 | 水上 優哉 （ミズカミ ユウヤ）
| ||
Last Updated :2024/05/15
コミュニケーション情報 byコメンテータガイド
コメント
骨代謝性疾患および骨修復・再生について。特に細胞外小胞との関連について。
研究活動情報
論文
- Kiyotaka Okada; Yuto Niwa; Kazusa Fukuhara; Takashi Ohira; Yuya Mizukami; Naoyuki Kawao; Osamu Matsuo; Hiroshi KajiJournal of Bone and Mineral Metabolism 2024年05月
- Yuya Mizukami; Naoyuki Kawao; Takashi Ohira; Daiki Hashimoto; Kiyotaka Okada; Osamu Matsuo; Hiroshi KajiCalcified tissue international 2024年03月 [査読有り]
Heterotopic ossification (HO) is the process by which ectopic bone forms at an extraskeletal site. Inflammatory conditions induce plasminogen activator inhibitor 1 (PAI-1), an inhibitor of fibrinolysis, which regulates osteogenesis. In the present study, we investigated the roles of PAI-1 in the pathophysiology of HO induced by trauma/burn treatment using PAI-1-deficient mice. PAI-1 deficiency significantly promoted HO and increased the number of alkaline phosphatase (ALP)-positive cells in Achilles tendons after trauma/burn treatment. The mRNA levels of inflammation markers were elevated in Achilles tendons of both wild-type and PAI-1-deficient mice after trauma/burn treatment and PAI-1 mRNA levels were elevated in Achilles tendons of wild-type mice. PAI-1 deficiency significantly up-regulated the expression of Runx2, Osterix, and type 1 collagen in Achilles tendons 9 weeks after trauma/burn treatment in mice. In in vitro experiments, PAI-1 deficiency significantly increased ALP activity and mineralization in mouse osteoblasts. Moreover, PAI-1 deficiency significantly increased ALP activity and up-regulated osteocalcin expression during osteoblastic differentiation from mouse adipose-tissue-derived stem cells, but suppressed the chondrogenic differentiation of these cells. In conclusion, the present study showed that PAI-1 deficiency promoted HO in Achilles tendons after trauma/burn treatment partly by enhancing osteoblast differentiation and ALP activity in mice. Endogenous PAI-1 may play protective roles against HO after injury and inflammation. - Takashi Ohira; Yoko Ino; Naoyuki Kawao; Yuya Mizukami; Kiyotaka Okada; Osamu Matsuo; Hisashi Hirano; Yayoi Kimura; Hiroshi KajiJournal of applied physiology (Bethesda, Md. : 1985) 2024年02月 [査読有り]
Mechanical-unloading-induced skeletal muscle atrophy results in physical frailty and disability. Elucidating its mechanism is required to establish effective countermeasures for this muscle adaptation. First, we analyzed the proteome profile in the gastrocnemius (Gast) and soleus muscles of space-flown mice raised under microgravity or artificial 1-g for 30 days, and found that the expression levels of fibrinolysis-related proteins were significantly elevated in the mechanical-unloaded muscles. Next, we investigated the roles of the fibrinolytic system in skeletal muscle atrophy induced by mechanical unloading on the ground. Eight-week-old male mice with plasminogen gene deficiency (Plg-/-) and their wild-type littermates were divided into control and hindlimb-suspended groups, and were raised for 21 days. Plasminogen deficiency significantly enhanced the decrease in muscle mass at the lower limbs of mice following hindlimb unloading, and the Gast muscle atrophy was more prominent in Plg-/- mice. Additionally, plasminogen deficiency significantly increased the expression of autophagy-related markers, beclin1 mRNA and LC3B protein, in the mechanical-unloaded Gast muscles, but did not affect the increase in the gene expression of ubiquitin ligases, atrogin-1 and MuRF1. Neither plasminogen deficiency nor hindlimb unloading affected the Akt/mechanistic target of rapamycin pathway in the Gast muscles. These results suggested that plasminogen deficiency might accelerate protein breakdown via the autophagy-lysosome, but not the ubiquitin-proteasome, system in the mechanical-unloaded Gast muscles. In conclusion, we first showed that plasminogen deficiency exacerbated the Gast muscle atrophy in hindlimb-unloaded mice. Plasminogen and the fibrinolysis system might play some protective roles against muscle atrophy induced by mechanical unloading in developing mice. - Naoyuki Kawao; Daichi Matsumura; Ayaka Yamada; Katsumi Okumoto; Takashi Ohira; Yuya Mizukami; Daiki Hashimoto; Hiroshi KajiBone 181 117040 - 117040 2024年02月 [査読有り]
