The Role of Zinc Homeostasis in the Prevention of Diabetes Mellitus and Cardiovascular Diseases.

Zinc is an essential micronutrient for human health and is involved in various biological functions, such as growth, metabolism, and immune function. In recent years, research on intracellular zinc dynamics has progressed, and it has become clear that zinc transporters strictly control intracellular zinc localization, zinc regulates the functions of various proteins and signal transduction pathways as a second messenger similar to calcium ions, and intracellular zinc dyshomeostasis is associated with impaired insulin synthesis, secretion, sensitivity, lipid metabolism, and vascular function. Numerous animal and human studies have shown that zinc deficiency may be associated with the risk factors for diabetes and cardiovascular diseases (CVDs) and zinc administration might be beneficial for the prevention and treatment of these diseases. Therefore, an understanding of zinc biology may help the establishment of novel strategies for the prevention and treatment of diabetes and CVDs. This review will summarize the current knowledge on the role of zinc homeostasis in the pathogenesis of diabetes and atherosclerosis and will discuss the potential of zinc in the prevention of these diseases.

Once-weekly teriparatide reduces serum sclerostin levels in postmenopausal women with osteoprosis.

Once-weekly teriparatide treatment is widely used in the treatment of osteoporosis in Japan but the mechanisms causing the increase in bone mineral density (BMD) of the lumbar spine remain unknown. METHODS: This prospective study examined the effects of once-weekly teriparatide treatment on the serum levels of sclerostin, osteocalcin, and bone formation markers as well as BMD of the lumbar spine and femoral neck in 32 postmenopausal women with osteoporosis. RESULTS: The mean age of subjects was 76.3 ± 7.0 years old. Teriparatide significantly reduced serum sclerostin levels at 12 and 18 months in postmenopausal women with osteoporosis, and significantly increased serum osteocalcin levels at 3,12 and 18 months and PINP levels at 1 and 3 months, respectively. Teriparatide treatment significantly increased BMD of the lumbar spine at 6, 12, and 18 months, but did not affect BMD of the femoral neck. Examination of the relationships between percent changes in bone metabolic indices and BMD of the lumbar spine during the teriparatide treatment showed serum sclerostin changes at 3 months were negatively correlated with BMD changes of the lumbar spine at 6, 12, and 18 months. Serum osteocalcin changes were not correlated with BMD changes in the lumbar spine at 12 months. CONCLUSIONS: The present study showed that once-weekly teriparatide treatment reduced serum sclerostin levels in postmenopausal women with osteoporosis. The effects of teriparatide on sclerostin may be associated with the response of the BMD of the lumbar spine.

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.

Role of plasminogen activator inhibitor-1 in glucocorticoid-induced muscle change in mice.

We recently revealed that plasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor, is involved in diabetes, osteoporosis and muscle wasting induced by glucocorticoid (GC) treatment in mice. In the present study, we investigated the detailed mechanisms by which GC induces muscle wasting through PAI-1 in vivo and in vitro. PAI-1 deficiency suppressed the mRNA levels of atrogin1 and muscle RING-Finger Protein 1 (MuRF1), ubiquitin ligases leading to muscle degradation, elevated by GC treatment in the gastrocnemius muscle of mice. In vitro study revealed that active PAI-1 treatment augmented the increase in atrogin1 mRNA levels enhanced by dexamethasone (Dex) in mouse myoblastic C2C12 cells. Moreover, a reduction in endogenous PAI-1 level by siRNA suppressed the mRNA levels of atrogin1 and MuRF1 enhanced by Dex in C2C12 cells. In contrast, a reduction in endogenous PAI-1 levels and active PAI-1 did not affect the phosphorylations of Akt and p70S6 kinase nor myogenic differentiation with or without Dex in C2C12 cells. In addition, PAI-1 deficiency blunted IGF-1 mRNA levels decreased by GC treatment in the gastrocnemius muscle of mice, although neither active PAI-1 nor a reduction in endogenous PAI-1 levels affected the levels of IGF-1 mRNA in C2C12 cells in the presence of Dex. In conclusion, our data suggest that paracrine PAI-1 is involved in GC-induced muscle wasting through the enhancement of muscle degradation in mice.

