Effects of Enzamin, a Microbial Product, on Alterations of Intestinal Microbiota Induced by a High-Fat Diet.

In the human intestinal tract, there are more than 100 trillion microorganisms classified into at least 1000 different species. The intestinal microbiota contributes to the regulation of systemic physiologic functions and the maintenance of homeostasis of the host. It has been reported that the alteration of the intestinal microbiota is involved in metabolic syndromes, including type II diabetes and dyslipidemia, inflammatory bowel disease, allergic disease, and cancer growth. It has been reported that a microbial product from Paenibacillus polymyxa AK, which was named Enzamin, ameliorated adipose inflammation with impaired adipocytokine expression and insulin resistance in db/db mice. In order to investigate the effect of Enzamin on the intestinal microbiota and inflammation induced by obesity, mice were fed with a high-fat diet and 1% Enzamin for 4 weeks. Enzamin improved the Firmicutes-to-Bacteroidetes ratio and altered the intestinal microbiota in mice fed the high-fat diet. In addition, Enzamin suppressed the decreased expression of claudin-4 and the increased serum LPS level in mice fed with the high-fat diet. Modulating the intestinal microbiota with Enzamin may cause a decrease in serum LPS level. Based on these results, Enzamin may improve inflammation and metabolic disorders by regulating the intestinal microbiota in obese mice.

Protective Effects of Eicosapentaenoic Acid on the Glomerular Endothelium via Inhibition of EndMT in Diabetes.

Diabetes-induced endothelial pathologies are hypothesized to lead to the progression of diabetic kidney disease (DKD). The endothelial to mesenchymal transition (EndMT) possibly induces fibrosis, leading to glomerulosclerosis in the kidney. Furthermore, this could lead to albuminuria in diabetic nephropathy due to glomerular endothelial dysfunction. Eicosapentaenoic acid (EPA), purified from fish oil, decreases inflammatory cytokine levels in glomerulonephritis. Here, we aimed at finding whether ethyl eicosapentaenoate (EPA-E) exerts renal protective effects via EndMT inhibition. To find out whether EPA inhibits EndMT in vitro, the changes in CD31 expression were studied in cultured mouse endothelial cells. The addition of the conditioned medium from the adipocyte culture significantly decreased the protein levels of CD31, while the addition of EPA-E partially reversed this inhibition. Further, EndMT inhibition by EPA-E treatment might occur via the inhibition of the protein kinase Cß (PKCß)/transforming growth factor-ß (TGF-ß)/plasminogen activator inhibitor-1 (PAI-1) signaling and not via microRNAs. Streptozotocin-induced diabetic mice fed a high-fat diet (60% from fat) exhibited mesangial expansion and albuminuria. Induction of EPA-E ameliorated the mesangial expansion and decreased albuminuria without affecting blood pressure, triglyceride and free fatty acid levels, and intraperitoneal glucose. These findings suggest that EPA-E exerts renal protective effects on endothelial cells, by normalizing EndMT followed by the PKCß/TGF-ß/PAI-1 signaling. Thus, EPA-E has the potential for imparting renal protection by regulating EndMT in DKD.

Effects of Coriandrum sativum on Migration and Invasion Abilities of Cancer Cells.

Coriandrum sativum (coriander) is an annual herb in the Apiaceae family. Its leaves and seeds are used for cooking. Coriander has several beneficial functions such as anti-inflammatory, analgesic and anti-cancer effects. Although anti-carcinogenic potential of coriander has been known well, the effects of coriander on cancer metastasis have not yet been fully elucidated. In the present study, the effects of coriander on migration and invasion were investigated in vitro and in vivo by using human hepatocellular carcinoma cell line (HepG2) and mouse melanoma cell line (B16F10). The migration and invasion abilities of cancer cells had been evaluated by trans-well double chamber and these abilities were significantly impaired by treatment of cancer cells with coriander extract whose concentration did not affect proliferation. The treatment of cancer cells with coriander extract significantly reduced both matrix metalloproteinase 2 (MMP-2) and urokinase-type plasminogen activator (u-PA) activities, which were involved in cell migration and invasion, in their conditioned media. Furthermore, coriander extract suppressed the phosphorylation of Erk 1 or IkB in B16F10 cells, and inhibited the expression of MMP-2 or u-PA mRNA. After injection of B16F10 cells into the tail vein of C57BL/6J mice, the number of metastatic regions in lungs were counted. Mice fed with diet containing coriander possessed a smaller number of metastatic regions than those fed with control diet. It was suggested that coriander extract might have the abilities to suppress cancer cell migration and invasion, indicating that coriander provides the improvement of cancer prognosis.

