Effect of aging on the tendon structure and tendon-associated gene expression in mouse foot flexor tendon.

To evaluate the biological changes in tendons during the aging process, the present study examined the effect of aging on the tendon structure, distribution of collagen types I and III, and expression of tendon-associated genes, using flexor tendons in a mouse model. Histological assessment of the tendon structure and distribution of collagen types I and III were performed, and the expression of tendon-associated genes was evaluated in flexor digitorium longus tendons of young (8 weeks) and aged (78 weeks) female C57BL/6 mice. The results indicated that the Soslowsky score, based on the analysis of cellularity, fibroblastic changes, and collagen fiber orientation and disruption, was significantly increased, or worsened, in the tendons of the aged group compared with those in the young group. Furthermore, in the aged group, the distribution of type I collagen was decreased and the distribution of type III collagen was relatively increased compared with the young group. Finally, the mRNA expression levels of collagen (type I and type III) and tenogenic markers (Mohawk homeobox, tenomodulin and scleraxis BHLH transcription factor) were significantly decreased in the aged group compared with the young group. The present observations demonstrated that the structure of the tendons, distribution of types I and III collagen and the expression of tendon-associated genes were modulated by aging in the flexor tendon, and that these changes may contribute to the degeneration of tendons in tendinopathy.

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

Grafting ß-tricalcium phosphate (TCP) is a well-established method for restoring bone defects; however, there is concern that the mechanical stability of the grafted ß-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 ß-TCP using a rabbit bone defect model. Bone defects (5×15 mm) were created in the distal femoral condyle of Japanese white rabbits, and ß-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 ß-TCP-grafted rabbits. Furthermore teriparatide increased the degradation of ß-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 ß-TCP. The results of the present study demonstrate that teriparatide is effective in maintaining the mechanical stability of grafted ß-TCP, possibly by promoting new bone formation.

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 ß-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.

Angular acceleration and angular jerk of elbow extension-flexion movement as parameters for discriminating a sequential transform of spontaneous movements in early infants.

Objective: We assessed the root mean square (RMS) of angular acceleration and the RMS of an angular jerk as expressions of the transformation of spontaneous movements in early infancy. Methods: During 36-56 weeks post-menstrual age (PMA), 15 premature infants (6 male, 9 female; 36 weeks PMA>) were measured every 4 weeks. A three-dimensional motion analyzer (Fastrak system; Polhemus Inc.) was used to measure spontaneous movements of the upper right limb in the supine infants. Upper limb position data were used to calculate the RMS of angular acceleration and the RMS of angular jerk at the elbow. The calculated data were classified into three terms: 36th and 40th week PMA (termⅠ), the 44th and 48th week PMA (termⅡ), and the 52nd and 56th week PMA (termⅢ). The typical value was the mean value for each term. Results: The RMS of angular acceleration in termⅡ was significantly less than that of termⅠ (p<0.05). The RMS values of angular jerk in termⅡ and termⅢ were significantly less than that of termⅠ (p<0.05). Conclusion: The RMS of angular acceleration and the RMS of the angular jerk are useful for expressing changes in the strength of spontaneous movements of elbow extension-flexion movements.

Effect of glucosamine on expression of type II collagen, matrix metalloproteinase and sirtuin genes in a human chondrocyte cell line.

Glucosamine (GlcN) has been widely used to treat osteoarthritis (OA) in humans. However, the effects of GlcN on genes related to cartilage metabolism are still unknown. In the present study, to elucidate the chondroprotective action of GlcN on OA, we examined the effects of GlcN (0.1-10 mM) on the expression of the sirtuin (SIRT) genes as well as type II collagen and matrix metalloproteinases (MMPs) using a human chondrocyte cell line SW 1353. SW 1353 cells were incubated in the absence or presence of GlcN. RT-PCR analyses revealed that GlcN markedly increased the mRNA expression of type II collagen (COL2A1). By contrast, the levels of MMP-1 and MMP-9 mRNA were only slightly increased by GlcN. Furthermore, western blot analyses revealed that GlcN significantly increased the protein level of COL2A1. Importantly, GlcN enhanced the mRNA expression and protein level of SIRT1, an upstream-regulating gene of COL2A1. Moreover, a SIRT1 inhibitor suppressed GlcN-induced COL2A1 gene expression. Together these observations suggest that GlcN enhances the mRNA expression and protein level of SIRT1 and its downstream gene COL2A1 in chondrocytes, thereby possibly exhibiting chondroprotective action on OA.

