Force-sensing treadmill gait analysis system can detect gait abnormalities in haemophilia patients without arthropathy.

BACKGROUND: Joint damage in patients with haemophilia (PwH) is commonly assessed by imaging, but few reports have described how structural changes in joints, for example, haemophilic arthropathy (HA)-affect gait ability. OBJECTIVES: We evaluated gait function among PwH with HA, PwH without HA, and people without haemophilia (non-PwH) using a Zebris FDM-T treadmill (FDM-T), an easy-to-use gait assessment instrument with a force sensor matrix. METHODS: The following gait parameters were collected: centre of pressure trajectory intersection (COPi) anterior/posterior variability, COPi lateral variability, COPi anterior/posterior symmetry, COPi lateral symmetry, single-limb support line (SLSL) length, and SLSL variability. Participants walked at their typical gait speed. The physical function of the PwH was assessed by the Hemophilia Joint Health Score (HJHS). Parameters were compared among the three groups. RESULTS: Twelve PwH with HA, 28 PwH without HA, and 12 non-PwH were enrolled. Gait speed significantly differed between groups (non-PwH, 3.1 ± 0.7; PwH without HA, 2.0 ± 0.7; PwH with HA; 1.5 ± 0.4). The COPi anterior/posterior variability, COPi lateral variability, SLSL length, and SLSL variability were greater in the PwH groups than in the non-PwH group. The COPi lateral symmetry differed between PwH with HA and the other groups. The HJHS was not correlated with gait parameters among PwH with HA. CONCLUSIONS: Gait parameters and speed were abnormal in both PwH with HA and PwH without HA. The FDM-T can be used to identify early stages of physical dysfunction that cannot be detected by conventional functional assessments such as the HJHS.

The pathogenesis of cancer-associated thrombosis.

Patients with cancer have a higher risk of venous thromboembolism (VTE), including deep vein thrombosis (DVT) and pulmonary embolism (PE), compared to the general population. Cancer-associated thrombosis (CAT) is a thrombotic event that occurs as a complication of cancer or cancer therapy. Major factors determining VTE risk in cancer patients include not only treatment history and patient characteristics, but also cancer type and site. Cancer types can be broadly divided into three groups based on VTE risk: high risk (pancreatic, ovarian, brain, stomach, gynecologic, and hematologic), intermediate risk (colon and lung), and low risk (breast and prostate). This implies that the mechanism of VTE differs between cancer types and that specific VTE pathways may exist for different cancer types. This review summarizes the specific pathways that contribute to VTE in cancer patients, with a particular focus on leukocytosis, neutrophil extracellular traps (NETs), tissue factor (TF), thrombocytosis, podoplanin (PDPN), plasminogen activator inhibitor-1 (PAI-1), the intrinsic coagulation pathway, and von Willebrand factor (VWF).

Terminus-Selective Covalent Immobilization of Heparin on a Thermoresponsive Surface Using Click Chemistry for Efficient Binding of Basic Fibroblast Growth Factor.

Cell therapy using endothelial cells (ECs) has great potential for the treatment of congenital disorders, such as hemophilia A. Cell sheet technology utilizing a thermoresponsive culture dish is a promising approach to efficiently transplant donor cells. In this study, a new method to prepare terminus-selective heparin-immobilized thermoresponsive culture surfaces is developed to facilitate the preparation of EC sheets. Alkynes are introduced to the reducing terminus of heparin via reductive amination. Cu-catalyzed azide-alkyne cycloaddition (CuAAC) facilitates efficient immobilization of the terminus of heparin on a thermoresponsive surface, resulting in a higher amount of immobilized heparin while preserving its function. Heparin-immobilized thermoresponsive surfaces prepared using CuAAC exhibit good adhesion to human endothelial colony-forming cells (ECFCs). In addition, upon further binding to basic fibroblast growth factor (bFGF) on heparin-immobilized surfaces, increased proliferation of ECFCs on the surface is observed. The confluent ECFC monolayer cultured on bFGF-bound heparin-immobilized thermoresponsive surfaces exhibits relatively high fibronectin accumulation and cell number and detaches at 22 °C while maintaining the sheet-like structure. Because heparin has an affinity for several types of bioactive molecules, the proposed method can be applied to facilitate efficient cultures and sheet formations of various cell types.