Linagliptin affects IRS1/Akt signaling and prevents high glucose-induced apoptosis in podocytes.

Diabetes-induced podocyte apoptosis is considered to play a critical role in the pathogenesis of diabetic kidney disease (DKD). We proposed that hyperglycaemia can induce podocyte apoptosis by inhibiting the action of podocyte survival factors, thus inactivating the cellular effects of insulin signalling. In this study, we aimed to determine the effects of linagliptin on high glucose-induced podocyte apoptosis. Linagliptin reduced the increase in DNA fragmentation as well as the increase in TUNEL-positive cells in podocytes induced by high-glucose condition. Furthermore, linagliptin improved insulin-induced phosphorylation of insulin receptor substrate 1 (IRS1) and Akt, which was inhibited in high-glucose conditions. Adenoviral vector-mediated IRS1 overexpression in podocytes partially normalised DNA fragmentation in high-glucose conditions, while downregulation of IRS1 expression using small interfering RNA increased DNA fragmentation even in low-glucose conditions. Because reactive oxygen species inhibit glomerular insulin signalling in diabetes and Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway is one of the most important intrinsic antioxidative systems, we evaluated whether linagliptin increased Nrf2 in podocytes. High-glucose condition and linagliptin addition increased Nrf2 levels compared to low-glucose conditions. In summary, linagliptin offers protection against DKD by enhancing IRS1/Akt insulin signalling in podocytes and partially via the Keap1/Nrf2 pathway. Our findings suggest that linagliptin may induce protective effects in patients with DKD, and increasing IRS1 levels could be a potential therapeutic target in DKD.

Antithrombotic Effect of Oral Administration of Mozuku (Cladosiphon okamuranus, Brown Seaweed) Extract in Rat.

Dysfunction of vascular endothelial cells causes the risk of thrombosis. Aim of this study is to evaluate the antithrombotic effect of Okinawa mozuku (brown seaweed, Cladosiphon okamuranus) extract by using cultured vascular endothelial cells and rat carotid arterial thrombosis model induced by ferric chloride (FeCl3). The cell line (TKM-33) established from human umbilical vein endothelial cells were cultured with or without Okinawa mozuku extract. After incubation for 24 h, the conditioned medium was collected to evaluate urokinase-type plasminogen activator (u-PA) activity. Next, rats were fed with water or water containing 5% of Okinawa mozuku extract for 8 wk. After 8 wk of treatments, the rats were provided for the carotid arterial thrombosis model, and fibrinolytic factor and coagulation factor in blood were measured. Okinawa mozuku extract significantly augmented u-PA activity in the conditioned medium. The decrease of carotid artery blood flow induced by 40% FeCl3 injury in rats fed with Okinawa mozuku extract was less than that in control rats. Thus, oral administration of Okinawa mozuku extract prevented thrombus formation in this model. Oral administration of Okinawa mozuku extract significantly increased u-PA activity in euglobulin fraction, compared with control group. On the other hand, platelet aggregation activity, activated partial thromboplastin time, and active PAI-1 level in plasma exhibited no significant differences between control and Okinawa mozuku groups. These results indicate that oral administration of Okinawa mozuku enhances fibrinolytic activity in plasma and prevents thrombus formation which is induced by injury of vascular endothelial cells.

Obesity-associated glomerular inflammation increases albuminuria without renal histological changes.

