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 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.

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.

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.

Plasminogen activator inhibitor-1 deficiency suppresses osteoblastic differentiation of mesenchymal stem cells in mice.

Plasminogen activator inhibitor-1 (PAI-1) is known as an inhibitor of fibrinolytic system. Previous studies suggest that PAI-1 is involved in the pathogenesis of osteoporosis induced by ovariectomy, diabetes, and glucocorticoid excess in mice. However, the roles of PAI-1 in early-stage osteogenic differentiation have remained unknown. In the current study, we investigated the roles of PAI-1 in osteoblastic differentiation of mesenchymal stem cells (MSCs) using wild-type (WT) and PAI-1-deficient (PAI-1 KO) mice. PAI-1 mRNA levels were increased with time during osteoblastic differentiation of MSCs or mesenchymal ST-2 cells. However, the increased PAI-1 levels declined at the mineralization phase in the experiment using MC3T3-E1 cells. PAI-1 deficiency significantly blunted the expression of osteogenic gene, such as osterix and alkaline phosphatase enhanced by bone morphogenetic protein (BMP)-2 in bone marrow-derived MSCs (BM-MSCs), adipose-tissue-derived MSCs (AD-MSCs), and bone marrow stromal cells of mice. Moreover, a reduction in endogenous PAI-1 levels by small interfering RNA significantly suppressed the expression of osteogenic gene in ST-2 cells. Plasmin did not affect osteoblastic differentiation of AD-MSCs induced by BMP-2 with or without PAI-1 deficiency. PAI-1 deficiency and a reduction in endogenous PAI-1 levels did not affect the phosphorylations of receptor-specific Smads by BMP-2 and transforming growth factor-ß in AD-MSCs and ST-2 cells, respectively. In conclusion, we first showed that PAI-1 is crucial for the differentiation of MSCs into osteoblasts in mice.

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.

Enhancement of Tissue Factor Expression in Monocyte-Derived Dendritic Cells by Pentraxin 3 and Its Modulation by C1 Esterase Inhibitor.

BACKGROUND: We have previously shown that human monocyte-derived dendritic cells (moDCs) may participate in immune system-mediated hypercoagulable state through enhanced tissue factor (TF) expression and that the complement system may be involved in this process. OBJECTIVES: The aim of this study was to explore the role of pentraxin 3 (PTX3) and the complement system in enhanced TF expression in moDCs. METHODS: moDCs were generated from isolated human monocytes. PTX3 levels in whole human blood supplemented with moDCs were determined after lipopolysaccharide (LPS) stimulation. PTX3 release by the generated moDCs upon LPS stimulation was also assessed. The effect of PTX3 on whole blood coagulation was investigated using thromboelastometric analysis. TF expression in stationary moDCs treated with LPS and/or PTX3 was determined by measuring TF activity. The effect of complement inhibitors on TF activity in moDCs treated with LPS and/or PTX3 under low-shear conditions was evaluated. RESULTS: PTX3 levels were higher in whole blood supplemented with moDCs than in the presence of monocytes and were further elevated by LPS stimulation. PTX3 release from generated moDCs was also increased by LPS stimulation. PTX3 reduced whole blood coagulation time in a dose-dependent manner. However, PTX3 did not increase TF expression in stationary moDCs. Under low-shear conditions, PTX3 increased TF expression in moDCs. C1 esterase inhibitor (C1-inh) suppressed this effect. CONCLUSIONS: PTX3 might have a thrombophilic activity and enhance TF expression in moDCs under low-shear conditions. Furthermore, suppression of moDC-associated hypercoagulability by C1-inh might be partly ascribed to its inhibitory effect on PTX3.