Single-facility study of the effectiveness of rehabilitation therapy using wearable hybrid assistive limb for patients with bleeding disorders: study protocol for a randomised controlled trial.

INTRODUCTION: Haemophilic arthropathy, a serious complication of haemophilia, results from recurrent joint bleeding, causing progressive joint damage and severely impacting patient quality of life. Rehabilitation therapy (RT) effectively addresses declining physical function due to joint degradation, but pain during RT can hinder its success. Therefore, an effective pain-alleviating treatment method is required. The single-joint hybrid assistive limb (HAL-SJ), a powered exoskeleton, measures bioelectric potential during muscle contraction and provides motorised support, potentially alleviating pain. OBJECTIVE: This study outlines our protocol for a randomised, prospective, single-blind (evaluator) trial aimed to investigate the effects of HAL-SJ on pain reduction during RT, kinesiophobia and other physical functions in patients with haemophilia. METHODS AND ANALYSIS: This two-group comparison intervention study will include 24 male patients aged 12-85 years diagnosed with a bleeding disorder necessitating RT for pain and physical function improvement. The primary outcome measures pain changes during the first and second RT session in patients receiving HAL-SJ-assisted RT compared with traditional RT without HAL-SJ. The secondary outcomes include kinesiophobia (Japanese version of the Tampa Scale for Kinesiophobia), standing position gait (zebris FDM-T treadmill), range of motion (manual goniometer) and body surface temperature (infrared thermography camera) during the study period of up to 3 months or until the end of 10 RTs. RT intensity remains below that required to move the affected joint against gravity, given HAL-SJ's muscular support. The follow-up period extends to 1 month after the last RT. Intergroup study variables are compared by an unpaired t-test or Mann-Whitney test. Intragroup comparisons of secondary outcomes are analysed by a paired t-test or Wilcoxon signed-rank test. ETHICS AND DISSEMINATION: This study was approved by the accreditation committee of Nara Medical University Hospital. The study results will disseminate through publication in a peer-reviewed journal. TRIAL REGISTRATION NUMBER: jRCTs052220076.

Regulation of von Willebrand factor by ADAMTS13 ameliorates lipopolysaccharide-induced lung injury in mice.

The relationship between von Willebrand factor (VWF) and inflammation has attracted considerable attention in recent years. VWF, which is stored in the Weibel-Palade bodies (WPBs) of endothelial cells (ECs), is released from WPBs in response to inflammatory stimuli and is thought to contribute to inflammation by promoting leukocyte extravasation. In this study, lung injury model mice were produced by intratracheal injection with lipopolysaccharides. The severity of lung inflammation was evaluated in mice with different genotypes (wild-type, Vwf-/-, Adamts13-/-) and mice treated with drugs that inhibit VWF function. Lung inflammation was significantly ameliorated in Vwf-/- mice compared with wild-type mice. Furthermore, inflammation was significantly suppressed in wild-type mice treated with anti-VWF A1 antibody or recombinant human ADAMTS13 compared with the untreated control group. The underlying mechanism appears to be an increased VWF/ADAMTS13 ratio at the site of inflammation and the interaction between blood cell components, such as leukocytes and platelets, and the VWF A1 domain, which promotes leukocyte infiltration into the lung. This study suggested that ADAMTS13 protein and other VWF-targeting agents may be a novel therapeutic option for treatment of pulmonary inflammatory diseases.

Generation of functional liver sinusoidal endothelial-like cells from human bone marrow-derived mesenchymal stem cells.

Introduction: Liver sinusoidal endothelial cells (LSECs) are specialized vascular endothelial cells that play an important role in the maintenance of biological homeostasis. However, the lack of versatile human LSECs has hindered research on LSECs and development of medical technologies for liver diseases including hemophilia A. In this study, we developed a technique to induce LSEC differentiation from human bone marrow-derived mesenchymal stem cells (BM-MSCs). Methods: To induce LSECs from human BM-MSCs, cytokines and chemical compounds associated with signaling implicated in LSEC differentiation and liver development were screened. Then LSEC-related genes and proteins expression in the differentiated cells were analyzed by qPCR and flow cytometry analysis, respectively. LSEC-related functions of the differentiated cells were also examined. Results: We found that the gene expression of LSEC markers, such as LYVE1, was considerably increased by culturing human BM-MSCs with bone morphogenetic protein 4, fibroblast growth factor 8b, transforming growth factor-ß signal inhibitor, and cyclic AMP. Furthermore, the differentiated cells expressed LSEC marker proteins and clearly demonstrated LSEC-specific functions, such as the uptake of hyaluronic acid. Conclusions: Our result indicate that the functional LSEC-like cells were successfully generated from human BM-MSCs using our established protocol.