Obesity is one of risk factors for chronic kidney disease (CKD), but the precise mechanism involved is unclear. This study characterizes the effect of obesity-induced glomerular inflammation, oxidative stress, and albuminuria in obese rats. Glomerular samples were collected from fatty (ZF) and lean (ZL) Zucker rats. After 2 months of feeding, body weight and albuminuria were significantly increased in ZF rats when compared to ZL rats. Expression of the inflammatory markers TNF-α and CCR2 was significantly increased in the glomeruli of ZF rats. However, expression of IL-6 mRNA was not increased. Analysis of renal pathology showed no glomerular expansion. As inflammatory and oxidative stress markers are associated with NF-κB, we evaluated whether NF-κB activation was increased in the glomeruli of mice on a high-fat diet. Immunohistochemistry showed increased NF-κB activation in the glomeruli when transgenic mice overexpressing an NF-κB-dependent enhanced green fluorescent protein were fed with a high-fat diet. These results suggest that obesity of only 2 months duration can cause albuminuria, due to increased inflammation or oxidative stress, but may not be long enough to develop renal pathological changes.

Role of GPx4 in human vascular endothelial cells, and the compensatory activity of brown rice on GPx4 ablation condition.

Oxidative stress is implicated in the pathologies of vascular endothelial cells. However, the importance of specific antioxidant enzymes in vascular endothelial cells is not fully understood. The purpose of this study was to elucidate the importance of Glutathione peroxidase 4 (GPx4), and the involvement of ferroptosis on cell death induced by GPx4 loss in human vascular endothelial cells. In addition, we examined the compensatory activity of brown rice on GPx4 ablation condition. Human umbilical vein endothelial cells were transfected with GPx4 or scramble control siRNA. GPx4 knockdown caused the increase in the levels of lipid oxidation, and induced cytotoxicity. On the other hand, α-tocopherol (vitamin E) and extract of brown rice, ameliorated lipid peroxidation, cytotoxicity, and delay of proliferation induced by GPx4 knockdown. Furthermore, ferrostatin-1, inhibitor of ferroptosis, also prevented cytotoxicity and delay of proliferation. In conclusion, our data demonstrated that GPx4 is an essential antioxidant enzyme for protecting lipid peroxidation, and is a regulator of ferroptosis in vascular endothelial cells. Furthermore, vitamin E rich food, such as brown rice, can compensate for GPx4 loss by protecting cells against lipid peroxidation.

α2AP mediated myofibroblast formation and the development of renal fibrosis in unilateral ureteral obstruction.

Renal fibrosis is the final common pathway of a wide variety of chronic kidney diseases. Myofibroblast formation via the differentiation of from tissue-resident fibroblasts and bone marrow-derived mesenchymal stem cells (MSCs), and epithelial-to-mesenchymal transition (EMT) is known to play a pivotal role in the development of renal fibrosis. However, the detailed mechanisms underlying this disorder remain unclear. We herein investigated the role of alpha 2-antiplasmin (α2AP) in myofibroblast formation and the development of renal fibrosis. We observed the development of renal fibrosis using unilateral ureteral obstruction (UUO). α2AP had accumulated in the UUO-induced obstructed kidneys and α2AP deficiency attenuated UUO-induced renal fibrosis in mice. The degree of myofibroblast formation in the obstructed kidneys of α2AP(-/-) mice was less than that in α2AP(+/+) mice. In vitro, α2AP induced myofibroblast formation in renal tubular epithelial cells (RTECs), renal fibrosblasts, and bone marrow-derived mesenchymal stem cells (MSCs). α2AP also induced the production of TGF-ß, which is known to be a key regulator of myofibroblast formation and fibrosis. α2AP-induced the TGF-ß production was significantly reduced by SP600125, c-Jun N-terminal kinase (JNK) specific inhibitor. Our findings suggest that α2AP induces myofibroblast formation in the obstructed kidneys, and mediates the development of renal fibrosis.

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.

Lack of both α2-antiplasmin and plasminogen activator inhibitor type-1 induces high IgE production.

