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Irisin is a proteolytic product of fibronectin type II domain-containing 5, which is related to the improvement in glucose metabolism. Numerous studies have suggested that irisin is a crucial myokine linking muscle to bone in physiological and pathophysiological states.We examined the effects of local irisin administration with gelatin hydrogel sheets and intraperitoneal injection of irisin on the delayed femoral bone repair caused by streptozotocin (STZ)-induced diabetes in female mice. We analyzed the femurs of mice using quantitative computed tomography and histological analyses and then measured the mRNA levels in the damaged mouse tissues.Local irisin administration significantly blunted the delayed bone repair induced by STZ 10 days after a femoral bone defect was generated. Local irisin administration significantly blunted the number of Osterix-positive cells that were suppressed by STZ at the damaged site 4 days after a femoral bone defect was generated, although it did not affect the mRNA levels of chondrogenic and adipogenic genes 4 days after bone injury in the presence or absence of diabetes. On the other hand, intraperitoneal injection of irisin did not affect delayed bone repair induced by STZ 10 days after bone injury. Irisin significantly blunted the decrease in Osterix mRNA levels induced by advanced glycation end products or high-glucose conditions in ST2 cells in the presence of bone morphogenetic protein-2.We first showed that local irisin administration with gelatin hydrogel sheets improves the delayed bone repair induced by diabetic state partially by enhancing osteoblastic differentiation.

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.

Androgen deficiency plays a crucial role in the pathogenesis of male osteoporosis and sarcopenia. Myokines have recently been identified as humoral factors that are involved in the interactions between muscle and bone; however, the influence of androgen deficiency on these interactions remains unclear. Therefore, we herein investigated the roles of humoral factors linking muscle to bone using orchidectomized mice with sarcopenia and osteopenia. Orchidectomy (ORX) significantly reduced muscle mass, grip strength, and trabecular bone mineral density (BMD) in mice. Among the myokines examined, ORX only significantly reduced fibronectin type III domain-containing 5 (Fndc5) mRNA levels in both the soleus and gastrocnemius muscles of mice. In simple regression analyses, Fndc5 mRNA levels in the soleus muscle positively correlated with trabecular BMD, but not cortical BMD. The administration of irisin, a product of Fndc5, significantly protected against the decrease induced in trabecular BMD, but not muscle mass, by androgen deficiency in mice. In conclusion, the present results demonstrated that androgen deficiency decreases the expression of irisin in the skeletal muscle of mice. Irisin may be involved in muscle/bone relationships negatively affected by androgen deficiency.

Glucocorticoid (GC) treatments induce osteoporosis and chronic GC treatments have been suggested to induce delayed bone repair; however, the mechanisms by which GC induces delayed bone repair remain unclear. We herein investigated the roles of plasminogen activator inhibitor-1 (PAI-1) in GC-induced effects on bone repair after femoral bone injury using female mice with a PAI-1 deficiency and their wild-type counterparts. Dexamethasone (Dex) increased plasma PAI-1 levels as well as PAI-1 mRNA levels in the adipose tissues and muscles of wild-type mice. PAI-1 deficiency significantly blunted Dex-induced delayed bone repair in mice. Moreover, PAI-1 deficiency significantly blunted Runx2 mRNA levels suppressed by Dex as well as Dex-induced osteoblast apoptosis at the damaged site 7 days after bone injury in mice. On the other hand, PAI-1 deficiency did not affect adipogenic gene expression enhanced by Dex at the damaged site 7 days after bone injury in mice. In conclusion, we herein showed for the first time that PAI-1 is involved in delayed bone repair after bone injury induced by GC in mice. PAI-1 may influence early stage osteoblast differentiation and apoptosis during the osteoblastic restoration phase of the bone repair process.

The vestibular system controls balance, posture, blood pressure, and gaze. However, the roles of the vestibular system in energy and glucose metabolism remain unknown. We herein examined the roles of the vestibular system in obesity and impaired glucose metabolism using mice with vestibular lesions (VL) fed a high-sucrose/high-fat diet (HSHFD). VL was induced by surgery or arsenic. VL significantly suppressed body fat enhanced by HSHFD in mice. Glucose intolerance was improved by VL in mice fed HSHFD. VL blunted the levels of adipogenic factors and pro-inflammatory adipokines elevated by HSHFD in the epididymal white adipose tissue of mice. A β-blocker antagonized body fat and glucose intolerance enhanced by HSHFD in mice. The results of an RNA sequencing analysis showed that HSHFD induced alterations in genes, such as insulin-like growth factor-2 and glial fibrillary acidic protein, in the vestibular nuclei of mice through the vestibular system. In conclusion, we herein demonstrated that the dysregulation of the vestibular system influences an obese state and impaired glucose metabolism induced by HSHFD in mice. The vestibular system may contribute to the regulation of set points under excess energy conditions.