The intermittent administration of parathyroid hormone (PTH) exerts potent bone anabolic effects, which increase bone mineral density (BMD) and reduce fracture risk in osteoporotic patients. However, the underlying mechanisms remain unclear. Tmem119 has been proposed as a factor that is closely linked to the osteoblast phenotype, and we previously reported that PTH enhanced the expression of Tmem119 in mouse osteoblastic cells. However, roles of Tmem119 in the bone anabolic effects of PTH in vivo remain unknown. We herein investigated the roles of Tmem119 in bone anabolic effects of PTH using Tmem119-deficient mice. Tmem119 deficiency significantly reduced PTH-induced increases in trabecular bone volume and cortical BMD of femurs. Effects of Tmem119 deficiency on bone mass seemed predominant in female mice. Histomorphometric analyses with calcein labeling showed that Tmem119 deficiency significantly attenuated PTH-induced increases in the rates of bone formation and mineralization as well as numbers of osteoblasts. Moreover, Tmem119 deficiency significantly blunted PTH-induced decreases in phosphorylation of β-catenin and increases in alkaline phosphatase activity in osteoblasts. In conclusion, the present results indicate that Tmem119 is involved in bone anabolic effects of PTH through osteoblastic bone formation partly related to canonical Wnt-β-catenin signaling in mice. - Matsumura D; Kawao N; Okumoto K; Ohira T; Mizukami Y; Akagi M; Kaji HPLoS One 18 6 e0287541 2023年06月 [査読有り]
- Yuya Mizukami; Naoyuki Kawao; Yoshimasa Takafuji; Takashi Ohira; Kiyotaka Okada; Jun-Ichiro Jo; Yasuhiko Tabata; Hiroshi KajiPLOS ONE 18 4 e0284258 - e0284258 2023年04月 [査読有り]
Matrix vesicles (MtVs) are one of the extracellular vesicles (EVs) secreted by osteoblasts. Although MtVs have a classically-defined function as an initiator of ossification and recent findings suggest a role for MtVs in the regulation of bone cell biology, the effects of MtVs on bone repair remain unclear. In the present study, we employed collagenase-released EVs (CREVs) containing abundant MtVs from mouse osteoblasts. CREVs were administered locally in gelatin hydrogels to damaged sites after a femoral bone defect in mice. CREVs exhibited the characteristics of MtVs with a diameter <200 nm. The local administration of CREVs significantly promoted the formation of new bone with increases in the number of alkaline phosphatase (ALP)-positive cells and cartilage formation at the damaged site after the femoral bone defect. However, the addition of CREVs to the medium did not promote the osteogenic differentiation of ST2 cells or the ALP activity or mineralization of mouse osteoblasts in vitro. In conclusion, we herein showed for the first time that MtVs enhanced bone repair after a femoral bone defect partly through osteogenesis and chondrogenesis in mice. Therefore, MtVs have potential as a tool for bone regeneration. - Yuto Takada; Yoshimasa Takafuji; Yuya Mizukami; Takashi Ohira; Naoyuki Kawao; Kiyotaka Okada; Hiroshi KajiCalcified tissue international 1 - 12 2022年12月 [査読有り]
Extracellular vesicles (EVs) play crucial roles in physiological and pathophysiological processes. Although studies have described muscle-bone interactions via humoral factors, we reported that EVs from C2C12 muscle cells (Myo-EVs) suppress osteoclast formation. Current clinical evidence suggests that inflammation induces both sarcopenia and osteoporosis. Although tumor necrosis factor-α (TNF-α) is a critical proinflammatory factor, the influences of TNF-α on muscle-bone interactions and Myo-EVs are still unclear. In the present study, we investigated the effects of TNF-α stimulation of C2C12 cells on osteoclast formation and osteoblastic differentiation modulated by Myo-EVs in mouse cells. TNF-α significantly decreased the protein amount in Myo-EVs, but did not affect the Myo-EV size distribution. TNF-α treatment of C2C12 myoblasts significantly decreased the suppression of osteoclast formation induced by Myo-EVs from C2C12 myoblasts in mouse bone marrow cells. Moreover, TNF-α treatment of C2C12 myoblasts in mouse preosteoclastic Raw 264.7 cells significantly limited the Myo-EV-induced suppression of osteoclast formation and decreased the Myo-EV-induced increase in mRNA levels of osteoclast formation-related genes. On the other hand, TNF-α treatment of C2C12 muscle cells significantly decreased the degree of Myo-EV-promoted mRNA levels of Osterix and osteocalcin, as well as ALP activity in mouse mesenchymal ST-2 cells. TNF-α also significantly decreased miR196-5p level in Myo-EVs from C2C12 myoblasts in quantitative real-time PCR. In conclusion, TNF-α stimulation of C2C12 muscle cells blunts both the osteoclast formation suppression and the osteoblastic differentiation promotion that occurs due to Myo-EVs in mouse cells. Thus, TNF-α may disrupt the muscle-bone interactions by direct Myo-EV modulation. - Takashi Ohira; Naoyuki Kawao; Yoshimasa Takafuji; Yuya Mizukami; Hiroshi KajiExperimental and clinical endocrinology & diabetes : official journal, German Society of Endocrinology [and] German Diabetes Association 2022年12月 [査読有り]