Vitamin D deficiency aggravates diabetes-induced muscle wasting in female mice.

We recently reported that vitamin D deficiency aggravates diabetic bone loss in mice. Although vitamin D affects both muscle and bone, the role of the vitamin D state in diabetic muscle loss and muscle-bone relationships remains unclear. In the present study, we examined the effects of vitamin D deficiency on muscle mass, muscle differentiation and muscle-derived humoral factors linking muscle to bone in diabetic female mice. Diabetes was induced in mice by streptozotocin (STZ) injection after feeding with a normal or vitamin D-deficient diet for 6 weeks. Quantitative computed tomography analysis showed that tibial muscle mass was significantly decreased in diabetic mice compared with control mice 4 weeks after induction of diabetes. Vitamin D deficiency accelerated muscle loss in diabetic mice. Vitamin D deficiency augmented the decreases in Pax7 mRNA levels and the increases in muscle RING-Finger Protein-1 and atrogin-1 mRNA levels induced by diabetes in the gastrocnemius muscle of mice. Moreover, vitamin D deficiency decreased the mRNA levels of insulin-like growth factor-1, fibroblast growth factor-2 and osteoglycin in muscle of diabetic mice. In conclusion, we demonstrated that vitamin D deficiency aggravates muscle loss induced by diabetes in female mice. Vitamin D may exert significant effects on the maintenance of the musculoskeletal system partly through the muscle-bone relationships in diabetic state.

The vestibular system is critical for the changes in muscle and bone induced by hypergravity in mice.

Gravity changes concurrently affect muscle and bone as well as induce alterations in vestibular signals. However, the role of vestibular signals in the changes in muscle and bone induced by gravity changes remains unknown. We therefore investigated the effects of vestibular lesions (VL) on the changes in muscle and bone induced by 3 g hypergravity for 4 weeks in C57BL/6J mice. Quantitative computed tomography analysis revealed that hypergravity increased muscle mass surrounding the tibia and trabecular bone mineral content, adjusting for body weight in mice. Hypergravity did not affect cortical bone and fat masses surrounding the tibia. Vestibular lesions blunted the increases in muscle and bone masses induced by hypergravity. Histological analysis showed that hypergravity elevated the cross-sectional area of myofiber in the soleus muscle. The mRNA levels of myogenic genes such as MyoD, Myf6, and myogenin in the soleus muscle were elevated in mice exposed to hypergravity. Vestibular lesions attenuated myofiber size and the mRNA levels of myogenic differentiation markers enhanced by hypergravity in the soleus muscle. Propranolol, a ß-blocker, antagonized the changes in muscle induced by hypergravity. In conclusion, this study is the first to demonstrate that gravity changes affect muscle and bone through vestibular signals and subsequent sympathetic outflow in mice.

Novel roles of FKBP5 in muscle alteration induced by gravity change in mice.

Skeletal muscle hypertrophy and wasting are induced by hypergravity and microgravity, respectively. However, the mechanisms by which gravity change regulates muscle mass still remain unclear. We previously reported that hypergravity increases muscle mass via the vestibular system in mice. In this study, we performed comparative DNA microarray analysis of the soleus muscle from mice kept in 1 or 3 g environments with or without vestibular lesions. Mice were kept in 1 g or 3 g environment for 4 weeks by using a centrifuge 14 days after surgical bilateral vestibular lesions. FKBP5 was extracted as a gene whose expression was enhanced by hypergravity through the vestibular system. Stable FKBP5 overexpression increased the phosphorylations of Akt and p70 S6 kinase (muscle protein synthesis pathway) and myosin heavy chain, a myotube gene, mRNA level in mouse myoblastic C2C12 cells, although it reduced the mRNA levels of atrogin-1 and MuRF1, muscle protein degradation-related genes. In conclusion, we first showed that FKBP5 is induced by hypergravity through the vestibular system in anti-gravity muscle of mice. Our data suggest that FKBP5 might increase muscle mass through the enhancements of muscle protein synthesis and myotube differentiation as well as an inhibition of muscle protein degradation in mice.