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.

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–β (TGF-β) elevated by bone injury. TGF-β 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-β, and SDF-1 at the damaged site during bone repair in mice., Highlights • We investigated the roles of plasminogen in bone marrow stem cell changes after bone injury in mice. • Plasminogen deficiency blunted a decrease in the number of HSCs after bone injury. • Plasminogen deficiency blunted the expression of SDF-1, TGF-β and MMP-9 at the damaged site. • TGF-β signaling inhibition blunted a decrease in the number of HSCs after bone injury. • Plasminogen is involved in HSC change through TGF-β and SDF-1 at the damaged site during bone repair.

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)-1β elevated by OVX. α2-AP treatment elevated the levels of IL-1β 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-1β 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-1β and TNF-α in monocytes.

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-β (TGF-β), 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-β 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.

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-1β 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.

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.

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.

Unrecognizable exposure to estrogenic substance may cause estrogen-dependent diseases, endometriosis and cancer. Pregnant mice (ICR/Jcl, CLEA) were exposed to 0.01 mg ethinyl estradiol (EE2 )/kg per day or vehicle (olive oil) through oral intubation from day 11 to 17 of gestation. They delivered their offspring and raised them. When the experimental female F1 mice were at 8 weeks of age, they were not exposed to EE2 or to the same dose of EE2 or to vehicle twice a week until 20 weeks of age. The control female F1 mice were exposed to the same dose of EE2 or vehicle alone, similarly. All mice were killed at 28 weeks of age. The resected uteri and ovaries were processed for microscopic examinations and for determination of the aromatase mRNA levels and aromatase protein through quantitative RT-PCR and Western blotting, respectively. Adenomyosis and adenocarcinomatous changes were significantly discernible in the EE2 -exposed uteri, and incidence of ectopic glands and serous cysts were significantly increased in the prenatally EE2 -exposed ovaries as compared with respective controls. Significant upregulation of the aromatase mRNA was seen in the prenatally EE2 -exposed uteri and in the EE2 -exposed ovaries. The aromatase protein was identified in all ovaries examined, and in EE2 -exposed uteri but not in controls and confirmed its localization in eutopic and ectopic glands, abnormally proliferated lesions and the lining of the cysts. Taken together, continuous EE2 exposure may cause endometriotic and precancerous lesions due to excessive estrogen synthesis in both target organs.

Before establishment of feto-placental circulation, decidua can synthesize hemoproteins to maintain oxygen homeostasis in situ. Using the human decidua of induced abortions ranging from 5 to 8 weeks of gestation, we determined the expression levels of erythropoietin, erythropoietin receptor, cytoglobin, myoglobin, embryonic-, fetal- and adult hemoglobin mRNA by quantitative RT-PCR analysis and identified their proteins by Western blot and immunohistochemical analyses. Erythropoietin signaling was demonstrated in phosphatidylinositol-3-kinase/protein kinase B pathway by Western blot, and the transcriptional factors for erythroid and non-erythroid heme synthesis were examined by RT-PCR analysis. In decidua, erythropoietin and its receptor mRNAs, erythropoietin receptor protein and phosphatidylinositol-3-kinase, were expressed with a peak at 6 weeks of gestation. Moreover, the decidua during 5 to 8 weeks of gestation expressed embryonic, fetal and adult hemoglobins additionally cytoglobin and myoglobin at transcriptional and protein levels. The heme portion of these hemoproteins is considered to be synthesized by non-erythroid δ-aminolevulinate synthase. These hemoproteins were discernible especially in decidual cells concomitant with cytotrophoblast cells and macrophage in these developing decidua. Considering the different capacity for oxygen binding and dissociation among hemoglobins with the oxygen storage capacity for cytoglobin and myoglobin, these hemoproteins appear to play a role in oxygen demand in decidua in situ before development of feto-placental circulation under the control of erythropoietin signaling.

SummaryUrokinase-type plasminogen activator (u-PA) and plasminogen play a primary role in liver repair through the accumulation of macrophages and alteration of their phenotype. However, it is still unclear whether u-PA and plasminogen mediate the activation of macrophage phagocytosis during liver repair. Herein, we investigated the morphological changes in macrophages that accumulated at the edge of damaged tissue induced by a photochemical reaction or hepatic ischaemia-reperfu-sion in mice with u-PA (u-PA−/− ) or plasminogen (Plg−/− ) gene deficiency by using transmission electron and fluorescence microscopy. In wild-type mice, the macrophages aligned at the edge of the damaged tissue and extended a large number of long pseudopodia. These macrophages clearly engulfed cellular debris and showed well-developed organelles, including lysosome-like vacuoles, nuclei, and Golgi complexes. In wild-type mice, the distribution of the Golgi complex in these macrophages was biased towards the direction of the damaged tissue, indicating the extension of their pseudopodia in this direction. Conversely, in u-PA−/− and Plg−/− mice, the macrophages located at the edge of the damaged tissue had few pseudopodia and less developed organelles. The Golgi complex was randomly distributed in these macrophages in u-PA−/− mice. Furthermore, interferon γ and IL-4 were expressed at a low level at the border region of the damaged tissue in u-PA−/− mice. Our data provide novel evidence that u-PA and plasminogen are essential for the phagocytosis of cellular debris by macrophages during liver repair. Furthermore, u-PA plays a critical role in the induction of macrophage polarity by affecting the microenvironment at the edge of damaged tissue.