The interactions between muscle and bone are noted for the clinical relationships between sarcopenia and osteoporosis. Myokines secreted from the skeletal muscles play roles in the muscle-bone interactions related to various physiological and pathophysiological states. Although numerous evidence suggested that growth hormone (GH) influences both muscle and bone, the effects of GH on the muscle-bone interactions have remained unknown. We therefore investigated the influences of GH administration for 8 weeks on muscle and bone, including myokine expression, in mice with or without ovariectomy (OVX). GH administration significantly increased muscle mass in the whole body and lower limbs, as well as tissue weights of the extensor digitorum longus (EDL) and soleus muscles, in mice with or without OVX. Moreover, it markedly increased grip strength in both mice. As for femurs, GH administration significantly increased cortical thickness and area in mice with or without OVX. Moreover, GH significantly blunted the decrease in the ratio of bone volume to tissue volume at trabecular bone in mice with OVX. GH administration significantly decreased follistatin mRNA levels in the EDL, but not the soleus, muscles in mice with or without OVX, although it did not affect the other myokines examined. However, GH administration significantly elevated serum follistatin levels in mice. In conclusion, our study indicated that GH administration increases skeletal muscle mass and grip strength, as well as cortical and trabecular bone-related parameters obtained by µCT analyses, in mice. However, myokine regulation might not be critical for the effects of GH on muscle and bone. - Yuya Mizukami; Takuma Yamaguchi; Miki Shiono; Yuki Takahashi; Kazunori Shimizu; Satoshi Konishi; Yoshinobu Takakura; Makiya NishikawaEuropean Polymer Journal 181 111671 - 111671 2022年12月 [査読有り]
- Kawao N; Kawaguchi M; Ohira T; Ehara H; Mizukami Y; Takafuji Y; Kaji HJ Cachexia Sarcopenia Muscle. 14 1 661 - 662 2022年11月 [招待有り]
- Yuko Kinoshita; Yoshimasa Takafuji; Katsumi Okumoto; Yuto Takada; Hiroki Ehara; Yuya Mizukami; Naoyuki Kawao; Jun-Ichiro Jo; Yasuhiko Tabata; Hiroshi KajiJournal of bone and mineral metabolism 2022年08月 [査読有り]
INTRODUCTION: Irisin is a proteolytic product of fibronectin type II domain-containing 5, which is related to the improvement in glucose metabolism. Numerous studies have suggested that irisin is a crucial myokine linking muscle to bone in physiological and pathophysiological states. MATERIALS AND METHODS: We examined the effects of local irisin administration with gelatin hydrogel sheets and intraperitoneal injection of irisin on the delayed femoral bone repair caused by streptozotocin (STZ)-induced diabetes in female mice. We analyzed the femurs of mice using quantitative computed tomography and histological analyses and then measured the mRNA levels in the damaged mouse tissues. RESULTS: Local irisin administration significantly blunted the delayed bone repair induced by STZ 10 days after a femoral bone defect was generated. Local irisin administration significantly blunted the number of Osterix-positive cells that were suppressed by STZ at the damaged site 4 days after a femoral bone defect was generated, although it did not affect the mRNA levels of chondrogenic and adipogenic genes 4 days after bone injury in the presence or absence of diabetes. On the other hand, intraperitoneal injection of irisin did not affect delayed bone repair induced by STZ 10 days after bone injury. Irisin significantly blunted the decrease in Osterix mRNA levels induced by advanced glycation end products or high-glucose conditions in ST2 cells in the presence of bone morphogenetic protein-2. CONCLUSIONS: We first showed that local irisin administration with gelatin hydrogel sheets improves the delayed bone repair induced by diabetic state partially by enhancing osteoblastic differentiation. - Extracellular vesicles secreted from mouse muscle cells improve delayed bone repair in diabetic miceYoshimasa Takafuji; Naoyuki Kawao; Takashi Ohira; Yuya Mizukami; Kiyotaka Okada; Jun-Ichiro Jo; Yasuhiko Tabata; Hiroshi KajiEndocrine Journal 2022年08月 [査読有り]
