Shigeru Ueshima

The Absence of uPAR Is Associated with the Progression of Dermal Fibrosis

The fibrinolytic system is considered to play an important role in the degradation of extracellular matrices (ECM). However, the detailed mechanism regarding how this system affects fibrosis remains unclear. Urokinase-type plasminogen activator receptor (uPAR) not only functions as a proteinase receptor but also plays a role in cellular adhesion, differentiation, proliferation, and migration through intracellular signaling. To investigate the effect of uPAR on dermal fibrosis, the skin of wild-type mice was compared with uPAR-deficient (uPAR(-/-)) mice. The results showed that the absence of uPAR increases dermal thickness. In addition, collagen synthesis as well as the number of myofibroblasts was greater in the skin of uPAR(-/-) mice than in the skin of uPAR(+/+) mice. Moreover, we showed that the absence of uPAR attenuates the activity of matrix metalloproteinases (MMP)-2, 9 in the skin. In conclusion, this study suggests that the absence of uPAR not only regulates fibrosis-related gene expression and MMP activity but also results in ECM deposition. Therefore, the absence of uPAR induces dermal fibrosis. These findings provide new insights into the role of uPAR on dermal fibrosis.

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 uPAR attenuates insulin-induced vascular smooth muscle cell migration and proliferation

Excess vascular smooth muscle cell (VSMC) proliferation and migration are features of diabetic vascular disease. In diabetes, there is an abnormal metabolic environment including high glucose, high free fatty acids, and either high insulin or an insulin deficiency. Diabetic conditions have been shown to lead to accelerated atherosclerosis [1]. Because insulin stimulates the proliferation and migration of VSMC in culture, insulin might exert an atherogenic role by promoting the proliferation of SMC in the intima which is considered as one of the most important initial steps in atherogenesis [2,3].

Urokinase-type plasminogen activator and plasminogen mediate activation of macrophage phagocytosis during liver repair in vivo

Urokinase-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-reperfusion 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.

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.

Alpha2-Antiplasmin Is a Critical Regulator of Angiotensin II–Mediated Vascular Remodeling

Objective—Alpha2-antiplasmin (&agr;2-AP) is the major circulating inhibitor of plasmin, which plays a determining role in the regulation of intravascular fibrinolysis. We investigated the role of &agr;2-AP on vascular remodeling in response to angiotensin II (Ang II). Methods and Results—&agr;2-AP–deficient mice were performed. Ang II and N&ohgr;-nitro- L-arginine methyl ester (L-NAME)–induced perivascular fibrosis was significantly decreased in &agr;2-AP−/− mice compared with wild-type mice. In situ gelatinolytic activity analysis shows that perivascular gelatinolytic activity was increased in &agr;2-AP−/− mice, which was responsible for decreased perivascular fibrosis in response to Ang II and L-NAME. Ang II–induced arterial wall thickening, vascular cell proliferation, apoptosis, c-Myc, and collagen &Igr; expression were significantly decreased in &agr;2-AP−/− mice compared with wild-type mice. Further analysis shows that increased p53 and p21 expression were responsible for inhibition of Ang II–induced vascular remodeling in &agr;2-AP−/− mice. Conclusion—The results show that &agr;2-AP is a critical regulator for vascular remodeling by inhibiting p53/p21 pathway, suggesting that &agr;2-AP is proposed to be a potential therapeutic target for vascular remodeling.

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

AIMSnWe 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.nnnMAIN METHODSnThe 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.nnnKEY FINDINGSnPlasma 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.nnnSIGNIFICANCEnThe 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.

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.

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.

A synthetic peptide derived from staphylokinase enhances plasminogen activation by tissue-type plasminogen activator

Summary.u2002 Background: A synthetic nonadecapeptide (SP; GPYLMVNVTGVDGKGNELL) previously enhanced the activation of plasminogen by the SAK/plasmin complex. Objectives: To identify the binding site for SP on plasminogen and elucidate the effects of SP on plasminogen activation by the tissue‐type plasminogen activator (t‐PA). Methods: The effects of SP on plasminogen activation were estimated using a chromogenic substrate and from the cleavage of plasmin on SDS‐PAGE under reduced conditions. The binding to SP of various peptides derived from the amino acid sequence of plasminogen was analyzed with an IAsys biosensor. The SP‐mediated structural change to plasminogen was analyzed by circular dichroism (CD) spectroscopy. The thrombolytic effects of SP were examined using a mouse model of thrombosis. Results: SP enhanced the activation of plasminogen by t‐PA. The catalytic efficiency (kcat/Km) of Glu‐plasminogen activation by t‐PA was 11.4‐fold higher in the presence than absence of SP. The binding of SP to plasminogen was greatly inhibited by a synthetic peptide, FEKDKYILQGVTSWGLG, located close to the C‐terminal of the plasminogen B region. Near‐ultraviolet CD spectra of the complex between SP and Glu‐plasminogen significantly differed from those of Glu‐plasminogen. When SP was administered in a mouse model of thrombosis, early recanalization was observed in a dose‐dependent manner. However, SP did not cause recanalization in t‐PA gene‐deficient mice. Conclusions: SP bound to the B region and promoted the activation of plasminogen by t‐PA, and then induced effective thrombolysis.