The use of infrared thermography for non-invasive detection of bleeding and musculoskeletal abnormalities in patients with hemophilia: an observational study.

BACKGROUND: In patients with hemophilia (PwH), bleeding often occurs in joints and muscles, and early detection of hemorrhage is important to prevent the onset and progression of mobility impairment. Complex-Image analysis such as ultrasonography, computed tomography, and magnetic resonance imaging are used to detect bleeding. On the other hand, no simple and rapid method to detect the active bleeding has been reported. Local inflammatory responses occur when blood leaks from damaged vessels, and the temperature at the site of active bleeding could be expected to increase in these circumstances, leading to an increase in surrounding skin temperature. Therefore, the purpose of this study was to investigate whether the measurement of skin temperature using infrared thermography (IRT) can be used as a diagnostic aid to detect active bleeding. METHODS: Fifteen PwH (from 6 to 82 years old) complaining of discomfort such as pain were examined. Thermal images were obtained simultaneously at the affected sides and comparable unaffected sides. The average skin temperature of the affected side and of the unaffected side were measured. The temperature differences were calculated by subtracting the average skin temperature at the unaffected side from the affected side. RESULTS: In eleven cases with active bleeding, the skin temperature at the affected side was more than 0.3 °C higher (0.3 °C to 1.4 °C) compared to the unaffected side. In two cases without active bleeding, there were no significant differences in skin temperature between the affected and unaffected sides. In two cases with previous rib or thumb bone fracture, the skin temperature at the affected side was 0.3 °C or 0.4 °C lower than that of the unaffected side, respectively. In two cases with active bleeding in which longitudinal evaluation was conducted, the difference in skin temperature decreased after hemostatic treatment. CONCLUSION: The analysis of skin temperature deference using IRT was a useful supportive tool to readily assess musculoskeletal abnormalities and bleeding in PwH as well as to determine the success of the hemostatic treatment.

Involvement of monocyte-derived extracellular vesicle-associated tissue factor activity in convallatoxin-induced hypercoagulability.

OBJECTIVES: Convallatoxin (CNT) is a natural cardiac glycoside extracted from lily of the valley ( Convallaria majalis ). Although it is empirically known to cause blood coagulation disorders, the underlying mechanism remains unclear. CNT exerts cytotoxicity and increases tissue factor (TF) expression in endothelial cells. However, the direct action of CNT on blood coagulation remains unclear. Therefore, herein, we investigated the effects of CNT on whole blood coagulation system and TF expression in monocytes. METHODS: Blood samples were collected from healthy volunteers to measure plasma thrombin-antithrombin complex (TAT) concentration using ELISA and to perform rotational thromboelastometry (ROTEM) and whole-blood extracellular vesicle (EV)-associated TF (EV-TF) analysis. The effects of CNT were also investigated using the monocytic human cell line THP-1. Quantitative real-time PCR and western blotting were performed, and PD98059, a mitogen-activated protein kinase (MAPK) inhibitor, was used to elucidate the action mechanism of CNT-mediated TF production. RESULTS: CNT treatment increased EV-TF activity, shortened the whole blood clotting time in rotational thromboelastometry analysis, and increased TAT levels, which is an index of thrombin generation. Furthermore, CNT increased TF mRNA expression in THP-1 cells and EV-TF activity in the cell culture supernatant. Therefore, CNT may induce a hypercoagulable state with thrombin generation, in which elevated EV-TF activity derived from monocytes might be involved. These procoagulant effects of CNT were reversed by PD98059, suggesting that CNT-induced TF production in monocytes might be mediated by the MAPK pathway. CONCLUSIONS: The findings of the present study have further clarified the procoagulant properties of CNT.

Myeloid Protease-Activated Receptor-2 Contributes to Influenza A Virus Pathology in Mice.