Reevaluation of antithrombotic fruits and vegetables: great variation between varieties.

In the quest for prevention of atherothrombotic diseases, an antithrombotic diet may offer a promising approach. The major stumbling block in finding an effective diet is the lack of pathophysiological relevant techniques to detect potential antithrombotic effects of various diet components. Platelet function and coagulation/fibrinolysis tests currently in use do not allow assessment of global thrombotic status and their value in screening diet-components for antithrombotic effects. Recently, we combined the point-of-care shear-induced ex vivo thrombosis test (Global Thrombosis Test-GTT) with the Flow-mediated Vasodilation (FMV) in vivo test and found that the combination improved the assessment of thrombotic status in humans and could be used for screening diet-components for antithrombotic effects. In the present experiments, a combination of GTT, hemostatometry, laser-induced thrombosis tests and FMV were employed for screening. The results show that the overall antithrombotic effect is determined by the effect on thrombus formation and endogenous thrombolytic activities. This study showed a great variation in the observed antithrombotic effect between the tested varieties. Antithrombotic activities were independent from polyphenolic content or antioxidant activities. The presented experimental techniques seem to be suitable for establishing an antithrombotic diet, which may be effective in the prevention of atherothrombotic cardiovascular diseases in humans.

Stromal cell-derived factor-1 mediates changes of bone marrow stem cells during the bone repair process.

Osteoblasts, osteoclasts, chondrocytes, and macrophages that participate in the bone repair process are derived from hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). However, the roles of these stem cells during the repair of injured bone tissue are still unclear. In the present study, we examined the effects of bone defect on HSCs and MSCs in bone marrow and spleen in 75 mice and its mechanism. We analyzed the HSC and MSC populations in these tissues of a mouse with femoral bone damage by using flow cytometry. The number of HSCs in the bone marrow of mice with damaged femurs was significantly lower than the number of these cells in the bone marrow of the contralateral intact femurs on day 2 after injury. Meanwhile, the number of MSCs in the bone marrow of mice with damaged femurs was significantly higher than that of the contralateral femurs. Both intraperitoneal administration of AMD3100, a C-X-C chemokine receptor 4 (CXCR4) antagonist, and local treatment with an anti-stromal cell-derived factor-1 (SDF-1) antibody blunted the observed decrease in HSC and increase in MSC populations within the bone marrow of injured femurs. In conclusion, the present study revealed that there is a concurrent decrease and increase in the numbers of HSCs and MSCs, respectively, in the bone marrow during repair of mouse femoral bone damage. Furthermore, the SDF-1/CXCR4 system was implicated as contributing to the changes in these stem cell populations upon bone injury.

Role of osteoclasts in heterotopic ossification enhanced by fibrodysplasia ossificans progressiva-related activin-like kinase 2 mutation in mice.

Fibrodysplasia ossificans progressiva (FOP) is a disorder of skeletal malformations and progressive heterotopic ossification. The constitutively activating mutation (R206H) of the bone morphogenetic protein type 1 receptor, activin-like kinase 2 (ALK2), is responsible for the pathogenesis of FOP. Although transfection of the causal mutation of FOP into myoblasts enhances osteoclast formation by transforming growth factor-ß (TGF-ß), the role of osteoclasts in heterotopic ossification is unknown. We therefore examined the effects of alendronate, SB431542 and SB203580 on heterotopic ossification induced by the causal mutation of FOP. Total bone mineral content as well as numbers of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated and alkaline phosphatase (ALP)-positive cells in heterotopic bone were significantly higher in muscle tissues implanted with ALK2 (R206H)-transfected mouse myoblastic C2C12 cells than in the tissues implanted with empty vector-transfected cells in nude mice. Alendronate, an aminobisphosphonate, did not affect total mineral content or numbers of TRAP-positive multinucleated and ALP-positive cells in heterotopic bone, which were enhanced by the implantation of ALK2 (R206H)-transfected C2C12 cells, although it significantly decreased serum levels of cross-linked C-telopeptide of type I collagen, a bone resorption index. Moreover, neither SB431542, an inhibitor of TGF-ß receptor type I kinase, nor SB203580, an inhibitor of p38 mitogen-activated protein kinase, affected the increase in heterotopic ossification due to the implantation of ALK2 (R206H)-transfected C2C12 cells. In conclusion, the present study indicates that osteoclast inhibition does not affect heterotopic ossification enhanced by FOP-related mutation.