AIMS: We investigated the pathophysiological changes in mice lacking α2-antiplasmin (α2-AP) and plasminogen activator inhibitor type-1 (PAI-1) genes, and elucidated the involvement of these inhibitors for fibrinolysis in immune response. MAIN METHODS: The pathophysiological changes induced by a lack of both α2-AP and PAI-1 were investigated using double knockout (KO) mice. The lung, liver, kidney and spleen tissues from α2-AP/PAI-1-double KO mice were compared with those from wild-type (WT) mice. Furthermore, the bone marrow cells from α2-AP/PAI-1-double KO mice were transplanted into 10-Gy X ray irradiated WT mice, and then the effects of the transplantation were studied. KEY FINDINGS: Plasma IgE levels in the α2-AP/PAI-1-double KO mice increased with age and exceeded 1000 ng/mL after 6 months of age. The plasma cells that produced IgE were detected in perivascular assembled lymphocytes. In the α2-AP/PAI-1-double KO mice, perivascular lymphocyte infiltration was observed in the lung, liver, and kidneys and peribronchial lymphocyte infiltration was present in the lung. When the bone marrow cells from α2-AP/PAI-1-double KO mice were transplanted into 10-Gy X ray irradiated WT mice, the phenotypes of the recipients were similar to those of α2-AP/PAI-1-double KO mice. SIGNIFICANCE: The simultaneous expression of both the α2-AP and PAI-1 genes contributes to the maintenance of immunological functions that are related to IgE. Moreover, it is suggested that both α2-AP and PAI-1 are involved in the recruitment of lymphocytes in the peripheral tissues.

Involvement of α2-antiplasmin in dendritic growth of hippocampal neurons.

The α2-Antiplasmin (α2AP) protein is known as a principal physiological inhibitor of plasmin, but we previously demonstrated that it acts as a regulatory factor for cellular functions independent of plasmin. α2AP is highly expressed in the hippocampus, suggesting a potential role for α2AP in hippocampal neuronal functions. However, the role for α2AP was unclear. This study is the first to investigate the involvement of α2AP in the dendritic growth of hippocampal neurons. The expression of microtubule-associated protein 2, which contributes to neurite initiation and neuronal growth, was lower in the neurons from α2AP⁻/⁻ mice than in the neurons from α2AP⁺/⁺ mice. Exogenous treatment with α2AP enhanced the microtubule-associated protein 2 expression, dendritic growth and filopodia formation in the neurons. This study also elucidated the mechanism underlying the α2AP-induced dendritic growth. Aprotinin, another plasmin inhibitor, had little effect on the dendritic growth of neurons, and α2AP induced its expression in the neurons from plaminogen⁻/⁻ mice. The activation of p38 MAPK was involved in the α2AP-induced dendritic growth. Therefore, our findings suggest that α2AP induces dendritic growth in hippocampal neurons through p38 MAPK activation, independent of plasmin, providing new insights into the role of α2AP in the CNS.

Alpha2-antiplasmin regulates the development of dermal fibrosis in mice by prostaglandin F(2α) synthesis through adipose triglyceride lipase/calcium-independent phospholipase A(2).

OBJECTIVE: Systemic sclerosis (SSc) is characterized by fibrosis of the skin and visceral organs. Patients with SSc have enhanced plasma levels of the plasmin-α2-antiplasmin (α2AP) complex, and we recently implicated α2AP in the development of fibrosis through transforming growth factor ß (TGFß) production. This study was undertaken to clarify how α2AP induces TGFß production and the development of fibrosis. METHODS: To clarify the detailed mechanism by which α2AP induces TGFß production, we focused on adipose triglyceride lipase (ATGL)/calcium-independent phospholipase A(2) (iPLA(2)) and examined whether ATGL/ iPLA(2) is associated with α2AP-induced TGFß production. The mouse model of bleomycin-induced SSc was used to evaluate the role of α2AP in the development of fibrosis. Dermal thickness and collagen content were determined in mouse skin treated with phosphate buffered saline or bleomycin. Moreover, we cultured SSc-like fibroblasts from the bleomycin-treated mouse skin and examined the production of TGFß and prostaglandin F(2α) (PGF(2α)). RESULTS: We found that α2AP binding to ATGL promoted PGF(2α) synthesis through iPLA(2) in fibroblasts, and the PGF(2α) synthesis that was promoted by α2AP induced TGFß production in fibroblasts. In addition, the neutralization of α2AP attenuated the production of TGFß and PGF(2α) in SSc-like fibroblasts from mice. The α2AP deficiency attenuated bleomycin-induced fibrosis and PGF(2α) synthesis, while the administration of PGF(2α) to α2AP-deficient mice facilitated α2AP deficiency-attenuated fibrosis. CONCLUSION: These findings suggest that α2AP regulates the development of fibrosis by PGF(2α) synthesis through ATGL/iPLA(2). The inhibition of α2AP-initiated pathways might provide a novel therapeutic approach to fibrotic diseases.

