Kohei Tatsumi

Role of follistatin in muscle and bone alterations induced by gravity change in mice

Interactions between muscle and bone have been recently noted. We reported that the vestibular system plays crucial roles in the changes in muscle and bone induced by hypergravity in mice. However, the details of the mechanisms by which gravity change affects muscle and bone through the vestibular system still remain unknown. Here, we investigated the roles of humoral factors linking muscle to bone and myostatin‐related factors in the hypergravity‐induced changes in muscle and bone in mice with vestibular lesions (VL). Hypergravity elevated serum and mRNA levels of follistatin, an endogenous inhibitor of myostatin, in the soleus muscle of mice. VL blunted the hypergravity‐enhanced levels of follistatin in the soleus muscle of mice. Simulated microgravity decreased follistatin mRNA level in mouse myoblastic C2C12 cells. Follistatin elevated the mRNA levels of myogenic genes as well as the phosphorylation of Akt and p70S6 kinase in C2C12 cells. As for bone metabolism, follistatin antagonized the mRNA levels of osteogenic genes suppressed by activin A during the differentiation of mesenchymal cells into osteoblastic cells. Moreover, follistatin attenuated osteoclast formation enhanced by myostatin in the presence of receptor activator of nuclear factor‐κB ligand in RAW 264.7 cells. Serum follistatin levels were positively related to bone mass in mouse tibia. In conclusion, the present study provides novel evidence that hypergravity affects follistatin levels in muscle through the vestibular system in mice. Follistatin may play some roles in the interactions between muscle and bone metabolism in response to gravity change.

Hepatocyte Transplantation: Cell Sheet Technology for Liver Cell Transplantation

Purpose of ReviewWe will review the recent developments of cell sheet technology as a feasible tissue engineering approach. Specifically, we will focus on the technological advancement for engineering functional liver tissue using cell sheet technology, and the associated therapeutic effect of cell sheets for liver diseases, highlighting hemophilia.Recent FindingsCell-based therapies using hepatocytes have recently been explored as a new therapeutic modality for patients with many forms of liver disease. We have developed a cell sheet technology, which allows cells to be harvested in a monolithic layer format. We have succeeded in fabricating functional liver tissues in mice by stacking the cell sheets composed of primary hepatocytes. As a curative measure for hemophilia, we have also succeeded in treating hemophilia mice by transplanting of cells sheets composed of genetically modified autologous cells.SummaryTissue engineering using cell sheet technology provides the opportunity to create new therapeutic options for patients with various types of liver diseases.

Roles of Irisin in the Linkage from Muscle to Bone During Mechanical Unloading in Mice

Mechanical unloading induces disuse muscle atrophy and bone loss, but the details in mechanism involved in those pathophysiological conditions are not fully understood. Interaction between muscle and bone has been recently noted. Here, we investigated the roles of humoral factors linking muscle to bone during mechanical unloading using mice with hindlimb unloading (HU) and sciatic neurectomy (SNX). HU and SNX reduced muscle volume surrounding the tibia, tissue weights of soleus and gastrocnemius muscle, and trabecular bone mineral density (BMD) in the tibia of mice. Among humoral factors linking muscle to bone, HU and SNX reduced fibronectin type III domain-containing 5 (FNDC5) mRNA levels in the soleus muscle of mice. Simple regression analysis revealed that FNDC5 mRNA levels in the soleus muscle were positively related to trabecular BMD in the tibia of control and HU mice as well as sham and SNX mice. Moreover, FNDC5 mRNA levels were negatively correlated with receptor activator of nuclear factor-κB ligand (RANKL) mRNA levels in the tibia of control and HU mice. Irisin, a product of FNDC5, suppressed osteoclast formation from mouse bone marrow cells and RANKL mRNA levels in primary osteoblasts. FNDC5 mRNA levels elevated by fluid shear stress were antagonized by bone morphogenetic protein (BMP) and phosphatidylinositol 3-kinase (PI3K) signaling inhibitors in myoblastic C2C12 cells. In conclusion, the present study first showed that mechanical unloading reduces irisin expression in the skeletal muscle of mice presumably through BMP and PI3K pathways. Irisin might be involved in muscle/bone relationships regulated by mechanical stress in mice.