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

Objectives: Interleukin (IL)-1ß 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-1ß on the chondrocytes using wild-type and PAI-1-deficient mice.Results: IL-1ß significantly elevated PAI-1 mRNA levels in the chondrocytes from wild-type mice. PAI-1 deficiency significantly blunted the mRNA levels of TGF-ß and IL-6 enhanced by IL-1ß 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-1ß in the chondrocytes. In addition, PAI-1 deficiency significantly reversed type II collagen mRNA levels suppressed by IL-1ß 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-1ß in murine chondrocytes. PAI-1 might be crucial for the cartilage matrix degradation and the impaired chondrogenesis by IL-1ß in mice.

Protease-activated receptor 1 activation enhances doxorubicin-induced cardiotoxicity.

OBJECTIVE: The anti-cancer anthracycline drug Doxorubicin (Dox) causes cardiotoxicity. We investigated the role of protease-activated receptor 1 (PAR-1) in Dox-induced cardiotoxicity. METHODS AND RESULTS: In vitro experiments revealed that PAR-1 enhanced Dox-induced mitochondrial dysfunction, reactive oxygen species and cell death of cardiac myocytes and cardiac fibroblasts. The contribution of PAR-1 to Dox-induced cardiotoxicity was investigated by subjecting PAR-1-/- mice and PAR-1+/+ mice to acute and chronic exposure to Dox. Heart function was measured by echocardiography. PAR-1-/- mice exhibited significant less cardiac injury and dysfunction compared to PAR-1+/+ mice after acute and chronic Dox administration. PAR-1-/- mice had reduced levels of nitrotyrosine, apoptosis and inflammation in their heart compared to PAR-1+/+ mice. Furthermore, inhibition of PAR-1 in wild-type mice with vorapaxar significantly reduced the acute Dox-induced cardiotoxicity. CONCLUSION: Our results indicate that activation of PAR-1 contributes to Dox-induced cardiotoxicity. Inhibition of PAR-1 may be a new approach to reduce Dox-induced cardiotoxicity in cancer patients.

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.

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).

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.

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.

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

BACKGROUND: Subchondral 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. METHODS: Wild type (WT) and PAI-1-deficient (KO) mice were ovariectomized (OVX), and then destabilization of the medial meniscus (DMM) surgery was performed. RESULTS: DMM 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-1ß in mouse primary osteoblasts, although it did not affect osteoblast differentiation. Moreover, PAI-1 deficiency significantly increased osteoclast formation from mouse bone marrow cells. CONCLUSION: We 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.

Hepatocyte tissue factor contributes to the hypercoagulable state in a mouse model of chronic liver injury.

BACKGROUND & AIMS: Patients with chronic liver disease and cirrhosis have a dysregulated coagulation system and are prone to thrombosis. The basis for this hypercoagulable state is not completely understood. Tissue factor (TF) is the primary initiator of coagulation in vivo. Patients with cirrhosis have increased TF activity in white blood cells and circulating microparticles. The aim of our study was to determine the contribution of TF to the hypercoagulable state in a mouse model of chronic liver injury. METHODS: We measured levels of TF activity in the liver, white blood cells and circulating microparticles, and a marker of activation of coagulation (thrombin-antithrombin complexes (TATc)) in the plasma of mice subjected to bile duct ligation for 12days. We used wild-type mice, mice with a global TF deficiency (low TF mice), and mice deficient for TF in either myeloid cells (TF(flox/flox),LysMCre mice) or in hepatocytes (TF(flox/flox),AlbCre). RESULTS: Wild-type mice with liver injury had increased levels of white blood cell, microparticle TF activity and TATc compared to sham mice. Low TF mice and mice lacking TF in hepatocytes had reduced levels of TF in the liver and in microparticles and exhibited reduced activation of coagulation without a change in liver fibrosis. In contrast, mice lacking TF in myeloid cells had reduced white blood cell TF but no change in microparticle TF activity or TATc. CONCLUSIONS: Hepatocyte TF activates coagulation in a mouse model of chronic liver injury. TF may contribute to the hypercoagulable state associated with chronic liver diseases in patients.