Evaluation of antithrombotic effect: Importance of testing components and methodologies.

The beneficial antithrombotic effect of some dietary components may offer the most promising approach of prevention of cardiovascular diseases and arterial thrombosis. The major stumbling block in finding effective dietary components is the lack of physiologically relevant techniques which can detect potential antithrombotic effect in humans. The presently used platelet function and coagulation tests do not allow the assessment of global thrombotic status and their value in screening dietary components for antithrombotic effect is questionable. Most of these in vitro tests ignore the effect of flow and shear stress, thrombin generation and vascular endothelium, the major contributors to arterial thrombogenesis in humans. As a gold standard, we employed the helium-neon (He-Ne) laser-induced thrombosis test in murine carotid artery and mesenteric microvessels, as the pathomechanism of this test closely reflects arterial thrombogenesis in humans. Results obtained with laser thrombosis test were compared with various shear-induced in vitro platelet function tests which use native blood (Haemostatometry, Thrombotic Status Analyser, Global Thrombosis Test-GTT). Contribution of vascular endothelium to thrombogenesis was assessed by measuring flow-mediated vasodilation (FMV) in vivo. The combination of the two shear-induced ex vivo thrombosis tests (Haemostatometry and GTT) with FMV correlated most closely with the laser-thrombosis test. Our findings suggest that combining the commercially available point-of-care GTT with the FMV test could provide a better assessment of the overall thrombotic status than either of the two tests alone.

The Prostaglandin E2 Receptor EP4 Regulates Obesity-Related Inflammation and Insulin Sensitivity.

With increasing body weight, macrophages accumulate in adipose tissue. There, activated macrophages secrete numerous proinflammatory cytokines and chemokines, giving rise to chronic inflammation and insulin resistance. Prostaglandin E2 suppresses macrophage activation via EP4; however, the role of EP4 signaling in insulin resistance and type 2 diabetes mellitus remains unknown. In this study, we treated db/db mice with an EP4-selective agonist, ONO-AE1-329, for 4 weeks to explore the role of EP4 signaling in obesity-related inflammation in vivo. Administration of the EP4 agonist did not affect body weight gain or food intake; however, in the EP4 agonist-treated group, glucose tolerance and insulin resistance were significantly improved over that of the vehicle-treated group. Additionally, administration of the EP4 agonist inhibited the accumulation of F4/80-positive macrophages and the formation of crown-like structures in white adipose tissue, and the adipocytes were significantly smaller. The treatment of the EP4 agonist increased the number of anti-inflammatory M2 macrophages, and in the stromal vascular fraction of white adipose tissue, which includes macrophages, it markedly decreased the levels of proinflammatory cytokines and chemokines. Further, EP4 activation increased the expression of adiponectin and peroxidase proliferator-activated receptors in white adipose tissue. Next, we examined in vitro M1/M2 polarization assay to investigate the impact of EP4 signaling on determining the functional phenotypes of macrophages. Treatment with EP4 agonist enhanced M2 polarization in wild-type peritoneal macrophages, whereas EP4-deficient macrophages were less susceptible to M2 polarization. Notably, antagonizing peroxidase proliferator-activated receptor δ activity suppressed EP4 signaling-mediated shift toward M2 macrophage polarization. Thus, our results demonstrate that EP4 signaling plays a critical role in obesity-related adipose tissue inflammation and insulin resistance by regulating macrophage recruitment and polarization. The activation of EP4 signaling holds promise for treating obesity and type 2 diabetes mellitus.