Plasminogen activator inhibitor-1 is involved in interleukin-1β-induced matrix metalloproteinase expression in murine chondrocytes

Abstract Objectives: Interleukin (IL)-1β and matrix metalloproteinases (MMPs) play important roles in the pathogenesis of osteoarthritis. On the other hand, plasminogen activator inhibitor-1 (PAI-1), an inhibitor of fibrinolysis, exerts functions in the pathogenesis of various diseases. However, the functional roles of PAI-1 in the chondrocytes have been still remained unknown. Methods: In the present study, we investigated the roles of PAI-1 in the effects of IL-1β on the chondrocytes using wild-type and PAI-1-deficient mice. Results: IL-1β significantly elevated PAI-1 mRNA levels in the chondrocytes from wild-type mice. PAI-1 deficiency significantly blunted the mRNA levels of TGF-β and IL-6 enhanced by IL-1β in murine chondrocytes. Moreover, PAI-1 deficiency significantly decreased the mRNA levels of MMP-13, -3 and -9 as well as MMP-13 activity enhanced by IL-1β in the chondrocytes. In addition, PAI-1 deficiency significantly reversed type II collagen mRNA levels suppressed by IL-1β in the chondrocytes. On the other hand, active PAI-1 treatment significantly enhanced the mRNA levels of MMP-13, -3 and -9 as well as decreased type II collagen mRNA levels in the chondrocytes from wild-type mice. Conclusion: We first demonstrated that PAI-1 is involved in MMP expression enhanced by IL-1β in murine chondrocytes. PAI-1 might be crucial for the cartilage matrix degradation and the impaired chondrogenesis by IL-1β in mice.

Experimental hypercoagulable state induced by tissue factor expression in monocyte-derived dendritic cells and its modulation by C1 inhibitor

The crosstalk between immune and coagulation systems plays pivotal roles in host defense, which may involve monocyte-derived dendritic cells (moDCs). Our objectives were to elucidate the role of moDCs in coagulation under inflammatory conditions and the involvement of the complement system. We assessed the effects of lipopolysaccharide (LPS)-stimulated moDCs on coagulation using whole blood thromboelastometry in the presence of complement inhibitors. The sum of clotting time and clot formation time (CT plus CFT) in whole blood thromboelastometry was significantly more reduced in the presence of moDCs than in the absence of monocytes or moDCs and in the presence of monocytes, indicating a more potent coagulability of moDCs. The mRNA expression of coagulation-related proteins in moDCs was analyzed by quantitative PCR, which showed an increase only in the mRNA levels of tissue factor (TF). TF protein expression was assessed by western blot analysis and an activity assay, revealing higher TF expression in moDCs than that in monocytes. The in vitro moDC-associated hypercoagulable state was suppressed by a TF-neutralizing antibody, whereas LPS enhanced the in vitro hypercoagulation further. C1 inhibitor suppressed the in vitro LPS-enhanced whole blood hypercoagulability in the presence of moDCs and the increased TF expression in moDCs. These results suggest a significant role of moDCs and the complement system through TF expression in a hypercoagulable state under inflammatory conditions and demonstrate the suppressive effects of C1 inhibitor on moDC-associated hypercoagulation.

Serpina3n, dominantly expressed in female osteoblasts, suppresses the phenotypes of differentiated osteoblasts in mice