Background: Innate immune responses to influenza A virus (IAV) infection are initiated in part by toll-like receptor 3 (TLR3). TLR3-dependent signaling induces an antiviral immune response and an NFκB-dependent inflammatory response. Protease-activated receptor 2 (PAR2) inhibits the antiviral response and enhances the inflammatory response. PAR2 deficiency protected mice during IAV infection. However, the PAR2 expressing cell-types contributing to IAV pathology in mice and the mechanism by which PAR2 contributes to IAV infection is unknown. Methods: IAV infection was analyzed in global (Par2-/- ), myeloid (Par2fl/fl;LysMCre+) and lung epithelial cell (EpC) Par2 deficient (Par2fl/fl ;SPCCre+) mice and their respective controls (Par2+/+ and Par2fl/fl). In addition, the effect of PAR2 activation on polyinosinic-polycytidylic acid (poly I:C) activation of TLR3 was analyzed in bone marrow-derived macrophages (BMDM). Lastly, we determined the effect of PAR2 inhibition in wild-type (WT) mice. Results: After IAV infection, Par2-/- and mice with myeloid Par2 deficiency exhibited increased survival compared to infected controls. The improved survival was associated with reduced proinflammatory mediators and reduced cellular infiltration in bronchoalveolar lavage fluid (BALF) of Par2-/- and Par2fl/fl;LysMCre+ 3 days post infection (dpi) compared to infected control mice. Interestingly, Par2fl/fl;SPCCre+ mice showed no survival benefit compared to Par2fl/fl . In vitro studies showed that Par2-/- BMDM produced less IL6 and IL12p40 than Par2+/+ BMDM after poly I:C stimulation. In addition, activation of PAR2 on Par2+/+ BMDM increased poly I:C induction of IL6 and IL12p40 compared to poly I:C stimulation alone. Importantly, PAR2 inhibition prior to IAV infection protect WT mice. Conclusion: Global Par2 or myeloid cell but not lung EpC Par2 deficiency was associated with reduced BALF inflammatory markers and reduced IAV-induced mortality. Our study suggests that PAR2 may be a therapeutic target to reduce IAV pathology.

Role of tissue factor in delayed bone repair induced by diabetic state in mice.

BACKGROUND: Tissue factor (TF) is the primary activator of the extrinsic coagulation protease cascade. Although TF plays roles in various pathological states, such as thrombosis, inflammatory diseases, cancer, and atherosclerosis, its involvement in bone metabolism remains unknown. MATERIALS AND METHODS: The present study examined the roles of TF in delayed bone repair induced by a diabetic state in mice using wild-type (WT) and low TF-expressing (LTF) male mice. A diabetic state was induced by intraperitoneal injections of streptozotocin (STZ). RESULTS: A prolonged diabetic state significantly reduced total and trabecular bone mineral densities (BMD) as well as cortical bone thickness in WT and LTF mice; these BMD parameters were similar between WT and LTF mice treated with or without STZ. The diabetic state induced in WT mice delayed the repair of the femur following injury. The diabetic state induced in LTF mice was associated with further delays in bone repair. In in vitro experiments, TF significantly decreased receptor activator of nuclear factor-κB ligand-induced osteoclast formation and osteoclastogenic gene expression in RAW264.7 cells. However, it did not affect the gene expression levels of runt-related transcription factor 2 and osterix as well as alkaline phosphatase activity in mouse primary osteoblasts. CONCLUSION: Low TF state was associated with enhanced bone repair delay induced by diabetic state in mice. The TF-induced suppression of bone remodeling may be a contributing factor to the protective effects of TF against delayed bone repair in a diabetic state.

Development of alternative gene transfer techniques for ex vivo and in vivo gene therapy in a canine model.