Microvesicle Tissue Factor Activity and Interleukin-8 Levels are Associated with Mortality in Patients with Influenza A/H1N1 Infection.

OBJECTIVES: To identify plasma biomarkers that can be early predictors of mortality in critically ill patients with primary influenza A/H1N1. DESIGN: A prospective, multicenter, case-cohort pilot study. SETTING: Three academic ICUs. PATIENTS: Fifteen patients with primary influenza A/H1N1 that included seven survivors and eight nonsurvivors. For comparison, age- and gender-matched healthy controls (n = 27) were also studied. INTERVENTIONS: Plasma was prepared from whole blood drawn on ICU admission in patients with influenza (ICU day 1). Microvesicle tissue factor activity, thrombin-antithrombin complexes, and D-dimers were measured as procoagulant markers and markers of activation of coagulation. Plasma cytokine levels were measured on the same blood samples in a subset of 12 patients with influenza using the Luminex Multi-Analyte Profiling system (Luminex Corporation, DeSoto, TX). Patients were followed up for the primary outcome of 28-day mortality. MEASUREMENTS AND MAIN RESULTS: The average admission Acute Physiology and Chronic Health Evaluation II score of the patients was 25.5 ± 9.3, 60% of patients had shock, and the 28-day mortality rate was 53.3% (n = 8/15). Patients with influenza had dysregulated indices of coagulation and inflammation compared with controls. Among the markers of activation of coagulation measured on ICU day 1, only increased microvesicle tissue factor activity was significantly associated with subsequent influenza-related mortality (5.6 ± 1.2 pg/mL in nonsurvivors vs 1.8 ± 0.8 pg/mL in survivors; p < 0.05). Interleukin-8 was significantly higher in nonsurvivors compared with survivors (71.8 ± 29.1 pg/mL, n = 5 vs 17.3 ± 3.7 pg/mL, n = 7; p < 0.05). In addition, microvesicle tissue factor activity and interleukin-8 levels were significantly and positively correlated (r = 0.60; p = 0.003). Other cytokines, thrombin-antithrombin complexes, and D-dimer were not different between nonsurvivors and survivors and did not correlate with illness severity or mortality. CONCLUSIONS: This study identifies an association between plasma interleukin-8 and microvesicle tissue factor activity measured on admission in patients with severe, primary influenza A/H1N1 infection and subsequent mortality. Thus, these biomarkers may serve as very early prognostic markers for patients with influenza A/H1N1.

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.

Tissue factor triggers procoagulation in transplanted mesenchymal stem cells leading to thromboembolism.

Mesenchymal stem cells (MSCs) have shown extreme clinical promise as a therapeutic regenerative system in the treatment of numerous types of diseases. A recent report, however, documented lethal pulmonary thromboembolism in a patient following the administration of adipose-derived MSCs (ADSCs). In our study, we designed experiments to examine the role of tissue factor (TF), which is highly expressed at the level of mRNA and localized to the cell surface of cultured MSCs, as a triggering factor in the procoagulative cascade activated by infused MSCs. A high mortality rate of ~85% in mice was documented following intravenous infusion of mouse ADSCs within 24 h due to the observation of pulmonary embolism. Rotation thromboelastometry and plasma clotting assay demonstrated significant procoagulation by the cultured mouse ADSCs, and preconditioning of ADSCs with an anti-TF antibody or usage of factor VII deficient plasma in the assay successfully suppressed the procoagulant properties. These properties were also observed in human ADSCs, and could be suppressed by recombinant human thrombomodulin. In uncultured mouse adipose-derived cells (ADCs), the TF-triggered procoagulant activity was not observed and all mice infused with these uncultured ADCs survived after 24 h. This clearly demonstrated that the process of culturing cells plays a critical role in sensitizing these cells as a procoagulator through the induction of TF expression. Our results would recommend that clinical applications of MSCs to inhibit TF activity using anti-coagulant agents or genetic approaches to maximize clinical benefit to the patients.

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.