Katsumi Okumoto

Plasminogen Plays a Crucial Role in Bone Repair

The further development in research of bone regeneration is necessary to meet the clinical demand for bone reconstruction. Plasminogen is a critical factor of the tissue fibrinolytic system, which mediates tissue repair in the skin and liver. However, the role of the fibrinolytic system in bone regeneration remains unknown. Herein, we investigated bone repair and ectopic bone formation using plasminogen‐deficient (Plg–/–) mice. Bone repair of the femur is delayed in Plg–/– mice, unlike that in the wild‐type (Plg+/+) mice. The deposition of cartilage matrix and osteoblast formation were both decreased in Plg–/– mice. Vessel formation, macrophage accumulation, and the levels of vascular endothelial growth factor (VEGF) and transforming growth factor‐β (TGF‐β) were decreased at the site of bone damage in Plg–/– mice. Conversely, heterotopic ossification was not significantly different between Plg+/+ and Plg–/– mice. Moreover, angiogenesis, macrophage accumulation, and the levels of VEGF and TGF‐β were comparable between Plg+/+ and Plg–/– mice in heterotopic ossification. Our data provide novel evidence that plasminogen is essential for bone repair. The present study indicates that plasminogen contributes to angiogenesis related to macrophage accumulation, TGF‐β, and VEGF, thereby leading to the enhancement of bone repair.

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.

Plasminogen activator inhibitor-1 is involved in impaired bone repair associated with diabetes in female mice.

Previous studies suggest that fracture healing is impaired in diabetes; however, the underlying mechanism remains unclear. Here, we investigated the roles of plasminogen activator inhibitor-1 (PAI-1) in the impaired bone repair process by using streptozotocin (STZ)-induced diabetic female wild-type (PAI-1 +/+) and PAI-1-deficient (PAI-1 −/−) mice. Bone repair and the number of alkaline phosphatase (ALP)-positive cells at the site of a femoral bone damage were comparable in PAI-1 +/+ and PAI-1 −/− mice without STZ treatment. Although the bone repair process was delayed by STZ treatment in PAI-1 +/+ mice, this delayed bone repair was blunted in PAI-1 −/− mice. The reduction in the number of ALP-positive cells at the site of bone damage induced by STZ treatment was attenuated in PAI-1 −/− mice compared to PAI-1 +/+ mice. On the other hand, PAI-1 deficiency increased the levels of ALP and type I collagen mRNA in female mice with or without STZ treatment, and the levels of Osterix and osteocalcin mRNA, suppressed by diabetic state in PAI-1 +/+ mice, were partially protected in PAI-1 −/− mice. PAI-1 deficiency did not affect formation of the cartilage matrix and the levels of types II and X collagen and aggrecan mRNA suppressed by STZ treatment, although PAI-1 deficiency increased the expression of chondrogenic markers in mice without STZ treatment. The present study indicates that PAI-1 is involved in the impaired bone repair process induced by the diabetic state in part through a decrease in the number of ALP-positive cells.

Plasminogen activator inhibitor-1 is involved in streptozotocin-induced bone loss in female mice

In diabetic patients, the risk of fracture is high because of impaired bone formation. However, the details of the mechanisms in the development of diabetic osteoporosis remain unclear. In the current study, we investigated the role of plasminogen activator inhibitor (PAI)-1 in the pathogenesis of type 1 diabetic osteoporosis by using PAI-1–deficient mice. Quantitative computed tomography analysis showed that PAI-1 deficiency protected against streptozotocin-induced bone loss in female mice but not in male mice. PAI-1 deficiency blunted the changes in the levels of Runx2, osterix, and alkaline phosphatase in tibia as well as serum osteocalcin levels suppressed by the diabetic state in female mice only. Furthermore, the osteoclast levels in tibia, suppressed in diabetes, were also blunted by PAI-1 deficiency in female mice. Streptozotocin markedly elevated the levels of PAI-1 mRNA in liver in female mice only. In vitro study demonstrated that treatment with active PAI-1 suppressed the levels of osteogenic genes and mineralization in primary osteoblasts from female mouse calvaria. In conclusion, the current study indicates that PAI-1 is involved in the pathogenesis of type 1 diabetic osteoporosis in females. The expression of PAI-1 in the liver and the sensitivity of bone cells to PAI-1 may be an underlying mechanism.

Systemic transplantation of embryonic stem cells accelerates brain lesion decrease and angiogenesis.

As stem cells can regenerate damaged tissue, their therapeutic potential on brain damage has been investigated. In this study, the effects of embryonic stem cell transplantation on brain damage were investigated by using a photochemically induced thrombotic brain damage model. Mice with systemic transplantation of embryonic stem cells expressing enhanced green fluorescence protein on day 1 showed a smaller brain lesion size on day 8 than the control mice. The smaller lesion was accompanied by the increase in the number of microvessels at the border of the damaged area. Inside and around the damaged lesion, no EGFP-positive cells were observed. These findings suggested that embryonic stem cell transplantation reduced the brain lesion through the acceleration of angiogenesis by endogenous endothelial cells.