INTRODUCTION: Gene therapy have recently attracted much attention as a curative therapeutic option for inherited single gene disorders such as hemophilia. Hemophilia is a hereditary bleeding disorder caused by the deficiency of clotting activity of factor VIII (FVIII) or factor IX (FIX), and gene therapy for hemophilia using viral vector have been vigorously investigated worldwide. Toward further advancement of gene therapy for hemophilia, we have previously developed and validated the efficacy of novel two types of gene transfer technologies using a mouse model of hemophilia A. Here we investigated the efficacy and safety of the technologies in canine model. Especially, validations of technical procedures of the gene transfers for dogs were focused. METHODS: Green fluorescence protein (GFP) gene were transduced into normal beagle dogs by ex vivo and in vivo gene transfer techniques. For ex vivo gene transfer, blood outgrowth endothelial cells (BOECs) derived from peripheral blood of normal dogs were transduced with GFP gene using lentivirus vector, propagated, fabricated as cell sheets, then implanted onto the omentum of the same dogs. For in vivo gene transfer, normal dogs were subjected to GFP gene transduction with non-viral piggyBac vector by liver-targeted hydrodynamic injections. RESULTS: No major adverse events were observed during the gene transfers in both gene transfer systems. As for ex vivo gene transfer, histological findings from the omental biopsy performed 4 weeks after implantation revealed the tube formation by implanted GFP-positive BOECs in the sub-adipose tissue layer without any inflammatory findings, and the detected GFP signals were maintained over 6 months. Regarding in vivo gene transfer, analyses of liver biopsy samples revealed more than 90% of liver cells were positive for GFP signals in the injected liver lobes 1 week after gene transfers, then the signals gradually declined overtime. CONCLUSIONS: Two types of gene transfer techniques were successfully applied to a canine model, and the transduced gene expressions persisted for a long term. Toward clinical application for hemophilia patients, practical assessments of therapeutic efficacy of these techniques will need to be performed using a dog model of hemophilia and FVIII (or FIX) gene.

Convallatoxin, the primary cardiac glycoside in lily of the valley (Convallaria majalis), induces tissue factor expression in endothelial cells.

BACKGROUND: Convallotoxin (CNT), present in lily of the valley (Convallaria majalis), is a toxin that causes food poisoning among humans and companion animals. Although various symptoms of CNT poisoning have been well described, hypercoagulability owing to CNT is only empirically known among some veterinarians, and the underlying mechanism remains to be elucidated. CNT exerts cytotoxic effects on endothelial cells. OBJECTIVES: This study aimed to determine whether CNT induces the expression of tissue factor (TF), a potent initiator of the extrinsic coagulation cascade, in endothelial cells and leads to a hypercoagulable state. METHODS: Human umbilical vein endothelial cells (HUVECs) were used for in vitro experiments. HUVECs were treated with or without CNT (50 and 100 nM) for 4 h. Phosphate-buffered saline was used as a control. Cell viability was determined using the WST-8 assay. Quantitative real-time polymerase chain reaction was performed to determine TF mRNA expression. TF protein expression was observed using a laser scanning confocal microscope. RESULTS: The viability of HUVECs significantly reduced after CNT treatment compared with that of non-treated cells (p < 0.05). Moreover, a significant increase in TF mRNA and protein expression was observed after 4 h of CNT treatment. CNT elicited these effects in a dose-dependent manner. CONCLUSIONS: TF expression induced by CNT in endothelial cells can contribute to the development of a hypercoagulable state. The present study partially revealed the mechanisms underlying the CNT-induced hypercoagulable state. The findings can contribute to the development of a novel therapy for lily of the valley poisoning.

Role of plasminogen activator inhibitor-1 in muscle wasting induced by a diabetic state in female mice.

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.

Effects of fluid flow shear stress to mouse muscle cells on the bone actions of muscle cell-derived extracellular vesicless.

The interactions between skeletal muscle and bone have been recently noted, and muscle-derived humoral factors related to bone metabolism play crucial roles in the muscle/bone relationships. We previously reported that extracellular vesicles from mouse muscle C2C12 cells (Myo-EVs) suppress osteoclast formation in mice. Although mechanical stress is included in extrinsic factors which are important for both muscle and bone, the detailed roles of mechanical stress in the muscle/bone interactions have still remained unknown. In present study, we examined the effects of fluid flow shear stress (FFSS) to C2C12 cells on the physiological actions of muscle cell-derived EV. Applying FFSS to C2C12 cells significantly enhanced muscle cell-derived EV-suppressed osteoclast formation and several osteoclast-related gene levels in mouse bone marrow cells in the presence of receptor activator nuclear factor κB ligand (RANKL). Moreover, FFSS to C2C12 cells significantly enhanced muscle cell-derived EV-suppressed mitochondria biogenesis genes during osteoclast formation with RANKL treatment. In addition, FFSS to C2C12 cells significantly enhanced muscle cell-derived EV-suppressed osteoclast formation and several osteoclast-related gene levels in Raw264.7 cells in the presence of RANKL. Small RNA-seq-analysis showed that FFSS elevated the expression of miR196a-5p and miR155-5p with the suppressive actions of osteoclast formation and low expression in mouse bone cells. On the other hand, muscle cell-derived EVs with or without FFSS to C2C12 cells did not affect the expression of osteogenic genes, alkaline phosphatase activity and mineralization in mouse osteoblasts. In conclusion, we first showed that FFSS to C2C12 cells enhances the suppressive effects of muscle cell-derived EVs on osteoclast formation in mouse cells. Muscle cell-derived EVs might be partly involved in the effects of mechanical stress on the muscle/bone relationships.