Humoral factors that are secreted from skeletal muscles can regulate bone metabolism and contribute to muscle-bone relationships. Although extracellular vesicles (EVs) play important roles in physiological and pathophysiological processes, the roles of EVs that are secreted from skeletal muscles in bone repair have remained unclear. In the present study, we investigated the effects of the local administration of muscle cell-derived EVs on bone repair in control and streptozotocin-treated diabetic female mice. Muscle cell-derived EVs (Myo-EVs) were isolated from the conditioned medium from mouse muscle C2C12 cells by ultracentrifugation, after which Myo-EVs and gelatin hydrogel sheets were transplanted on femoral bone defect sites. The local administration of Myo-EVs significantly improved delayed bone repair that was induced by the diabetic state in mice 9 days after surgery. Moreover, this administration significantly enhanced the ratio of bone volume to tissue volume at the damaged sites 9 days after surgery in the control mice. Moreover, the local administration of Myo-EVs significantly blunted the number of Osterix-positive cells that were suppressed by the diabetic state at the damage sites after bone injury in mice. Additionally, Myo-EVs significantly blunted the mRNA levels of Osterix and alkaline phosphatase (ALP), and ALP activity was suppressed by advanced glycation end product 3 in ST2 cells that were treated with bone morphogenetic protein-2. In conclusion, we have shown for the first time that the local administration of Myo-EVs improves delayed bone repair that is induced by the diabetic state through an enhancement of osteoblastic differentiation in female mice. - Miku Kawaguchi; Naoyuki Kawao; Masafumi Muratani; Yoshimasa Takafuji; Masayoshi Ishida; Yuko Kinoshita; Yuto Takada; Yuya Mizukami; Takashi Ohira; Hiroshi KajiJournal of Cellular Physiology 237 5 2492 - 2502 2022年02月 [査読有り]
Exercise is important for the prevention and treatment of sarcopenia and osteoporosis. Although the interactions between skeletal muscles and bone have recently been reported, the myokines linking muscle to bone during exercise remain unknown. We previously revealed that chronic exercise using treadmill running blunts ovariectomy-induced osteopenia in mice. We herein performed an RNA sequence analysis of the gastrocnemius and soleus muscles of male mice with or without chronic exercise to identify the myokines responsible for the effects of chronic exercise on the muscle/bone relationship. We extracted peripheral myelin protein 22 (PMP22) as a humoral factor that was putatively induced by chronic exercise in the soleus and gastrocnemius muscles of mice from the RNA sequence analysis. Chronic exercise significantly enhanced the expression of PMP22 in the gastrocnemius and soleus muscles of female mice. PMP22 suppressed macrophage-colony stimulating factor and receptor activator factor κB ligand-induced increases in the expression of osteoclast-related genes and osteoclast formation from mouse bone marrow cells. Moreover, PMP22 significantly inhibited osteoblast differentiation, alkaline phosphatase activity, and mineralization in mouse osteoblast cultures; however, the overexpression of PMP22 did not affect muscle phenotypes in mouse muscle C2C12 cells. A simple regression analysis revealed that PMP22 mRNA levels in the gastrocnemius and soleus muscles were positively related to cortical bone mineral density at the femurs of mice with or without chronic exercise. In conclusion, we identified PMP22 as a novel myokine induced by chronic exercise in mice. We first showed that PMP22 suppresses osteoclast formation and the osteoblast phenotype in vitro. - Naoyuki Kawao; Miku Kawaguchi; Takashi Ohira; Hiroki Ehara; Yuya Mizukami; Yoshimasa Takafuji; Hiroshi KajiJournal of Cachexia, Sarcopenia and Muscle 13 1 758 - 771 2022年02月 [査読有り]
BACKGROUND: Chronic renal failure induces bone mineral disorders and sarcopenia. Skeletal muscle affects other tissues, including bone, by releasing myokines. However, the effects of chronic renal failure on the interactions between muscle and bone remain unclear. METHODS: We investigated the effects of renal failure on bone, muscle, and myokines linking muscle to bone using a mouse 5/6 nephrectomy (Nx) model. Muscle mass and bone mineral density (BMD) were analysed by quantitative computed tomography 8 weeks after Nx. RESULTS: Nephrectomy significantly reduced muscle mass in the whole body (12.1% reduction, P < 0.05), grip strength (10.1% reduction, P < 0.05), and cortical BMD at the femurs of mice (9.5% reduction, P < 0.01) 8 weeks after surgery, but did not affect trabecular BMD at the femurs. Among the myokines linking muscle to bone, Nx reduced the expression of irisin, a proteolytic product of fibronectin type III domain-containing 5 (Fndc5), in the gastrocnemius muscles of mice (38% reduction, P < 0.01). Nx increased myostatin mRNA levels in the gastrocnemius muscles