Inhibition of PAI-1 attenuates perirenal fat inflammation and the associated nephropathy in high-fat diet-induced obese mice.

Plasminogen activator inhibitor-1 (PAI-1) is increasingly recognized as a mediator in extracellular matrix (ECM) accumulation in diabetic nephropathy. Previous studies have implicated PAI-1 in adipose tissue (AT) expansion, while also contributing to insulin resistance. As inflammation is also known to occur in perirenal AT during obesity, we hypothesized that in a high-fat diet (HFD)-induced obese mouse model, PAI-1 contributes to macrophage-mediated inflammation and diabetic nephropathy. The HFD mice showed increased expression of PAI-1 in perirenal fat and also displayed increased fat weight and macrophage numbers. We found that the macrophage polarization, proinflammatory macrophage-M1-phenotype, including CD11c, IL-6, and monocyte chemoattractant protein-1, were increased by an HFD and decreased by either the genetic depletion of PAI-1 or treatment with the PAI-1 inhibitor, PAI-039. Similarly, an enhanced anti-inflammatory M2-phenotype, including CD206 and IL-10, was accompanied by either the genetic deletion of PAI-1 or PAI-039 treatment. Furthermore, the inhibition of PAI-1 reduced HFD-induced renal histological lesions and abated profibrotic/extracellular-matrix protein. Collectively, our findings provide support that PAI-1 contributes to the development of inflammation in perirenal fat and correlates with the development of diabetic nephropathy in HFD-induced obesity.

Reduced monocyte adhesion to aortae of diabetic plasminogen activator inhibitor-1 knockout mice.

OBJECTIVE AND DESIGN: To determine the requirement of plasminogen activator inhibitor-1-knockout (PAI-1) for monocyte adhesion in animals and cells under diabetic conditions. METHODS AND SUBJECTS: Monocyte adhesion assay, enzyme-linked immunosorbent assay, and Western blotting were used in analyzing samples from PAI-1-knockout (PAI-1-KO) mice or cultured human umbilical vein endothelial cells (HUVEC). TREATMENTS: Diabetes in PAI-1-KO and wild-type mice was induced by intraperitoneal injection of streptozotocin (STZ). HUVEC was transfected with short interference RNA (siRNA) against PAI-1, tumor necrosis factor-α (TNFα), or toll-like receptor (TLR4), and then was treated with glycated low-density lipoproteins (glyLDL). RESULTS: The adhesion of monocytes to aortic intima was reduced in PAI-1-KO mice, which was associated with decreased levels of TNFα and monocyte chemotactic protein-1 (MCP-1) in plasma and cardiovascular tissue, and increased abundances of urokinase plasminogen activator (uPA) and uPA receptor (uPAR) in cardiovascular tissue compared to wild-type mice. Significant reductions in monocyte adhesion, inflammatory, and fibrinolytic regulators were detected in cardiovascular tissue or plasma in diabetic PAI-1-KO mice compared to wild-type diabetic mice. Transfection of PAI-1, TNFα or TLR4 siRNA to HUVEC inhibited glyLDL-induced monocyte adhesion to EC. PAI-1 siRNA inhibited the abundances of TLR4 and TNFα in EC. CONCLUSION: The findings suggest that PAI-1 is required for diabetes-induced monocyte adhesion via interactions with uPA/uPAR, and it also regulates TLR4 and TNFα expression in vascular EC. Inhibition of PAI-1 potentially reduces vascular inflammation under diabetic condition.

Cilostazol inhibits HMGB1 release in LPS-activated RAW 264.7 cells and increases the survival of septic mice.

