcN has a potential to induce the osteoblastic cell differentiation and suppress the osteoclastic cell differentiation, thereby increasing bone matrix deposition and decreasing bone resorption to modulate bone metabolism in OA.

Evaluation of the effect of methionine and glucosamine on adjuvant arthritis in rats

In the present study, we evaluated the effects of individual administration of methionine or glucosamine (GlcN) and compared with the combined administration of methionine and GlcN on the adjuvant arthritis model of rheumatoid arthritis in rats. Adjuvant arthritis was induced in female Lewis rats by injecting Freund’s complete adjuvant (FCA) into the right hind paws, and methionine (200 mg/kg body weight/day) and/or GlcN (400 mg/kg/day) were orally administered for 21 days. The progression of the adjuvant arthritis was clinically evaluated for characteristic signs and symptoms by employing an arthritis score. The administration of methionine combined with GlcN suppressed the swelling of FCA-uninjected left hind paws and the arthritis score. Additionally, histopathological examination revealed that the combined administration of methionine and GlcN markedly suppressed synovial hyperplasia and the destruction of the cartilage surface and articular meniscus of the knee joints of FCA-injected right hind paws. Furthermore, combined methionine and GlcN administration suppressed the increase in the levels of nitric oxide, prostaglandin E2 and hyaluronic acid in the plasma of rats with adjuvant arthritis. By contrast, individual administration of methionine or GlcN suppressed arthritis only slightly. These observations suggest that the combined administration of methionine and GlcN is more effective compared with individual administrations of methionine or GlcN in suppressing the progression of adjuvant arthritis (identified as swelling of joints and arthritis score), possibly by synergistically inhibiting synovial inflammation (identified as synovial hyperplasia and the destruction of the cartilage surface and articular meniscus) and the production of inflammatory mediators.

Evaluation of the effect of flavangenol on serum lipid peroxide levels and development of atherosclerosis in spontaneously hyperlipidemic B6.KOR-Apoeshl mice.

Antioxidative flavonoids are used to reduce the risk of cardiovascular diseases in humans. However, the precise mechanism for the anti-atherosclerotic actions of flavonoids remains to be elucidated. In the present study, to assess the mechanism for the action of antioxidative flavonoids on atherosclerosis, we investigated the effect of flavangenol, one of the most potent antioxidants currently known, on spontaneously hyperlipidemic B6.KOR-Apoeshl mice. Flavangenol was orally administered to B6.KOR-Apoeshl mice ad libitum (6 mg flavangenol/mouse/day). After 6 months, serum levels of lipids (total cholesterol, triglyceride, HDL-cholesterol and LDL-cholesterol) and lipid peroxide were measured, and histopathological changes (lipid accumulation and inflammatory cell infiltration) in the aortic root were evaluated. Serum levels of total cholesterol and LDL-cholesterol were markedly increased, and HDL-cholesterol levels were decreased in B6.KOR-Apoeshl mice compared to C57BL/6 mice used as a control (p<0.001). Among these serum lipids, only HDL-cholesterol levels were significantly increased by flavangenol administration (p<0.05). Moreover, Oil Red O staining (lipid accumulation) was significantly increased in B6.KOR-Apoeshl mice compared to C57BL/6 mice (p<0.001). Notably, flavangenol administration significantly suppressed the increase in Oil Red O staining (p<0.01). Similarly, inflammatory cell infiltration into the intima was significantly increased in B6.KOR-Apoeshl mice compared to C57BL/6 mice (p<0.01), and flavangenol administration significantly suppressed the inflammatory cell infiltration (p<0.01). Importantly, flavangenol administration significantly reduced the increase of serum lipid peroxide levels in B6.KOR-Apoeshl mice (p<0.05). Together, these observations indicate that flavangenol, one of the most potent antioxidants, exerts its anti-atherosclerotic action on spontaneously hyperlipidemic and atherosclerotic B6.KOR-Apoeshl mice, possibly by increasing HDL-cholesterol levels and reducing lipid peroxide levels, thereby suppressing the lipid accumulation (formation of atherosclerotic lesions) and inflammatory cell infiltration (chronic inflammation) in the intima of the aortic root.

Effect of intermittent administration of teriparatide on the mechanical and histological changes in bone grafted with β‑tricalcium phosphate using a rabbit bone defect model

Grafting β-tricalcium phosphate (TCP) is a well-established method for restoring bone defects; however, there is concern that the mechanical stability of the grafted β-TCP is not maintained during bone translation. Teriparatide has an anabolic effect, stimulating bone formation and increasing bone mineral density for the treatment of osteoporosis. The aim of the present study was to evaluate the effect of intermittent teriparatide treatment on changes in bone grafted with β-TCP using a rabbit bone defect model. Bone defects (5×15 mm) were created in the distal femoral condyle of Japanese white rabbits, and β-TCP granules of two different total porosities were manually grafted. Teriparatide (40 µg/kg) or 0.2% rabbit serum albumin solution as a vehicle control was subcutaneously injected three times per week following the surgery. At 4 or 8 weeks post-surgery, serum samples were obtained and the levels of γ-carboxylated osteocalcin (Gla-OC) were quantified using ELISA. Histomorphometry was also performed using sections of graft sites following staining for tartrate resistant acid phosphatase. Activity and mechanical strength (maximum shear strength, maximum shear stiffness and total energy absorption) were evaluated using an axial push-out load to failure test. Teriparatide treatment significantly increased (P<0.05) the serum levels of Gla-OC, a specific marker for bone formation, suggesting that teriparatide enhances bone formation in β-TCP-grafted rabbits. Furthermore teriparatide increased the degradation of β-TCP by bone remodeling (P<0.05) and promoted the formation of new bone following application of the graft compared with the control group (P<0.01). Furthermore, teriparatide suppressed the reduction in mechanical strength (P<0.05) during bone translation in bone defects grafted with β-TCP. The results of the present study demonstrate that teriparatide is effective in maintaining the mechanical stability of grafted β-TCP, possibly by promoting new bone formation.