of mice (54% increase, P < 0.01). In simple regression analyses, cortical BMD, but not trabecular BMD, at the femurs was positively related to Fndc5 mRNA levels in the gastrocnemius muscles of mice (r = 0.651, P < 0.05). The weekly administration of recombinant irisin to mice ameliorated the decrease in cortical BMD, but not muscle mass or grip strength, induced by Nx (6.2% reduction in mice with Nx vs. 3.3% reduction in mice with Nx and irisin treatment, P < 0.05). CONCLUSIONS: The present results demonstrated that renal failure decreases the expression of irisin in the gastrocnemius muscles of mice. Irisin may contribute to cortical bone loss induced by renal failure in mice as a myokine linking muscle to bone. - Masayoshi Ishida; Naoyuki Kawao; Yuya Mizukami; Yoshimasa Takafuji; Hiroshi KajiBMC Musculoskeletal Disorders 22 1 398 - 398 2021年12月 [査読有り]
Abstract Background Osteoblasts and osteoclasts play important roles during the bone remodeling in the physiological and pathophysiological states. Although angiopoietin family Angiopoietin like proteins (Angptls), including Angptl1, have been reported to be involved in inflammation, lipid metabolism and angiogenesis, the roles of Angptl1 in bone have not been reported so far.Methods We examined the effects of Angptl1 on the osteoblast and osteoclast phenotypes using mouse cells.Results Angptl1 significantly inhibited the osteoclast formation and mRNA levels of tartrate-resistant acid phosphatase and cathepsin K enhanced by receptor activator of nuclear factor κB ligand in RAW 264.7 and mouse bone marrow cells. Moreover, Angptl1 overexpression significantly enhanced Osterix mRNA levels, alkaline phosphatase activity and mineralization induced by bone morphogenetic protein-2 in ST2 cells, although it did not affect the expression of osteogenic genes in MC3T3-E1 and mouse osteoblasts. On the other hand, Angptl1 overexpression significantly reduced the mRNA levels of peroxisome proliferator-activated receptor γ and adipocyte protein-2 as well as the lipid droplet formation induced by adipogenic medium in 3T3-L1 cells.Conclusions The present study first indicated that Angptl1 suppresses and enhances osteoclast formation and osteoblastic differentiation in mouse cells, respectively, although it inhibits adipogenic differentiation of 3T3-L1 cells. These data suggest the possibility that Angptl1 might be physiologically related to bone remodeling. - Yuya Mizukami; Yuki Takahashi; Kazunori Shimizu; Satoshi Konishi; Yoshinobu Takakura; Makiya NishikawaBiological and Pharmaceutical Bulletin 44 10 1458 - 1464 2021年10月 [査読有り]
- Masayoshi Ishida; Naoyuki Kawao; Yuya Mizukami; Yoshimasa Takafuji; Hiroshi KajiBiochemistry and Biophysics Reports 26 101004 - 101004 2021年07月 [査読有り]
Serpinb1a, a serine protease inhibitor family protein, has been implicated in immunoregulation and several metabolic disorders, such as diabetes and obesity; however, its roles in bone remain unknown. Therefore, we herein investigated the physiological functions of Serpinb1a in osteoclastic and osteoblastic differentiation using mouse cell lines. Serpinb1a overexpression markedly reduced the number of tartrate-resistant acid phosphatase (TRAP)- and calcitonin receptor-positive multinucleated cells increased by receptor activator nuclear factor κB ligand (RANKL) in mouse preosteoclastic RAW 264.7 cells. Moreover, it significantly decreased the mRNA levels of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), TRAP and cathepsin K in these cells. Regarding osteoblasts, Serpinb1a overexpression significantly reduced the mRNA levels of alkaline phosphatase (ALP) and osteocalcin as well as ALP activity induced by bone morphogenetic protein-2 (BMP-2) in mouse mesenchymal ST2 cells, although it did not alter osteoblast differentiation in mouse osteoblastic MC3T3-E1 cells. Concerning the pathophysiological relevance of Serpinb1a, Serpinb1a mRNA levels were decreased in the soleus and gastrocnemius muscles of mice 4 weeks after bilateral sciatic nerve resection. In conclusion, we herein revealed for the first time that Serpinb1a inhibited osteoclast formation induced by RANKL in RAW 264.7 cells and suppressed BMP-2-induced ALP activity in ST2 cells. - Naoyuki Kawao; Shunki Iemura; Miku Kawaguchi; Yuya Mizukami; Yoshimasa Takafuji; Hiroshi KajiJournal of Bone and Mineral Metabolism 39 4 547 - 557 2021年07月 [査読有り]