INTRODUCTION: Inflammation and coagulation play important roles in the pathogenesis of sepsis. Anticoagulants with anti-inflammatory action draw attention as therapeutic agent in sepsis. OBJECTIVE: Whether cilostazol (6-[4-(1-cyclohexyl-1H-tetrazol-5-yl) butoxy]-3,4-dihydro-2-(1H)-quinolinone), anticoagulant, protects mice against sepsis and underlying mechanism(s) were investigated. METHODS: Induction of heme oxygenase (HO)-1 protein, phosphorylation of 5' adenosine monophosphate-activated protein kinase (AMPK), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) luciferase activity, and release of high mobility group box 1 (HMGB1) were analyzed using signal inhibitors and transfection techniques. Survival and organ damage were compared in septic mice with and without cilostazol. RESULTS: In RAW264.7 cells, cilostazol increased phosphorylation of AMPK which was followed by HO-1 induction. Lipopolysaccharide (LPS)-activated HMGB1 release was reduced by cilostazol which was reversed by both SB203580 and silencing of HO-1 or AMPK RNA. Interestingly, silencing AMPK reduced HO-1 expression, whereas silencing HO-1 did not affect p-AMPK by cilostazol. Both compound C and zinc protoporphyrin IX (ZnPPIX) antagonized inhibitory effect of HMGB1 by cilostazol. Cilostazol inhibited NF-κB luciferase activity which was antagonized by SB203580. Finally, the administration of cilostazol increased the survival of endotoxemic mice but failed to do so when co-treated with rHMGB1. Cilostazol reduced circulating HMGB1, plasminogen activator inhibitor-1 (PAI-1) levels, organ damages and protein expression of PAI-1 in lung tissues of CLP-septic mice, which were antagonized by ZnPPIX. CONCLUSION: These findings suggest that HMGB1 can be a target molecule of cilostazol by 1) AMPK activation, and 2) induction of HO-1 by p38 MAPK and AMPK. Therefore, cilostazol may be useful for treatment of sepsis.

Innovative thrombolytic strategy using a heterodimer diabody against TAFI and PAI-1 in mouse models of thrombosis and stroke.

Circulating thrombin-activatable fibrinolysis inhibitor (TAFI) and plasminogen activator inhibitor-1 (PAI-1) are causal factors for thrombolytic failure. Therefore, we evaluated an antibody-engineered bispecific inhibitor against TAFI and PAI-1 (heterodimer diabody, Db-TCK26D6x33H1F7) in several mouse models of thrombosis and stroke. Prophylactic administration of the diabody (0.8 mg/kg) in a thromboplastin-induced model of thromboembolism led to decreased lung fibrin deposition. In a model of cerebral ischemia and reperfusion, diabody administration (0.8 mg/kg, 1 hour postocclusion) led to a mitigated cerebral injury with a 2.3-fold reduced lesion and improved functional outcomes. In a mouse model of thrombin-induced middle cerebral artery occlusion, the efficacy of the diabody was compared to the standard thrombolytic treatment with recombinant tissue-type plasminogen activator (tPA). Early administration of diabody (0.8 mg/kg) caused a twofold decrease in brain lesion size, whereas that of tPA (10 mg/kg) had a much smaller effect. Delayed administration of diabody or tPA had no effect on lesion size, whereas the combined administration of diabody with tPA caused a 1.7-fold decrease in lesion size. In contrast to tPA, the diabody did not increase accumulative bleeding. In conclusion, administration of a bispecific inhibitor against TAFI and PAI-1 results in a prominent profibrinolytic effect in mice without increased bleeding.

Spatiotemporal expression of SERPINE2 in the human placenta and its role in extravillous trophoblast migration and invasion.

BACKGROUND: SERPINE2, one of the potent serpins belonging to the plasminogen activator (PA) system, is involved in the tissue remodeling. We previously demonstrated the expression patterns of Serpine2 in the mouse placenta and uterus, indicating that Serpine2 is a major PA inhibitor in the placenta and uterus during the estrous cycle, pregnancy, and lactation. In this study, we further investigated the expression pattern of SERPINE2 in the human placenta and explored possible functional roles of SERPINE2 in regulating trophoblast activity. METHODS: Placental tissues from various trimesters were collected for real-time reverse-transcription polymerase chain reaction quantification. Immunohistochemical staining was performed in placental tissues to assure localization of SERPINE2. SERPINE2 small interfering (si) RNA was applied to suppress its expression in villous explants and extravillous trophoblast-like 3A cells. Subsequent experiments to evaluate SERPINE2 levels, villous outgrowth, trophoblast invasion, and tube formation were performed. RESULTS: SERPINE2 messenger RNA was detected in the human placenta during pregnancy with the highest levels in the third trimester. The SERPINE2 protein was present in villous syncytiotrophoblasts and trophoblasts of chorionic villi for anti-SERPINE2 immunostaining. Extravillous trophoblasts in the chorionic plate and basal plate confronting the invasive face of anchoring villi were also positive. In most decidual cells, SERPINE2 was observed in the cytoplasm. In addition, fibrinoid deposit was weakly immunoreactive. Introduction of SERPINE2 siRNA into villous explants and trophoblast cells led to significantly reduced villous outgrowth, and trophoblastic migration and invasion. Moreover, capillary-like network formation of 3A cells in Matrigel was greatly attenuated by SERPINE2 siRNA and SERPINE2 antiserum. CONCLUSIONS: These data identify the temporal and spatial SERPINE2 distribution in the human placenta and suggest its possible role in modulating tissue remodeling of extravillous trophoblasts in the placenta during pregnancy.