Evaluation of the effect of glucosamine administration on biomarkers of cartilage and bone metabolism in bicycle racers.

In the present study, the effect of glucosamine administration (1.5 or 3 g/day) on cartilage and bone metabolism was investigated in bicycle racers, using cartilage­ and bone­specific biomarkers, including C­terminal cross­linked telopeptides of type II collagen (CTX­II), C­terminal propeptides of type II procollagen (CPII), N­terminal telopeptides of bone­specific type I collagen (NTx) and bone alkaline phosphatase (BAP). The results indicate that CPII (a marker of type II collagen synthesis) was not substantially changed, however, CTX­II (a marker of type II degradation) was reduced by glucosamine administration, particularly at a dose of 3 g/day. Consistent with these observations, the ratio of CTX­II/CPII was reduced by glucosamine administration and the effect of glucosamine was dose­dependent. By contrast, the levels of NTx (a bone resorption marker) and BAP (a bone formation marker) were not altered by glucosamine administration. A previous study by this group reported that glucosamine exerts a chondroprotective action in soccer players by preventing type II collagen degradation but maintaining type II collagen synthesis. Together these observations indicate that glucosamine may exert a chondroprotective action by preventing type II collagen degradation in athletes of various sports, including soccer players and bicycle racers.

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 E(2) 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.

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.

Effect of glucosamine, a therapeutic agent for osteoarthritis, on osteoblastic cell differentiation.

Osteoarthritis (OA) is characterized by qualitative and quantitative changes in the architecture and composition of all the joint structures. Glucosamine (GlcN) has been used to treat OA in humans, because GlcN is present in the cartilage tissues as a component of glycosaminoglycans, and exhibits the symptom-modifying effect on OA by normalizing cartilage metabolism. On the other hand, the pathological change of subchondral bone is associated with the initiation and progression of cartilage damage in OA. However, the effect of GlcN on bone metabolism remains unsolved. In the present study, we determined the effect of GlcN on bone metabolism (osteoblastic cell differentiation) using mouse osteoblastic MC3T3-E1 cells by evaluating the expression of early (type I collagen and alkaline phosphatase), middle (osteopontin) and late (osteocalcin and mineralization) stage differentiation markers, and further compared its effects to those of N-acetyl-D-glucosamine (GlcNAc), a derivative of GlcN. The results indicated that the mineralization of mature osteoblasts was increased by treatment with GlcN and GlcNAc. Furthermore, reverse transcription-polymerase chain reaction (RT-PCR) analyses revealed that GlcN and GlcNAc substantially increased the expression of a middle stage marker and a late stage marker, although they did not essentially affect the expression of early stage markers. In addition, GlcN and GlcNAc suppressed the expression of receptor activator of NF-κB ligand (RANKL), a key factor involved in the osteoclastic cell differentiation and activation. Together these observations suggest that both GlcN and GlcNAc may have a potential not only to induce osteoblastic cell differentiation especially at middle-late stages, but also to suppress the osteoclastic cell differentiation, thereby possibly increasing bone matrix deposition and decreasing bone resorption, and eventually modulating bone metabolism in OA.

Effects of glucosamine derivatives and uronic acids on the production of glycosaminoglycans by human synovial cells and chondrocytes.