We examined the effect of blocking the erythropoietin (Epo) signaling using an anti-Epo antibody, soluble form of Epo receptor (sEpoR) capable of binding to Epo or EpoR antagonist, and proved to be effective against xenografts of female reproductive organ malignancies and of cancer cell lines in nude mice. We transfected seven cancer cell lines of various origins to express constitutively active sEpoR, and examined their tumorigenesis in nude mice. Suppression of the tumor growth, decrease in viable and proliferating cells and reduction of vascular density were seen individually in all xenografts of transfected cell lines compared with the controls. Quantitative RT-PCR analyses showed that expression levels of Epo, EpoR, ?1A-adrenaline receptor (?1A-ADR) and muscalinic acetylcholine receptor subunit 3 mRNAs (m3-AchR) were higher in the majority of the wild-type xenografts than in the corresponding cell lines except for A549. In some of the transfected xenografts, EpoR, ?1A-ADR and m3-AchR mRNAs were down-regulated. Western blot analyses revealed that the constitutively activated ERK1/2MAPK was discernible in the majority of non-transfected cell lines and was reduced in the transfected cell lines. However, it was regained after exposure to acetylcholine and/or noradrenaline. These findings suggest that constitutively active sEpoR can effectively destroy the xenografts but signals from the autonomic neurotransmitters of the host produced under stress may interfere with this antitumor activity.

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.

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.

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.

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.

The aim of this study is to explore a cause-oriented therapy for patients with uterine cervical cancer that expresses erythropoietin (Epo) and its receptor (EpoR). Epo, by binding to EpoR, stimulates the proliferation and differentiation of erythroid progenitor cells into hemoglobin-containing red blood cells. In this study, we report that the HeLa cells in the xenografts expressed ε, γ, and α globins as well as myoglobin (Mb) to produce tetrameric α2ε2 and α2γ2 and monomeric Mb, most of which were significantly suppressed with an EpoR antagonist EMP9. Western blotting revealed that the EMP9 treatment inhibited the AKT-pAKT, MAPKs-pMAPKs, and STAT5-pSTAT5 signaling pathways. Moreover, the treatment induced apoptosis and suppression of the growth and inhibited the survival through disruption of the harmonized hemoprotein syntheses in the tumor cells concomitant with destruction of vascular nets in the xenografts. Furthermore, macrophages and natural killer (NK) cells with intense HIF-1α expression recruited significantly more in the degenerating foci of the xenografts. These findings were associated with the enhanced expressions of nNOS in the tumor cells and iNOS in macrophages and NK cells in the tumor sites. The treated tumor cells exhibited a substantial number of perforations on the cell surface, which indicates that the tumors were damaged by both the nNOS-induced nitric oxide (NO) production in the tumor cells as well as the iNOS-induced NO production in the innate immune cells. Taken together, these data suggest that HeLa cells constitutively acquire ε, γ and Mb synthetic capacity for their survival. Therefore, EMP9 treatment might be a cause-oriented and effective therapy for patients with squamous cell carcinoma of the uterine cervix.

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)-1β elevated by OVX. α2-AP treatment elevated the levels of IL-1β 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-1β 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-1β and TNF-α in monocytes.

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.

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.

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.

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.

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.

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 β‐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.

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.

Before establishment of feto-placental circulation, decidua can synthesize hemoproteins to maintain oxygen homeostasis in situ. Using the human decidua of induced abortions ranging from 5 to 8 weeks of gestation, we determined the expression levels of erythropoietin, erythropoietin receptor, cytoglobin, myoglobin, embryonic-, fetal- and adult hemoglobin mRNA by quantitative RT-PCR analysis and identified their proteins by Western blot and immunohistochemical analyses. Erythropoietin signaling was demonstrated in phosphatidylinositol-3-kinase/protein kinase B pathway by Western blot, and the transcriptional factors for erythroid and non-erythroid heme synthesis were examined by RT-PCR analysis. In decidua, erythropoietin and its receptor mRNAs, erythropoietin receptor protein and phosphatidylinositol-3-kinase, were expressed with a peak at 6 weeks of gestation. Moreover, the decidua during 5 to 8 weeks of gestation expressed embryonic, fetal and adult hemoglobins additionally cytoglobin and myoglobin at transcriptional and protein levels. The heme portion of these hemoproteins is considered to be synthesized by non-erythroid δ-aminolevulinate synthase. These hemoproteins were discernible especially in decidual cells concomitant with cytotrophoblast cells and macrophage in these developing decidua. Considering the different capacity for oxygen binding and dissociation among hemoglobins with the oxygen storage capacity for cytoglobin and myoglobin, these hemoproteins appear to play a role in oxygen demand in decidua in situ before development of feto-placental circulation under the control of erythropoietin signaling.