α2-Antiplasmin is involved in bone loss induced by ovariectomy in mice.

The mechanism of postmenopausal osteoporosis is not fully understood. α2-Antiplasmin (α2-AP) is the primary inhibitor of plasmin in the fibrinolytic system, but is known to have activities beyond fibrinolysis. However, its role in bone metabolism and the pathogenesis of osteoporosis remains unknown. In the current study, we therefore examined the effects of α2-AP deficiency on ovariectomy (OVX)-induced bone loss by using wild-type and α2-AP-deficient mice. Quantitative computed tomography analysis revealed that α2-AP deficiency blunted OVX-induced trabecular bone loss in mice. Moreover, α2-AP deficiency significantly blunted serum levels of bone-specific alkaline phosphatase, cross-linked C-telopeptide of type I collagen, and interleukin (IL)-1ß elevated by OVX. α2-AP treatment elevated the levels of IL-1ß and tumor necrosis factor (TNF)-α mRNA in RAW 264.7 cells, although it suppressed osteoclast formation induced by receptor activator of nuclear factor-κB ligand. α2-AP treatment activated ERK1/2 and p38 MAP kinase pathways in RAW 264.7 cells, and these MAP kinase inhibitors antagonized the levels of IL-1ß mRNA elevated by α2-AP. The data demonstrate that α2-AP is linked to bone loss due to OVX, through a mechanism that depends in part on the production of IL-1ß and TNF-α in monocytes.

The Tissue Fibrinolytic System Contributes to the Induction of Macrophage Function and CCL3 during Bone Repair in Mice.

Macrophages play crucial roles in repair process of various tissues. However, the details in the role of macrophages during bone repair still remains unknown. Herein, we examined the contribution of the tissue fibrinolytic system to the macrophage functions in bone repair after femoral bone defect by using male mice deficient in plasminogen (Plg-/-), urokinase-type plasminogen activator (uPA-/-) or tissue-type plasminogen activator (tPA-/-) genes and their wild-type littermates. Bone repair of the femur was delayed in uPA-/- mice until day 6, compared with wild-type (uPA+/+) mice. Number of Osterix-positive cells and vessel formation were decreased in uPA-/- mice at the bone injury site on day 4, compared with those in uPA+/+ mice. Number of macrophages and their phagocytosis at the bone injury site were reduced in uPA-/- and Plg-/-, but not in tPA-/- mice on day 4. Although uPA or plasminogen deficiency did not affect the levels of cytokines, including TNF-α, IL-1ß, IL-6, IL-4 and IFN-γ mRNA in the damaged femur, the elevation in CCL3 mRNA levels was suppressed in uPA-/- and Plg-/-, but not in tPA-/- mice. Neutralization of CCL3 antagonized macrophage recruitment to the site of bone injury and delayed bone repair in uPA+/+, but not in uPA-/- mice. Our results provide novel evidence that the tissue fibrinolytic system contributes to the induction of macrophage recruitment and CCL3 at the bone injury site, thereby, leading to the enhancement of the repair process.

[Current Topics on Vitamin D. Influences of vitamin D on muscle cells and function].

Vitamin D deficiency is associated with sarcopenia, which is characterized by the decrease in muscle mass and the muscle weakness. Active form of vitamin D binds to nuclear or non-nuclear vitamin D receptor (VDR) and regulates the proliferation and differentiation of myoblasts through its genomic or non-genomic actions. Clinical evidence showed the beneficial effects of vitamin D treatment on muscle mass and function in older people. Recent studies suggest that vitamin D is associated with the preservation of muscle function related to the interactions between bone and muscle.

Role of plasminogen activator inhibitor-1 in glucocorticoid-induced diabetes and osteopenia in mice.