INTRODUCTION: Exercise is beneficial for the prevention and treatment of osteoporosis. Skeletal muscle affects other tissues via myokines, the release of which is regulated by acute exercise. However, the effects of chronic exercise on myokines linking muscle to bone have not been fully elucidated. Therefore, we investigated the effects of chronic exercise on bone and myokines using ovariectomized (OVX) mice. MATERIALS AND METHODS: Treadmill exercise with moderate intensity was performed for 8 weeks after OVX or sham surgery. We measured bone mineral density (BMD) at the femurs and tibias of mice by quantitative computed tomography and myokine mRNA levels in the gastrocnemius and soleus muscles. RESULTS: Treadmill exercise ameliorated decreases in trabecular and cortical BMD in the femurs of OVX mice. Irisin is a proteolytic product of fibronectin type III domain-containing 5 (Fndc5). Among the myokines examined, treadmill exercise increased irisin protein and Fndc5 mRNA levels in the gastrocnemius and soleus muscles of sham and OVX mice. Treadmill exercise increased peroxisome proliferator-activated receptor γ coactivator-1α mRNA levels in the gastrocnemius muscles of mice. Fndc5 mRNA levels in the gastrocnemius muscles positively correlated with trabecular BMD, but not with cortical BMD, at the femurs and tibias of mice in simple regression analyses. CONCLUSIONS: We demonstrated that chronic exercise elevated irisin expression in the gastrocnemius and soleus muscles of estrogen-deficient mice. Irisin might be related to increases in trabecular BMD in mice; however, further studies are needed to clarify the involvement of irisin in the effects of chronic exercise on muscle/bone interactions. - Hiroki Ehara; Yoshimasa Takafuji; Kohei Tatsumi; Kiyotaka Okada; Yuya Mizukami; Naoyuki Kawao; Osamu Matsuo; Hiroshi KajiEndocrine Journal 68 12 1421 - 1428 2021年 [査読有り]
Muscle wasting is a complication in patients with diabetes and leads to a reduced quality of life. However, the detailed mechanisms of diabetes-induced muscle wasting remain unknown. Plasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor that suppresses plasminogen activator activity, is involved in the pathophysiology of various diseases, including diabetes. In the present study, we examined the role of endogenous PAI-1 in the decrease in muscle mass and the impaired grip strength induced by the diabetic state by employing streptozotocin (STZ)-treated PAI-1-deficient female mice. The analyses of skeletal muscles and grip strength were performed in PAI-1-deficient and wild-type mice 4 weeks after the induction of a diabetic state by STZ administration. PAI-1 deficiency did not affect muscle mass in the lower limbs measured by quantitative computed tomography or tissue weights of the tibialis anterior, gastrocnemius and soleus muscles of female mice with or without STZ treatment. On the other hand, PAI-1 deficiency significantly aggravated grip strength decreased by STZ in female mice. PAI-1 deficiency did not affect the mRNA levels of Pax7, MyoD, myogenin or myosin heavy chain in either the tibialis anterior or soleus muscles of female mice with or without STZ treatment. In conclusion, we revealed for the first time that PAI-1 deficiency aggravates grip strength impaired by the diabetic state in female mice, although it did not affect diabetes-decreased muscle mass. - Yuya Mizukami; Ai Moriya; Yuki Takahashi; Kazunori Shimizu; Satoshi Konishi; Yoshinobu Takakura; Makiya NishikawaBiological and Pharmaceutical Bulletin 43 8 1220 - 1225 2020年08月 [査読有り]
The multicellular spheroid three-dimensional cell culture system can be used as a formulation for cell-based therapy. However, the viability and functions of the cells in the core region of the spheroid tend to decrease because of limited oxygen supply. In this study, we incorporated gelatin microspheres (GMS) into HepG2 human hepatocyte spheroids to allow oxygen to reach the spheroid core. GMS with an approximate diameter of 37 µm were fabricated by water-in-oil emulsification followed by freeze drying. GMS-containing HepG2 spheroids (GMS/HepG2 spheroids) were prepared by incubation of the cells with GMS at various mixing ratios in agarose gel-based microwells. Increasing the GMS ratio increased the diameter of the spheroids, and few spheroids formed with excess GMS. HepG2 cells in the GMS/HepG2 spheroids were more oxygenated than those in the GMS-free spheroids. GMS incorporation increased the viability of HepG2 cells in the spheroids and increased the CYP1A1 activity of the cells to metabolize 7-ethoxyresorufin, although mRNA expression of the CYP1A1 gene was hardly affected by GMS incorporation. These results indicate that incorporating GMS into HepG2 spheroids improves the hypoxic microenvironment in the spheroids and increases cell viability and CYP1A1 metabolic activity. - Tomoyuki Furuta; Yuya Mizukami; Lisa Asano; Kenjiro Kotake; Slava Ziegler; Hiroki Yoshida; Mizuki Watanabe; Shin-Ichi Sato; Herbert Waldmann; Makiya Nishikawa; Motonori UesugiACS chemical biology 144 9 1860 - 1865 2019年08月 [査読有り]