Glucosamine (GlcN) has been widely used to treat osteoarthritis (OA) in humans. However, its chondroprotective action on the joint is poorly understood. In this study, to elucidate the chondroprotective action of GlcN, we examined the effects of GlcN-derivatives (GlcN and N-acetyl-D-glucosamine) and uronic acids (D-glucuronic acid and D-galacturonic acid) (0.1-1 mM) on the production of glycosaminoglycans (GAG), such as hyaluronic acid (HA), keratan sulfate and sulfated GAG by human synovial cells and chondrocytes. The results indicate that among GlcN-derivatives and uronic acids, GlcN but not N-acetyl-D-glucosamine, D-glucuronic acid and D-galacturonic acid induce the production of HA by synovial cells and chondrocytes at >0.25 and >0.1 mM (p<0.05), respectively, and the production levels are much higher (>10-fold) in synovial cells compared to chondrocytes. In contrast, neither N-acetyl-D-glucosamine, D-glucuronic acid nor D-galacturonic acid affected the production of keratan sulfate and sulfated GAG by these cells. Moreover, the experiments with 3H-labeled GlcN indicated that GlcN can be incorporated and utilized for the production of GAG (including HA) by synovial cells and chondrocytes. In addition, GlcN (1 mM) up-regulates the expression of HA-synthesizing enzymes (hyaluronan synthases) in synovial cells and chondrocytes. Together these observations indicate that GlcN may exhibit chondroprotective action on joint diseases such as OA by modulating the expression of HA-synthesizing enzymes and inducing the production of HA (a major component of GAG contained in synovial fluid) especially by synovial cells.

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.

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/cm(2), 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.

A histone lysine methyltransferase activated by non-canonical Wnt signalling suppresses PPAR-gamma transactivation.

Histone modifications induced by activated signalling cascades are crucial to cell-lineage decisions. Osteoblast and adipocyte differentiation from common mesenchymal stem cells is under transcriptional control by numerous factors. Although PPAR-gamma (peroxisome proliferator activated receptor-gamma) has been established as a prime inducer of adipogenesis, cellular signalling factors that determine cell lineage in bone marrow remain generally unknown. Here, we show that the non-canonical Wnt pathway through CaMKII-TAK1-TAB2-NLK transcriptionally represses PPAR-gamma transactivation and induces Runx2 expression, promoting osteoblastogenesis in preference to adipogenesis in bone marrow mesenchymal progenitors. Wnt-5a activates NLK (Nemo-like kinase), which in turn phosphorylates a histone methyltransferase, SETDB1 (SET domain bifurcated 1), leading to the formation of a co-repressor complex that inactivates PPAR-gamma function through histone H3-K9 methylation. These findings suggest that the non-canonical Wnt signalling pathway suppresses PPAR-gamma function through chromatin inactivation triggered by recruitment of a repressing histone methyltransferase, thus leading to an osteoblastic cell lineage from mesenchymal stem cells.

Vitamin K induces osteoblast differentiation through pregnane X receptor-mediated transcriptional control of the Msx2 gene.

Vitamin K is a fat-soluble vitamin that serves as a coenzyme for vitamin K-dependent carboxylase. Besides its canonical action, vitamin K binds to the steroid and xenobiotic receptor (SXR)/pregnane X receptor (PXR) and modulates gene transcription. To determine if the osteoprotective action of vitamin K is the result of the PXR/SXR pathway, we screened by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis the PXR/SXR target genes in an osteoblastic cell line (MC3T3-E1) treated with a vitamin K2 (menaquinone 4 [MK4]). Osteoblastic differentiation of MC3T3-E1 cells was induced by MK4. Msx2, an osteoblastogenic transcription factor, was identified as an MK4-induced gene. Functional analysis of the Msx2 gene promoter mapped a vitamin K-responsive element (PXR-responsive element [PXRE]) that was directly bound by a PXR/retinoid X receptor alpha heterodimer. In a chromatin immunoprecipitation analysis, PXR was recruited together with a coactivator, p300, to the PXRE in the Msx2 promoter. MK4-bound PXR cooperated with estrogen-bound estrogen receptor alpha to control transcription at the Msx2 promoter. Knockdown of either PXR or Msx2 attenuated the effect of MK4 on osteoblastic differentiation. Thus, the present study suggests that Msx2 is a target gene for PXR activated by vitamin K and suggests that the osteoprotective action of MK4 in the human mediates, at least in part, a genomic pathway of vitamin K signaling.