Long-term use of glucocorticoids (GCs) causes numerous adverse effects, including glucose/lipid abnormalities, osteoporosis, and muscle wasting. The pathogenic mechanism, however, is not completely understood. In this study, we used plasminogen activator inhibitor-1 (PAI-1)-deficient mice to explore the role of PAI-1 in GC-induced glucose/lipid abnormalities, osteoporosis, and muscle wasting. Corticosterone markedly increased the levels of circulating PAI-1 and the PAI-1 mRNA level in the white adipose tissue of wild-type mice. PAI-1 deficiency significantly reduced insulin resistance and glucose intolerance but not hyperlipidemia induced by GC. An in vitro experiment revealed that active PAI-1 treatment inhibits insulin-induced phosphorylation of Akt and glucose uptake in HepG2 hepatocytes. However, this was not observed in 3T3-L1 adipocytes and C2C12 myotubes, indicating that PAI-1 suppressed insulin signaling in hepatocytes. PAI-1 deficiency attenuated the GC-induced bone loss presumably via inhibition of apoptosis of osteoblasts. Moreover, the PAI-1 deficiency also protected from GC-induced muscle loss. In conclusion, the current study indicated that PAI-1 is involved in GC-induced glucose metabolism abnormality, osteopenia, and muscle wasting in mice. PAI-1 may be a novel therapeutic target to mitigate the adverse effects of GC.

Enhanced pre-operative thrombolytic status is associated with the incidence of deep venous thrombosis in patients undergoing total knee arthroplasty.

BACKGROUND: Deep venous thrombosis (DVT), which is often associated with pulmonary embolism (PE), is a serious complication after total knee arthroplasty (TKA). In the present study, we examined the overall thrombotic and thrombolytic status using Global Thrombosis Test (GTT) in non-anticoagulated blood of patients undergoing TKA to develop the predictable marker for the incidence of DVT. METHODS: DVT was diagnosed using doppler ultrasonography a day after the surgery in 31 patients with osteoarthritis (n = 24), rheumatoid arthritis (n = 6) and ankylosing spondylitis (n = 1) by the well-trained operator. We measured overall thrombotic and thrombolytic status using GTT and other biomarkers, which is associated with blood coagulation and fibrinolysis, before and immediately after the surgery. RESULTS: Newly-generated DVT during the operation was detected in 11 of 31 patients (35.4%) 1 day after TKA. There were no differences in markers of coagulation (PT and APTT), platelet activity (platelet aggregation-induced by ADP and collagen) and fibrinolysis (FDP and D-dimer) between non-DVT and DVT group both before and after the surgery. Both Pre- and Post-operative GTT-occlusion times (OT), an index of platelet reactivity, were tended to be shorter, but not significant, in DVT group compared with non-DVT group. Pre-operative GTT-lysis time (LT), an index of thrombolytic activity, was significantly shorter in DVT group compared with non-DVT group, while there were no differences in post-operative value of this index between DVT group and non-DVT group, suggesting overall thrombolytic activity was enhanced in DVT group before surgery. CONCLUSIONS: Our data suggest that enhancement of pre-operative thrombolytic activity assessed by GTT may be a predictable marker for the incidence of DVT after TKA.

Tissue-type plasminogen activator deficiency delays bone repair: roles of osteoblastic proliferation and vascular endothelial growth factor.

Further development in research of bone regeneration is necessary to meet the clinical demand for bone reconstruction. Recently, we reported that plasminogen is crucial for bone repair through enhancement of vessel formation. However, the details of the role of tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) in the bone repair process still remain unknown. Herein, we examined the effects of plasminogen activators on bone repair after a femoral bone defect using tPA-deficient (tPA(-/-)) and uPA-deficient (uPA(-/-)) mice. Bone repair of the femur was delayed in tPA(-/-) mice, unlike that in wild-type (tPA(+/+)) mice. Conversely, the bone repair was comparable between wild-type (uPA(+/+)) and uPA(-/-) mice. The number of proliferative osteoblasts was decreased at the site of bone damage in tPA(-/-) mice. Moreover, the proliferation of primary calvarial osteoblasts was reduced in tPA(-/-) mice. Recombinant tPA facilitated the proliferation of mouse osteoblastic MC3T3-E1 cells. The proliferation enhanced by tPA was antagonized by the inhibition of endogenous annexin 2 by siRNA and by the inhibition of extracellular signal-regulated kinase (ERK)1/2 phosphorylation in MC3T3-E1 cells. Vessel formation as well as the levels of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) were decreased at the damaged site in tPA(-/-) mice. Our results provide novel evidence that tPA is crucial for bone repair through the facilitation of osteoblast proliferation related to annexin 2 and ERK1/2 as well as enhancement of vessel formation related to VEGF and HIF-1α at the site of bone damage.