It is well known that sex differences exist concerning the severity of osteoporosis and bone metabolism, suggesting that factors other than sex hormones might be responsible for sex differences of bone metabolism. We therefore examined sex differences of osteoblast phenotypes of mouse osteoblasts and then performed comparative gene expression analyses using a comprehensive DNA microarray between female and male osteoblasts. Alkaline phosphatase (ALP) activity, mineralization, and the expression of Osterix, ALP, and bone sialoprotein were significantly lower in mouse female osteoblasts compared with male osteoblasts. We identified Serpina3n, a novel serine protease inhibitor, as the gene whose expression has the highest ratio of females to males. A reduction in endogenous levels of Serpina3n by small interfering RNA significantly enhanced the mRNA levels of Runx2, ALP, osteocalcin, and type I collagen (Col1a1) in both male and female osteoblasts. Moreover, Serpina3n overexpression significantly suppressed the mRNA levels of Osterix, ALP, osteocalcin, and Col1a1 in MC3T3-E1 cells. Serpina3n overexpression did not affect Osterix, ALP, and osteocalcin mRNA levels enhanced by bone morphogenetic protein (BMP)-2 in ST2 cells, adipogenic differentiation in ST2 and 3T3-L1 cells, and receptor activator of nuclear factor κB ligand-induced osteoclast formation in RAW264.7 cells, although it significantly suppressed mineralization in ST2 cells differentiated into osteoblasts by BMP-2. In conclusion, we found Serpina3n as the most female osteoblast-dominant gene. Serpina3n exerts a suppression of the osteoblast phenotypes such as Col1a1 expression and ALP activity in differentiated osteoblasts, which might partly explain sex differences of the osteoblast phenotypes in mice.

Role of Macrophages and Plasminogen Activator Inhibitor-1 in Delayed Bone Repair in Diabetic Female Mice

Delayed fracture healing is a clinical problem in diabetic patients. However, the mechanisms of diabetic delayed bone repair remain unknown. Here, we investigate the role of macrophages in diabetic delayed bone repair after femoral bone injury in streptozotocin (STZ)-treated and plasminogen activator inhibitor-1 (PAI-1)-deficient female mice. STZ treatment significantly decreased the numbers of F4/80-positive cells (macrophages) but not granulocyte-differentiation antigen-1-positive cells (neutrophils) at the damaged site on day 2 after femoral bone injury in mice. It significantly decreased the messenger RNA (mRNA) levels of macrophage colony-stimulating factor, inducible nitric oxide synthase (iNOS), interleukin (IL)-6, and CD206 at the damaged site on day 2 after bone injury. Moreover, STZ treatment attenuated a decrease in the number of hematopoietic stem cells in bone marrow induced by bone injury. On the other hand, PAI-1 deficiency significantly attenuated a decrease in the number of F4/80-positive cells induced by STZ treatment at the damaged site on day 2 after bone injury in mice. PAI-1 deficiency did not affect the mRNA levels of iNOS and IL-6 in F4/80- and CD11b-double-positive cells from the bone marrow of the damaged femurs decreased by diabetes in mice. PAI-1 deficiency significantly attenuated the phagocytosis of macrophages at the damaged site suppressed by diabetes. In conclusion, we demonstrated that type 1 diabetes decreases accumulation and phagocytosis of macrophages at the damaged site during early bone repair after femoral bone injury through PAI-1 in female mice.

Roles of plasminogen in the alterations in bone marrow hematopoietic stem cells during bone repair

We previously revealed that stromal cell-derived factor-1 (SDF-1) is involved in the changes in the number of bone marrow stem cells during the bone repair process in mice. Moreover, we reported that plasminogen (Plg) deficiency delays bone repair and the accumulation of macrophages at the site of bone damage in mice. We investigated the roles of Plg in the changes in bone marrow stem cells during bone repair. We analyzed the numbers of hematopoietic stem cells (HSC) and mesenchymal stem cells (MSCs) within bone marrow from Plg-deficient and wild-type mice after a femoral bone injury using flow cytometric analysis. Plg deficiency significantly blunted a decrease in the number of HSCs after bone injury in mice, although it did not affect an increase in the number of MSCs. Plg deficiency significantly blunted the number of SDF-1- and Osterix- or SDF-1- and alkaline phosphatase-double-positive cells in the endosteum around the lesion as well as matrix metalloprotainase-9 (MMP-9) activity and mRNA levels of SDF-1 and transforming growth factor–β (TGF-β) elevated by bone injury. TGF-β signaling inhibition significantly blunted a decrease in the number of HSCs after bone injury. The present study showed that Plg is critical for the changes in bone marrow HSCs through MMP-9, TGF-β, and SDF-1 at the damaged site during bone repair in mice.