MicroRNA-196a-5p in Extracellular Vesicles Secreted from Myoblasts Suppresses Osteoclast-like Cell Formation in Mouse Cells.

Muscle/bone interaction has been recently noted. Extracellular vesicles (EVs) play a vital role in physiological and pathophysiological processes by transferring microRNA (miRNA) to distant tissues. We previously reported that EVs secreted from C2C12 myoblasts (Myo-EVs) suppress osteoclast differentiation. In the present study, we identified 4 miRNAs in Myo-EVs that suppressed osteoclast-like cell formation in Raw264.7 cells using small RNA sequencing analysis. Among them, miR-196a-5p expression was higher in C2C12 cells compared to mouse osteoblasts and bone marrow cells. Transfection of miR-196a-5p mimic suppressed the mRNA levels of osteoclast-related genes and mitochondrial energy metabolism induced by receptor activator of nuclear factor-κB ligand in Raw264.7 cells. In contrast, miR-196a-5p mimic enhanced osteoblastic differentiation in ST-2 cells and MC3T3-E1 cells. In conclusion, we demonstrated that miR-196-5p suppresses osteoclast-like cell formation and mitochondrial energy metabolism in mouse cells, suggesting that it might be a crucial factor for muscle/bone interaction via EVs.

PAR1 regulation of CXCL1 expression and neutrophil recruitment to the lung in mice infected with influenza A virus.

BACKGROUND: Protease-activated receptor 1 (PAR1) is expressed in various immune cells and in the lung. We showed that PAR1 plays a role in Coxsackievirus B3 infection by enhancing toll-like receptor 3-dependent interferon- ß expression in cardiac fibroblasts. OBJECTIVES: We investigated the role of PAR1 in a mouse model of influenza A virus (IAV) infection. METHODS: We used mice with either a global deficiency of PAR1, cell type-specific deficiencies of PAR1, or mutation of PAR1 at the R41 or R46 cleavage sites. RESULTS: PAR1-deficient mice had increased CXCL1 expression in the lung, increased neutrophil recruitment, increased protein levels in the bronchoalveolar lavage fluid, and increased mortality after IAV infection compared with control mice infected with IAV. Results from mice with cell type-specific deletion of PAR1 indicated that PAR1 expression by hematopoietic cells suppressed CXCL1 expression, whereas PAR1 expression by endothelial cells enhanced CXCL1 expression in response to IAV infection. PAR1 activation also enhanced polyinosinic:polycytodylic acid induction of interleukin-8 in a human endothelial cell line. Mutation of the R46 cleavage site of PAR1 was associated with increased CXCL1 expression in the lung in response to IAV infection, which suggested that R46 signaling suppresses CXCL1 expression. CONCLUSIONS: These results indicate that PAR1 expression by different cell types and activation by different proteases modulates the immune response during IAV infection.

Extracellular vesicles secreted from mouse muscle cells suppress osteoclast formation: Roles of mitochondrial energy metabolism.