Short- and long-term treatment with folic acid suppresses thrombus formation in atherogenic mice in vivo.

In the present study, we examined the effects of short- and long-term treatment with folic acid (FA) on thrombus formation in vivo in atherogenic mice to explore a novel agent for the prevention of atherothrombotic disease. Apolipoprotein E and low-density lipoprotein receptor double deficient (ApoE(-/-)LDLR(-/-)) mice were orally administrated a single bolus of FA (20mg/kg) or fed an atherogenic diet with or without FA (0.02, 0.5, and 1.5mg/kg) for 12 weeks. Thrombus formation and endothelial function were assessed in vivo using the He-Ne laser-induced carotid artery thrombus formation test and the flow-mediated vasodilation method. Platelet reactivity was assessed ex vivo using haemostatometry. Short-term treatment with FA markedly increased plasma folate levels and significantly suppressed laser-induced thrombus formation in apoE(-/-)LDLR(-/-) mice. Short-term treatment with FA suppressed platelet reactivity in apoE(-/-)LDLR(-/-) mice, but FA treatment did not affect endothelial function or plasma homocysteine levels. Long-term treatment with FA increased plasma folate levels dose-dependently. Thrombus formation and endothelial dysfunction were suppressed by treatment with 0.5 and 1.5mg/kg of FA, respectively, but not with 0.02mg/kg of FA, whereas platelet reactivity was not altered by treatment with any dose of FA. Long-term treatment with all doses of FA decreased the plasma homocysteine levels in apoE(-/-)LDLR(-/-) mice, although this result was not consistent with its anti-thrombotic action. In conclusion, our data showed that short- and long-term treatment with FA could suppress in vivo thrombus formation in an atherogenic setting, independent of its hypohomocysteinemic action.

Fibrodysplasia ossificans progressiva-related activated activin-like kinase signaling enhances osteoclast formation during heterotopic ossification in muscle tissues.

Fibrodysplasia ossificans progressiva is characterized by extensive ossification within muscle tissues, and its molecular pathogenesis is responsible for the constitutively activating mutation (R206H) of the bone morphogenetic protein type 1 receptor, activin-like kinase 2 (ALK2). In this study, we investigated the effects of implanting ALK2 (R206H)-transfected myoblastic C2C12 cells into nude mice on osteoclast formation during heterotopic ossification in muscle and subcutaneous tissues. The implantation of ALK2 (R206H)-transfected C2C12 cells with BMP-2 in nude mice induced robust heterotopic ossification with an increase in the formation of osteoclasts in muscle tissues but not in subcutaneous tissues. The implantation of ALK2 (R206H)-transfected C2C12 cells in muscle induced heterotopic ossification more effectively than that of empty vector-transfected cells. A co-culture of ALK2 (R206H)-transfected C2C12 cells as well as the conditioned medium from ALK2 (R206H)-transfected C2C12 cells enhanced osteoclast formation in Raw264.7 cells more effectively than those with empty vector-transfected cells. The transfection of ALK2 (R206H) into C2C12 cells elevated the expression of transforming growth factor (TGF)-ß, whereas the inhibition of TGF-ß signaling suppressed the enhanced formation of osteoclasts in the co-culture with ALK2 (R206H)-transfected C2C12 cells and their conditioned medium. In conclusion, this study demonstrated that the causal mutation transfection of fibrodysplasia ossificans progressiva in myoblasts enhanced the formation of osteoclasts from its precursor through TGF-ß in muscle tissues.