- Ippei Takashima; Kosuke Kusamori; Hayase Hakariya; Megumi Takashima; Thi Hue Vu; Yuya Mizukami; Noda Naotaka; Yukiya Takayama; Yousuke Katsuda; Shin-Ichi Sato; Yoshinobu Takakura; Makiya Nishikawa; Motonari UesugiACS chemical biology 14 4 775 - 783 2019年04月 [査読有り]
Cell-based therapy is a promising approach to restoring lost functions to compromised organs. However, the issue of inefficient cell engraftment remains to be resolved. Herein, we take a chemical approach to facilitate cell engraftment by using self-assembling molecules which modify two cellular traits: cell survival and invasiveness. In this system, the self assembling molecule induces syndecan-4 clusters on the cellular surface, leading to enhanced cell viability. Further integration with Halo-tag technology provided this self-assembly structure with matrix metalloproteinase-2 to functionalize cells with cell-invasion activity. In vivo experiments showed that the pretreated cells were able to survive injection and then penetrate and engraft into the host tissue, demonstrating that the system enhances cell engraftment. Therefore, cell-surface modification via an alliance between self-assembling molecules and ligation technologies may prove to be a promising method for cell engraftment. - Yuya Mizukami; Yuki Takahashi; Kazunori Shimizu; Satoshi Konishi; Yoshinobu Takakura; Makiya NishikawaTissue Engineering Part A 25 5-6 390 - 398 2019年02月 [査読有り]
- Yutaro Tanaka; Makiya Nishikawa; Yuya Mizukami; Kosuke Kusamori; Yuka Ogino; Shunsuke Nishimura; Kazunori Shimizu; Satoshi Konishi; Yuki Takahashi; Yoshinobu TakakuraJournal of Controlled Release 270 177 - 183 2018年01月 [査読有り]
Immune cell-based therapy is a promising approach for cancer immunotherapy. Macrophages can be used for this purpose if their tumoricidal activity and viability are properly controlled. In the present study, we aimed to enhance these properties of macrophages by constructing uniformly sized multicellular spheroids. Mouse macrophage-like J774.1 cells were selected as model macrophages, and poly(N-isopropylacrylamide)-coated polydimethylsiloxane-based microwell plates with an approximate diameter of 750 μm were used to prepare J774.1 spheroids. J774.1 spheroids were successfully generated, and the viability of cells in the spheroids was over 95%. J774.1 spheroids showed higher mRNA expression of induced nitric oxide synthase, a marker of M1-type activated macrophages, than monolayered J774.1 cells. The production of reactive oxygen species was also high in J774.1 spheroids, suggesting the existence of hypoxic regions in the spheroids. J774.1 spheroids released more tumor necrosis factor-α than monolayered cells upon stimulation with lipopolysaccharide. Moreover, J774.1 spheroids in the upper compartment of the Transwell system more efficiently inhibited the proliferation of mouse adenocarcinoma colon 26 cells in its lower compartment than monolayered J774.1 cells did. These results indicate that spheroid formation can be used to increase the tumoricidal activity of macrophages for use in cell-based cancer immunotherapy. - Tomoko Nishikawa; Yutaro Tanaka; Kosuke Kusamori; Narumi Mizuno; Yuya Mizukami; Yuka Ogino; Kazunori Shimizu; Satoshi Konishi; Yuki Takahashi; Yoshinobu Takakura; Makiya NishikawaBiotechnology Journal 12 8 2017年08月 [査読有り]
Previous studies demonstrated that multicellular spheroids developed using polydimethylsiloxane-based microwells exhibited superior functions, such as insulin secretion from pancreatic cells, over suspended cells. To successfully apply these spheroids, the effect of spheroid size on cellular functions must be determined. In this study, using murine adenocarcinoma colon26 cells, the authors examined whether such spheroids were useful for developing tumor-bearing animal models, which requires the efficient and stable engraftment of cancer cells at implanted sites and/or metastatic sites. The authors prepared microwells with widths of 360, 450, 560, and 770 μm through a micromolding technique, and obtained colon26 spheroids with average diameters of 169, 240, 272, and 341 μm, respectively. Small and medium spheroids were subsequently used. mRNA levels of integrin β1, CD44, and fibronectin, molecules involved in cell adhesion, increased with increasing colon26 spheroid size. Approximately 1.5 × 104 colon26 cells in suspension or in spheroids were intravenously inoculated into BALB/c mice. At 21 days after inoculation, the lung weight of both colon26 spheroid groups, especially the group injected with small spheroids, was significantly higher than that of mice in the suspended colon26 cell group. These results indicate that controlling cancer cell spheroid size is crucial for tumor development in tumor-bearing mouse models. - Tomoko Nishikawa; Yutaro Tanaka; Makiya Nishikawa; Yuka Ogino; Kosuke Kusamori; Narumi Mizuno; Yuya Mizukami; Kazunori Shimizu; Satoshi Konishi; Yuki Takahashi; Yoshinobu TakakuraBiological and Pharmaceutical Bulletin 40 3 334 - 338 2017年 [査読有り]