Plasminogen is essential for granulation tissue formation during the recovery process after liver injury in mice

Summary.  Background: The involvement of plasminogen in liver repair has been reported, but its exact role in promoting this process is unknown. Objective: To elucidate the underlying mechanism, we examined the dynamics of liver repair by using a reproducible liver injury model in plasminogen gene‐deficient mice and their wild‐type littermates. Methods: Liver injury was induced by photochemical reaction and the subsequent responses were histologically analyzed. Results: In wild‐type animals, the area of the damage successively decreased, and the repair process was associated with macrophage accumulation at its border. Neutrophils were also attracted to the damaged region on day 1 and were evident only at its border by day 4, which spatially and temporally coincided with the expression of macrophage chemoattractant protein‐1 (MCP‐1). Neutrophil depletion suppressed recruitment of macrophages at the border between the damaged and the normal tissues. These changes were followed by activated hepatic stellate cell accumulation, collagen fiber deposition and angiogenesis at the boundaries of the injured zone. In contrast, in plasminogen gene‐deficient mice, the decrease in the area of damage, macrophage accumulation, late‐phase neutrophil recruitment, hepatic stellate cell accumulation, collagen fiber deposition and angiogenesis were all impaired. Conclusion: Our data suggest that accumulated neutrophils at the border of the damaged area may contribute to macrophage accumulation at granulation tissue via the production of MCP‐1 after liver injury. The plasminogen system is critical for liver repair by facilitating macrophage accumulation and triggering a cascade of subsequent repair events.

Fibrodysplasia Ossificans Progressiva-related Activated Activin-like Kinase Signaling Enhances Osteoclast Formation during Heterotopic Ossification in Muscle Tissues

Background: The mutation (R206H) of bone morphogenetic protein type I receptor, activin-like kinase 2 (R206H), is the molecular pathogenesis of fibrodysplasia ossificans progressiva (FOP). Results: ALK2 (R206H) mutation increases osteoclast formation through TGF-β. Conclusion: The activation of activin-like kinase signaling enhanced osteoclast formation through TGF-β expression during heterotopic ossification in muscle tissues. Significance: An increase in the formation of osteoclasts may be involved in the pathogenesis of FOP. 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)-β, whereas the inhibition of TGF-β 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-β in muscle tissues.

Influence of diabetic state and vitamin D deficiency on bone repair in female mice

Type 1 diabetes is associated with an increased fracture risk, an impaired fracture healing, and an increased vitamin D insufficiency. However, the role of vitamin D in diabetic bone repair process remains unclear. We therefore examined the effects of vitamin D deficiency on the impaired bone repair in streptozotocin (STZ)-induced diabetes using female mice. Diabetes was induced by STZ injection into female mice after feeding with normal or vitamin D-deficient diet for 6weeks from the age of 4weeks. A femoral bone defect was induced in mice 4 weeks after induction of diabetes. The repair of damaged site on the femur was significantly delayed at days 7 and 10 after bone defect by diabetic state in mice, as assessed by quantitative computed tomography, while vitamin D deficiency did not affect the bone repair both in mice with normal and diabetic state. The decreases in bone mineral density (BMD) at cortical and trabecular bone by diabetic state were significantly augmented by vitamin D deficiency in tibia at the undamaged side in mice. Diabetic state blunted the levels of osteogenic and chondrogenic genes enhanced by vitamin D deficiency. Moreover, vitamin D deficiency significantly aggravated the decreases in osteocalcin and IGF-1 mRNA by diabetic state. In conclusion, our study showed that vitamin D deficiency aggravates the decrease in BMD by diabetic state in female mice, although vitamin D deficiency did not affect bone repair delayed by diabetic state.

Role of plasminogen in macrophage accumulation during liver repair

INTRODUCTION Although the involvement of plasminogen in liver repair has been reported, its roles are still poorly understood. Here, we investigated the role of plasminogen in accumulations of macrophages and neutrophils after liver injury in mice with gene deficient of plasminogen (Plg(-/-)) or its wild type (Plg(+/+)). MATERIALS AND METHODS Mice received traumatic liver injury caused by stabbing on the lobe or hepatic ischemia-reperfusion, and the damaged sites were histologically analyzed. RESULTS After the traumatic liver injury, both the stab wound and the damaged tissue were decreased until day 7 in the Plg(+/+) mice. In contrast, both the stab wound and the damaged tissue were still remained until day 7 in the Plg(-/-) mice. On day 4 after traumatic liver injury, macrophages were abundant at the surrounding area of the damaged site in the Plg(+/+) mice. However, the macrophage accumulation was impaired in the Plg(-/-) mice. After hepatic ischemia-reperfusion injury, macrophage accumulation and decrease in the damaged tissue were also observed in the Plg(+/+) mice until day 7. In contrast, these responses were also impaired in the Plg(-/-) mice. Furthermore, neutrophil accumulation at the surrounding area of the damaged site was also impaired in the Plg(-/-) mice on day 4 after both liver traumatic liver injury and hepatic ischemia-reperfusion injury. CONCLUSIONS Our data indicate that plasminogen plays a crucial role in macrophage accumulation together with the neutrophil accumulation after liver injury in both models, which may be essential for triggering the subsequent healing responses including decrease in the damaged tissue.

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.