Unrecognizable exposure to estrogenic substance may cause estrogen-dependent diseases, endometriosis and cancer. Pregnant mice (ICR/Jcl, CLEA) were exposed to 0.01 mg ethinyl estradiol (EE2 )/kg per day or vehicle (olive oil) through oral intubation from day 11 to 17 of gestation. They delivered their offspring and raised them. When the experimental female F1 mice were at 8 weeks of age, they were not exposed to EE2 or to the same dose of EE2 or to vehicle twice a week until 20 weeks of age. The control female F1 mice were exposed to the same dose of EE2 or vehicle alone, similarly. All mice were killed at 28 weeks of age. The resected uteri and ovaries were processed for microscopic examinations and for determination of the aromatase mRNA levels and aromatase protein through quantitative RT-PCR and Western blotting, respectively. Adenomyosis and adenocarcinomatous changes were significantly discernible in the EE2 -exposed uteri, and incidence of ectopic glands and serous cysts were significantly increased in the prenatally EE2 -exposed ovaries as compared with respective controls. Significant upregulation of the aromatase mRNA was seen in the prenatally EE2 -exposed uteri and in the EE2 -exposed ovaries. The aromatase protein was identified in all ovaries examined, and in EE2 -exposed uteri but not in controls and confirmed its localization in eutopic and ectopic glands, abnormally proliferated lesions and the lining of the cysts. Taken together, continuous EE2 exposure may cause endometriotic and precancerous lesions due to excessive estrogen synthesis in both target organs.

Preliminary findings of various types of globin expressed in the respiratory bronchiolar and alveolar epithelium prompted us to compare the expression of erythropoietin (Epo) and its receptor (EpoR) in normal (healthy) human lung tissues with that in malignant lung tissues. The expression of Epo and EpoR was examined at the transcriptional and protein levels in normal and malignant lung tissues by reverse transcription-PCR, western blot, and immunohistochemical analyses. EpoR mRNA, but not Epo mRNA, was detected in all samples. In normal tissues, EpoR was detected in the mesothelium, chondrocytes, alveolar cells, vascular endothelial cells, smooth muscle fibers, macrophages, and neutrophils, while in malignant foci, the cancer cells of five malignant types showed various intensities of EpoR immunoreactivity. The pattern of staining of EpoR protein was generally stronger in the malignant tissues than in the normal samples. Phosphorylation of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK-ERK1/2) was frequently seen in malignant cells, but not in the normal tissues, with the exception of macrophages. Based on the expression of Epo and EpoR mRNA with the EpoR in almost all cell components in normal tissues, we suggest that the normal lung may produce various types of globin through the autocrine and/or paracrine role of Epo. When the Epo signal is upregulated by hypoxic stress, the normal cells appear to transform into malignant cells and proliferate through activated MAPK signaling.

The severity of an ischemic stroke is variable in patients, because the occlusion position on the artery and the territory of distal vessels are individual. However, the relationship between the extent of initial brain lesion and the subsequent pathophysiological responses is poorly understood. Here, we studied the effects of the initial brain lesion size on the subsequent pathophysiological responses by using a photochemically induced thrombotic brain damage (PIT-BD) model, in which the brain lesion size can be well-reproducibly controlled than that induced by a middle cerebral artery occlusion (MCA-O) model. In the PIT-BD model, a large lesion, which comprised 4.9% of the whole brain on day 3, showed a 56% reduction until day 7. However, a small lesion, which comprised 1.3% of the whole brain, showed a 30% reduction. In addition, on day 5, the activation of both microglia and astrocytes was lesser in mice with small lesions than in mice with large lesions. Furthermore, we found that, smaller lesions in mice lacking gene of urokinase-receptor (uPAR(-/-)) than wild type (uPAR(+/+)) mice on day 3 showed less reduction until day 7 in MCA-O model, whereas lesions with comparable size in uPAR(-/-) mice showed comparable reduction with uPAR(+/+) mice in PIT-BD model. Thus it was indicated that the less reduction of the lesions in uPAR(-/-) mice in the MCA-O model did not result from the deficient gene but the difference of the initial lesion size. These findings suggested that the more severe the brain damage, the stronger the subsequent pathophysiological responses.

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-1β, 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.