Multicellular spheroids are useful as three-dimensional cell culture systems and for cell-based therapies. Their successful application requires an understanding of the consequences of spheroid size for cellular functions. In the present study, we prepared multicellular spheroids of different sizes using the human hepatoblastoma HepG2 cells, as hepatocytes are frequently used for in vitro drug screening and cell-based therapy. Precise polydimethylsiloxane-based microwells with widths of 360, 450, 560, and 770 μm were fabricated using a micromolding technique. Incubation of HepG2 cells in cell culture plates containing the microwells resulted in the formation of HepG2 spheroids with average diameters of 195, 320, 493, and 548 μm. The cell number per spheroid positively correlated with its diameter, and the viability of HepG2 cells was 94% or above for all samples. The smallest HepG2 spheroids showed the highest albumin secretion. On the other hand, the metabolic activity of 7-ethoxyresorufin, a fluorometric substrate for CYP1A1, increased with increasing spheroid size. These results indicate that controlling spheroid size is important when preparing HepG2 spheroids and that the size of HepG2 spheroids greatly influences the cellular function of HepG2 cells in the spheroids. - Kosuke Kusamori; Makiya Nishikawa; Narumi Mizuno; Tomoko Nishikawa; Akira Masuzawa; Yutaro Tanaka; Yuya Mizukami; Kazunori Shimizu; Satoshi Konishi; Yuki Takahashi; Yoshinobu TakakuraPharmaceutical Research 33 1 247 - 256 2016年01月 [査読有り]
Purpose: We previously have shown that multicellular spheroids containing insulin-secreting cells are an effective therapy for diabetic mice. Here we attempted to increase insulin secretion by incorporating other cell types into spheroids. Materials and Methods: Multicellular spheroids of mouse MIN6 pancreatic β cells were formed in microwells alone and with aortic vascular endothelial MAEC cells or embryo fibroblast NIH3T3 cells. mRNA expression of insulin genes and insulin secretion of MIN6 cells in each spheroid were measured by real-time PCR and an insulin ELIZA kit. Moreover, collagen IV expression in each spheroid was analyzed by western blot. Results: In all cases, uniformly sized (about 300 μm) multicellular spheroids were obtained. MAEC or NIH3T3 cell incorporation into MIN6 spheroids significantly increased mRNA expression of insulin genes and insulin secretion. In addition, collagen IV expression, which was reported to enhance insulin secretion from pancreatic β cells, also increased in their spheroids. Conclusions: The formation of mixed multicellular spheroids containing collagen IV-expressing cells can improve the insulin secretion from insulin-secreting MIN6 cells, and mixed multicellular spheroids can be a potent therapeutic option for patients with type I diabetes mellitus.
講演・口頭発表等
- 水上優哉; 高藤義正; 河尾直之; 大平宇志; 岡田清孝; 城潤一郎; 田畑泰彦; 梶博史第41回 日本骨代謝学会学術集会 2023年07月 口頭発表（一般）
- 水上優哉; 高藤義正; 河尾直之; 大平宇志; 岡田清孝; 城潤一郎; 田畑泰彦; 梶博史第22回 日本再生医療学会総会 2023年03月 口頭発表（一般）
- 水性二相分離法により作製したbFGF搭載メタクリル酸修飾ゼラチン微粒子の内包による脂肪由来幹細胞スフェロイドの高機能化水上優哉日本薬学会第140年会 2020年03月 その他
- 水性二相分離法を用いたbFGF封入メタクリル酸修飾ゼラチン粒子の開発 [通常講演]水上優哉第35回日本DDS学会学術集会 2019年07月 口頭発表（一般）
- 高機能細胞スフェロイドの作製を目指した酸素生成マイクロウェルの開発 [通常講演]水上優哉第12回次世代を担う若手医療薬科学シンポジウム 2018年09月 口頭発表（一般）
- ゼラチン微粒子の内包による肝細胞スフェロイドの細胞機能の向上 [通常講演]水上優哉日本薬剤学会第33年会 2018年05月
- Elucidation of factors determining the cell distribution in artificially fabricated mixed multicellular spheroids for improved insulin secretion. [通常講演]Yuya MizukamiGPEN conference 2016 2016年11月 ポスター発表
- 混合細胞スフェロイド中の細胞局在に及ぼす因子の解明 [通常講演]水上優哉第32回日本DDS学術集会 2016年06月 口頭発表（一般）
- 混合細胞スフェロイドを構成する細胞の局在化規定因子の解明 [通常講演]水上優哉日本薬剤学会第30年会 2015年05月 口頭発表（一般）
受賞
共同研究・競争的資金等の研究課題
- 日本学術振興会：科学研究費助成事業 研究活動スタート支援研究期間 : 2020年09月 -2022年03月代表者 : 水上 優哉本研究では、高い骨再生能を持つことが報告される間葉系幹細胞スフェロイドの骨修復・再生メカニズムの解明を目的に研究を行った。脂肪組織由来間葉系幹細胞（ADSCs）をスフェロイド化し、健常マウスおよびストレプトゾトシン誘発性糖尿病モデルマウスの大腿骨欠損部に移植した。しかし、ADSCsのスフェロイド化による有意な骨修復・再生効果はみられなかった。また、足場素材、移植細胞数、スフェロイドサイズの変更によっても、骨修復・再生効果に改善はみられなかった。以上の結果より、ADSCsスフェロイドでは予想された骨修復・再生の促進効果がみられなかったため、骨修復・再生促進メカニズムの解明に至らなかった。