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).

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.

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.

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.

Endothelial miR-30c suppresses tumor growth via inhibition of TGF-ß-induced Serpine1.

In tumors, extravascular fibrin forms provisional scaffolds for endothelial cell (EC) growth and motility during angiogenesis. We report that fibrin-mediated angiogenesis was inhibited and tumor growth delayed following postnatal deletion of Tgfbr2 in the endothelium of Cdh5-CreERT2 Tgfbr2fl/fl mice (Tgfbr2iECKO mice). ECs from Tgfbr2iECKO mice failed to upregulate the fibrinolysis inhibitor plasminogen activator inhibitor 1 (Serpine1, also known as PAI-1), due in part to uncoupled TGF-ß-mediated suppression of miR-30c. Bypassing TGF-ß signaling with vascular tropic nanoparticles that deliver miR-30c antagomiRs promoted PAI-1-dependent tumor growth and increased fibrin abundance, whereas miR-30c mimics inhibited tumor growth and promoted vascular-directed fibrinolysis in vivo. Using single-cell RNA-Seq and a NanoString miRNA array, we also found that subtypes of ECs in tumors showed spectrums of Serpine1 and miR-30c expression levels, suggesting functional diversity in ECs at the level of individual cells; indeed, fresh EC isolates from lung and mammary tumor models had differential abilities to degrade fibrin and launch new vessel sprouts, a finding that was linked to their inverse expression patterns of miR-30c and Serpine1 (i.e., miR-30chi Serpine1lo ECs were poorly angiogenic and miR-30clo Serpine1hi ECs were highly angiogenic). Thus, by balancing Serpine1 expression in ECs downstream of TGF-ß, miR-30c functions as a tumor suppressor in the tumor microenvironment through its ability to promote fibrin degradation and inhibit blood vessel formation.

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.

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.

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.

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.

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-ß (TGF-ß) elevated by bone injury. TGF-ß 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-ß, and SDF-1 at the damaged site during bone repair 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.

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.

Protease-Activated Receptor 1 Enhances Poly I:C Induction of the Antiviral Response in Macrophages and Mice.

The coagulation cascade is activated during viral infections as part of the host defense system. Coagulation proteases activate cells by cleavage of protease-activated receptors (PARs). Recently, we reported that the activation of PAR-1 enhanced interferon (IFN)ß and CXCL10 expression in cardiac fibroblasts and in the hearts of mice infected with Coxsackievirus B3. In this study, we used the double-stranded RNA mimetic polyinosinic:polycytidylic acid (poly I:C) to induce an antiviral response in macrophages and mice. Activation of PAR-1 enhanced poly I:C induction of IFNß and CXCL10 expression in the murine macrophage cell line RAW264.7, bone-marrow derived mouse macrophages (BMM) and mouse splenocytes. Next, poly I:C was used to induce a type I IFN innate immune response in the spleen and plasma of wild-type (WT) and PAR-1-/- mice. We found that poly I:C treated PAR-1-/- mice and WT mice given the thrombin inhibitor dabigatran etexilate exhibited significantly less IFNß and CXCL10 expression in the spleen and plasma than WT mice. These studies suggest that thrombin activation of PAR-1 contributes to the antiviral response in mice.

Therapeutic approaches for treating hemophilia A using embryonic stem cells.

Hemophilia A is an X-linked rescessive bleeding disorder that results from F8 gene aberrations. Previously, we established embryonic stem (ES) cells (tet-226aa/N6-Ainv18) that secrete human factor VIII (hFVIII) by introducing the human F8 gene in mouse Ainv18 ES cells. Here, we explored the potential of cell transplantation therapy for hemophilia A using the ES cells. Transplant tet-226aa/N6-Ainv18 ES cells were injected into the spleens of severe combined immunodeficiency (SCID) mice, carbon tetrachloride (CCl4)-pretreated wild-type mice, and CCl4-pretreated hemophilia A mice. F8 expression was induced by doxycycline in drinking water, and hFVIII-antigen production was assessed in all cell transplantation experiments. Injecting the ES cells into SCID mice resulted in an enhanced expression of the hFVIII antigen; however, teratoma generation was confirmed in the spleen. Transplantation of ES cells into wild-type mice after CCl4-induced liver injury facilitated survival and engraftment of transplanted cells without teratoma formation, resulting in hFVIII production in the plasma. Although CCl4 was lethal to most hemophilia A mice, therapeutic levels of FVIII activity, as well as the hFVIII antigen, were detected in surviving hemophilia A mice after cell transplantation. Immunolocalization results for hFVIII suggested that transplanted ES cells might be engrafted at the periportal area in the liver. Although the development of a safer induction method for liver regeneration is required, our results suggested the potential for developing an effective ES-cell transplantation therapeutic model for treating hemophilia A in the future.

Tissue Factor and Atherothrombosis.

Atherosclerosis is a progressive disease characterized by the accumulation of lipids in medium to large sized arteries. Atherothrombosis is a term used to describe formation of a thrombus after rupture of an atherosclerotic plaque. Thrombosis can lead to myocardial infarction and stroke. Risk factors for atherosclerosis include hyperlipidemia, diabetes, smoking and hypertension all of which increase tissue factor (TF) expression. High levels of TF are present in atherosclerotic plaques due to expression by macrophages and vascular smooth muscle cells and the presence of cell-derived TF-positive microvesicles (MVs). In addition, hyperlipidemia leads to the formation of oxidized LDL, which induces TF expression in circulating monocytes and the release of TF-positive MVs. The major source of TF that drives thrombosis after plaque rupture is TF within the plaque. However, TF in the blood on monocytes and MVs may also contribute the thrombosis. Inhibition of the TF/factor VIIa complex is unlikely to be an effective strategy to reduce atherothrombosis due the essential role of the complex in hemostasis. However, selective blockade of pathologic TF without affecting protective TF may be effective in reducing atherothrombosis. For instance, statins have been shown to reduce TF expression in the plaque and in circulating monocytes, which would be expected to reduce thrombosis. Further studies are needed to determine safe strategies to reduce pathologic TF expression and atherothrombosis.