Adult hepatocytes direct liver organogenesis through non-parenchymal cell recruitment in the kidney

BACKGROUND & AIMS Since the first account of the myth of Prometheus, the amazing regenerative capacity of the liver has fascinated researchers because of its enormous medical potential. Liver regeneration is promoted by multiple types of liver cells, including hepatocytes and liver non-parenchymal cells (NPCs), through complex intercellular signaling. However, the mechanism of liver organogenesis, especially the role of adult hepatocytes at ectopic sites, remains unknown. In this study, we demonstrate that hepatocytes alone spurred liver organogenesis to form an organ-sized complex 3D liver that exhibited native liver architecture and functions in the kidneys of mice. METHODS Isolated hepatocytes were transplanted under the kidney capsule of monocrotaline (MCT) and partial hepatectomy (PHx)-treated mice. To determine the origin of NPCs in neo-livers, hepatocytes were transplanted into MCT/PHx-treated green fluorescent protein transgenic mice or wild-type mice transplanted with bone marrow cells isolated from green fluorescent protein-mice. RESULTS Hepatocytes engrafted at the subrenal space of mice underwent continuous growth in response to a chronic hepatic injury in the native liver. More than 1.5 years later, whole organ-sized liver tissues with greater mass than those of the injured native liver had formed. Most remarkably, we revealed that at least three types of NPCs with similar phenotypic features to the liver NPCs were recruited from the host tissues including bone marrow. The neo-livers in the kidney exhibited liver-specific functions and architectures, including sinusoidal vascular systems, zonal heterogeneity, and emergence of bile duct cells. Furthermore, the neo-livers successfully rescued the mice with lethal liver injury. CONCLUSION Our data clearly show that adult hepatocytes play a leading role as organizer cells in liver organogenesis at ectopic sites via NPC recruitment. LAY SUMMARY The role of adult hepatocytes at ectopic locations has not been clarified. In this study, we demonstrated that engrafted hepatocytes in the kidney proliferated, recruited non-parenchymal cells from host tissues including bone marrow, and finally created an organ-sized, complex liver system that exhibited liver-specific architectures and functions. Our results revealed previously undescribed functions of hepatocytes to direct liver organogenesis through non-parenchymal cell recruitment and organize multiple cell types into a complex 3D liver at ectopic sites. Transcript profiling: Microarray data are deposited in GEO (GEO accession: GSE99141).

Plasminogen activator inhibitor-1 deficiency enhances subchondral osteopenia after induction of osteoarthritis in mice

BackgroundSubchondral osteopenia is important for the pathophysiology of osteoarthritis (OA). Although previous studies suggest that plasminogen activator inhibitor-1 (PAI-1), an inhibitor of fibrinolysis, is related to bone metabolism, its role in OA remains unknown. We therefore investigated the roles of PAI-1 in the subchondral bone in OA model mice.MethodsWild type (WT) and PAI-1-deficient (KO) mice were ovariectomized (OVX), and then destabilization of the medial meniscus (DMM) surgery was performed.ResultsDMM and OVX significantly decreased the trabecular bone mineral density of the subchondral bone evaluated by quantitative computed tomography in PAI-1 KO mice. The effects of OVX and/or PAI-1 deficiency on the OARSI score for the evaluation of the progression of knee degeneration were not significant. PAI-1 deficiency significantly augmented receptor activator nuclear factor κB ligand mRNA levels enhanced by IL-1β in mouse primary osteoblasts, although it did not affect osteoblast differentiation. Moreover, PAI-1 deficiency significantly increased osteoclast formation from mouse bone marrow cells.ConclusionWe showed that PAI-1 deficiency accelerates the subchondral osteopenia after induction of OA in mice. PAI-1 might suppress an enhancement of bone resorption and subsequent subchondral osteopenia after induction of OA in mice.