Recent reports have described the interactions of muscle and bone. Various muscle-derived humoral factors, known as myokines, affect bone. Although extracellular vesicles (EVs) play a vital role in physiological and pathophysiological processes by transferring their contents to distant tissues during bone metabolism, the roles of EVs in the muscle-bone interactions remain unknown. In the present study, we investigated the effects of EVs secreted from mouse muscle C2C12 cells on mouse bone cells and mitochondrial biogenesis. EVs secreted from C2C12 cells (Myo-EVs) were isolated from the conditioned medium of C2C12 cells by ultracentrifugation. Myo-EVs suppressed osteoclast formation as well as the expression of tartrate-resistant acid phosphatase, cathepsin K, nuclear factor of activated T-cells cytoplasmic 1 and dendritic cell-specific transmembrane protein induced by receptor activator of nuclear factor κB ligand (RANKL) in mouse bone marrow cells and preosteoclastic Raw264.7 cells. Moreover, Myo-EVs suppressed oxygen consumption and mRNA expression of the mitochondrial biogenesis markers enhanced by RANKL in these cells. However, Myo-EVs did not affect the phenotypes or mitochondrial biogenesis of mouse primary osteoblasts. In conclusion, the present study showed for the first time that Myo-EVs suppress osteoclast formation and mitochondrial energy metabolism in mouse bone marrow and Raw264.7 cells. EVs secreted from skeletal muscles might be a crucial mediator of muscle-bone interactions.

Adipose Tissue-Derived Stem Cell Sheet Improves Glucose Metabolism in Obese Mice.

Previous studies indicate that the administration of adipose tissue-derived stem cells (ADSCs) through the venous route improves insulin resistance partly through a reduction in the proinflammatory cytokines in diabetic animals. However, the effects of ADSC sheet transplantation for the treatment of diabetes and obesity still remained unknown. In this study, we investigated the effects of ADSC sheet transplantation into the subcutaneous sites on the diabetic state of mice fed high-fat and high-sucrose diet (HF/HSD). ADSCs were isolated and propagated from subcutaneous adipose tissues of non-diabetic intact mice. We used the thermoresponsive designated cell culture dishes to fabricate ADSC cell sheets. ADSC sheet transplantation into the subcutaneous sites significantly improved glucose intolerance induced by HF/HSD in mice. ADSC-conditioned medium (CM) augmented the phosphorylation of Akt with or without insulin in mouse C2C12 myotubes and mouse 3T3-L1 adipocytes. Plasma adiponectin and tumor necrosis factor-α (TNF-α) levels were significantly increased and decreased by ADSC sheet transplantation in mice with or without HF/HSD, respectively. Moreover, ADSC sheet enhanced adiponectin expression in the subcutaneous adipose tissues in HF/HSD-fed mice, whereas it reduced TNF-α expression in the visceral adipose tissues. ADSC-CM enhanced and reduced the protein levels of adiponectin and TNF-α in 3T3-L1 adipocytes, respectively. In conclusion, we first revealed that ADSC sheet transplantation into the subcutaneous sites improves glucose intolerance in mice fed with HF/HSD. Changes of adiponectin and TNF-α production from the host adipose tissues might be involved in the effects of ADSC sheet on glucose metabolism in mice. ADSC sheet transplantation therapy may be a novel clinical application for diabetes.

Protease-activated receptor 4 protects mice from Coxsackievirus B3 and H1N1 influenza A virus infection.

PAR4 is expressed by a variety of cells, including platelets, cardiac, lung and immune cells. We investigated the contribution of PAR4 to viral infections of the heart and lung. Toll-like receptor (TLR) 3-dependent immune responses were analyzed after co-stimulation of PAR4 in murine bone-marrow derived macrophages, embryonic fibroblasts and embryonic cardiomyocytes. In addition, we analyzed Coxsackievirus B3 (CVB3) or H1N1 influenza A virus (H1N1 IAV) infection of PAR4-/- (ΔPAR4) and wild-type (WT) mice. Lastly, we investigated the effect of platelet inhibition on H1N1 IAV infection. In vitro experiments revealed that PAR4 stimulation enhances the expression of TLR3-dependent CXCL10 expression and decreases TLR3-dependent NFκB-mediated proinflammatory gene expression. Furthermore, CVB3-infected ΔPAR4 mice exhibited a decreased anti-viral response and increased viral genomes in the heart leading to more pronounced CVB3 myocarditis compared to WT mice. Similarly, H1N1 IAV-infected ΔPAR4 mice had increased immune cell numbers and inflammatory mediators in the lung, and increased mortality compared with infected WT controls. The study showed that PAR4 protects mice from viral infections of the heart and lung.