Enzamin ameliorates adipose tissue inflammation with impaired adipocytokine expression and insulin resistance in db/db mice.

The effects of Enzamin on obesity-related metabolic disorders in obese db/db mice were examined to explore a novel agent for the prevention of insulin resistance. Db/db mice were treated with water containing Enzamin (0·1 and 1·0 %) for 8 weeks from 6 weeks of age. Enzamin treatment at 1·0 %, but not at 0·1 %, significantly decreased the fasting plasma glucose, serum total cholesterol and TAG levels in db/db mice, without affecting body weight gain and body fat composition. Furthermore, insulin sensitivity and glucose tolerance were improved by the treatment of db/db mice with 1·0 % Enzamin. Immunohistochemical studies and gene expression analysis showed that 1·0 % Enzamin treatment suppressed macrophage accumulation and inflammation in the adipose tissue. In addition, 1·0 % Enzamin treatment increased serum adiponectin in db/db mice. Treatment with 1·0 % Enzamin also significantly suppressed the expression of NADPH oxidase subunits, suggesting an antioxidative effect for Enzamin in the adipose tissue. Furthermore, in vitro experiments demonstrated that the lipopolysaccharide-induced inflammatory reaction was significantly suppressed by Enzamin treatment in macrophages. Enzamin treatment increased the expression of GLUT4 mRNA in muscle, but not GLUT2 mRNA in the liver of db/db mice. Enzamin also increased the mRNA expression of carnitine palmitoyltransferase 1a (CPT1a, muscle isoform) in db/db mice, whereas Enzamin treatment did not affect the mRNA expression of CPT1b (liver isoform) in db/db mice. In conclusion, our data indicate that Enzamin can improve insulin resistance by ameliorating impaired adipocytokine expression, presumably through its anti-inflammatory action, and that Enzamin possesses a potential for preventing the metabolic syndrome.

Spatiotemporal differences in vascular permeability after ischaemic brain damage.

To study spatiotemporal differences in vascular permeability, we histologically analysed tracer extravasation, neovessels and reactive astrocytes in a mouse ischaemic brain damage model. On day 1 after damage induction, the extravasation was not associated with the distribution of neovessels or reactive astrocytes. On day 7, the extravasation was limited within the infarct region in which neovessels, but not reactive astrocytes, were observed. However, the extravasation was not observed at peri-infarct region in which both neovessels and reactive astrocytes were observed, suggesting that neovessels had high permeability and reactive astrocytes prevented the extravasation from neovessels. Furthermore, the extravasation was denser in the regions near the surface than in those further in the infarct region, suggesting a spatial heterogeneity in neovascular permeability.

Profibrinolytic effect of Enzamin, an extract of metabolic products from Bacillus subtilis AK and Lactobacillus.

Fibrinolytic system impairment contributes to the development of thrombotic disease such as cardiovascular disease and stroke. Therefore, an agent that increases fibrinolytic activity may be useful for the prevention of these diseases. In this study, to explore novel profibrinolytic agents, we examined the profibrinolytic effect of Enzamin, an extract of metabolic products from Bacillus subtilis AK and Lactobacillus in vitro and in vivo. Enzamin directly enhanced plasmin activity generated by tissue-type plasminogen activator (t-PA) by twofold but not by urokinase-type plasminogen activator (u-PA) in vitro, which was measured employing both the chromogenic substrate H-D: -Val-Leu-Lys-pNA (S-2251) and fibrin plate. Enzamin also increased plasmin activity generated by t-PA in the cell lysate and culture medium of endothelial cells, measured by fibrin zymography. Furthermore, the oral administration of a 1% concentration of Enzamin increased plasmin activity generated by t-PA by 1.7-fold but not by u-PA in the euglobulin fraction of mouse plasma. In conclusion, Enzamin has a unique ability to enhance the fibrinolytic activity through an increase in endogenous plasmin activity generated by t-PA released from endothelial cells, and may be a beneficial supplement for the prevention of thrombotic episodes.

Plasminogen/plasmin modulates bone metabolism by regulating the osteoblast and osteoclast function.

The contribution of plasminogen (Plg)/plasmin, which have claimed to be the main fibrinolytic regulators in the bone metabolism, remains unclear. This study evaluated how the absence of Plg affects the function of osteoblast (OB) and osteoclast (OC). There was a larger population of pre-OCs in bone marrow-derived cells from the Plg(-/-) mice than the population of that from the WT mice. In addition, the absence of Plg suppressed the expression of osteoprotegerin in OBs. Moreover, an exogenous plasmin clearly induced the osteoprotegerin expression in Plg(-/-) OBs. The osteoclastogenesis of RAW264.7 mouse monocyte/macrophage lineage cells in co-culture with OBs from the Plg(-/-) mice was significantly accelerated in comparison with that in co-culture with OBs from the WT mice. Intriguingly, the accelerated OC differentiation of RAW264.7 cells co-cultured with Plg(-/-) OBs was clearly suppressed by the treatment of an exogenous plasmin. Consequently, Plg(-/-) mice display decreased bone mineral density. These findings could eventually lead to the development of new clinical therapies for bone disease caused by a disorder of the fibrinolytic system.

The absence of urokinase-type plasminogen activator receptor plays a role in the insulin-independent glucose metabolism.

The urokinase-type plasminogen activator receptor (uPAR) is a glycosylphosphatidylinositol-anchored membrane protein with multiple functions. In the present study, we examined whether the uPAR plays any role in the regulation of glucose metabolism. The experiments were performed using male wild-type (uPAR) and uPAR knockout (uPAR) C57BL/6J mice. The blood glucose levels after the intraperitoneal injection of glucose were significantly decreased in uPAR mice compared with uPAR mice. On the other hand, there were no differences in the insulin secretion induced by glucose injection and the reactivity of insulin between uPAR and uPAR mice. The expression levels of glucose transporter 2 (GLUT2) in the liver and GLUT4 in the skeletal muscles from the uPAR mice were significantly increased compared with those of the uPAR mice. In addition, we found that the level of phosphorylation of AMP-activated protein kinase in skeletal muscles and myoblasts from the uPAR mice increased compared with those in uPAR mice. These data suggest that the increase in the GLUT2 and GLUT4 expression and the activation of AMP-activated protein kinase by uPAR deficiency enhances the glucose intake. These findings therefore provide new insights into the role of uPAR in the glucose metabolism.

Urokinase-type plasminogen activator receptor is associated with the development of adipose tissue.

Urokinase-type plasminogen activator receptor (uPAR) plays a role in cellular responses which include cellular adhesion, differentiation, proliferation and migration. The aim of this study was to clarify the role of uPAR on the development of adipose tissue. To clarify the role of uPAR on adipogenesis, we examined the effect of uPAR overexpression and uPAR deficiency on the adipocyte differentiation. Adipocyte differentiation was induced by incubation of 3T3-L1 cells with differentiation media containing insulin, dexamethasone, and 1-methyl-3-isobutyl-xanthin. uPAR overexpression by transfection of uPAR expression vector induced adipocyte differentiation. In addition, we examined the difference in adipocyte differentiation of mesenchymal stem cells from wild-type mice and uPAR knockout (uPAR-/-) mice. The uPAR deficiency attenuated differentiation media-induced adipocyte differentiation. Moreover, we found that the inhibition of phosphatidylinositol 3-kinase (PI3K) pathway attenuated uPAR overexpression-induced adipocyte differentiation, and uPAR overexpression induced the activation of Akt. We also found that an increase of the adipose tissue mass in uPAR-/- mice was less than that observed in wild-type mice. The present results suggest that uPAR plays a pivotal role in the development of adipose tissue through PI3K/Akt pathway.

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.

Initial brain lesion size affects the extent of subsequent pathophysiological responses.

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.