Protective roles for fibrin, tissue factor, plasminogen activator inhibitor-1, and thrombin activatable fibrinolysis inhibitor, but not factor XI, during defense against the gram-negative bacterium Yersinia enterocolitica.

Septic infections dysregulate hemostatic pathways, prompting coagulopathy. Nevertheless, anticoagulant therapies typically fail to protect humans from septic pathology. The data reported in this work may help to explain this discrepancy by demonstrating critical protective roles for coagulation leading to fibrin deposition during host defense against the Gram-negative bacterium Yersinia enterocolitica. After i.p. inoculation with Y. enterocolitica, fibrinogen-deficient mice display impaired cytokine and chemokine production in the peritoneal cavity and suppressed neutrophil recruitment. Moreover, both gene-targeted fibrinogen-deficient mice and wild-type mice treated with the anticoagulant coumadin display increased hepatic bacterial burden and mortality following either i.p. or i.v. inoculation with Y. enterocolitica. Mice with low tissue factor activity succumb to yersiniosis with a phenotype similar to fibrin(ogen)-deficient mice, whereas factor XI-deficient mice show wild-type levels of resistance. Mice deficient in plasminogen activator inhibitor-1 or thrombin-activatable fibrinolysis inhibitor display modest phenotypes, but mice deficient in both plasminogen activator inhibitor-1 and thrombin-activatable fibrinolysis inhibitor succumb to yersiniosis with a phenotype resembling fibrin(ogen)-deficient mice. These findings demonstrate critical protective roles for the tissue factor-dependent extrinsic coagulation pathway during host defense against bacteria and caution that therapeutics targeting major thrombin-generating or antifibrinolytic pathways may disrupt fibrin-mediated host defense during Gram-negative sepsis.

Platelet protease nexin-1, a serpin that strongly influences fibrinolysis and thrombolysis.

BACKGROUND: Protease nexin-1 (PN-1) is a serpin that inhibits plasminogen activators, plasmin, and thrombin. PN-1 is barely detectable in plasma, but we have shown recently that PN-1 is present within the α-granules of platelets. METHODS AND RESULTS: In this study, the role of platelet PN-1 in fibrinolysis was investigated with the use of human platelets incubated with a blocking antibody and platelets from PN-1-deficient mice. We showed by using fibrin-agar zymography and fibrin matrix that platelet PN-1 inhibited both the generation of plasmin by fibrin-bound tissue plasminogen activator and the activity of fibrin-bound plasmin itself. Rotational thromboelastometry and laser scanning confocal microscopy were used to demonstrate that PN-1 blockade or deficiency resulted in increased clot lysis and in an acceleration of the lysis front. Protease nexin-1 is thus a major determinant of the lysis resistance of platelet-rich clots. Moreover, in an original murine model in which thrombolysis induced by tissue plasminogen activator can be measured directly in situ, we observed that vascular recanalization was significantly increased in PN-1-deficient mice. Surprisingly, general physical health, after tissue plasminogen activator-induced thrombolysis, was much better in PN-1-deficient than in wild-type mice. CONCLUSIONS: Our results reveal that platelet PN-1 can be considered as a new important regulator of thrombolysis in vivo. Inhibition of PN-1 is thus predicted to promote endogenous and exogenous tissue plasminogen activator-mediated fibrinolysis and may enhance the therapeutic efficacy of thrombolytic agents.

Spatiotemporal expression of the serine protease inhibitor, SERPINE2, in the mouse placenta and uterus during the estrous cycle, pregnancy, and lactation.

BACKGROUND: SERPINE2, also known as glia-derived nexin or protease nexin-1, belongs to the serine protease inhibitor (SERPIN) superfamily. It is one of the potent serpins that modulates the activity of the plasminogen activator (PA) and was implicated in tissue remodeling. In this study, we investigated the expression patterns of SERPINE2 in the mouse placenta and uterus during the estrous cycle, pregnancy, and lactation. METHODS: SERPINE2 was purified from mouse seminal vesicle secretion using liquid chromatography (LC) and identified by LC/tandem mass spectrometry. The antiserum against the SERPINE2 protein was raised in rabbits. To reveal the uterine and placental expression of SERPINE2, tissues at various stages were collected for real-time PCR quantification, Western blotting, and immunohistochemical staining. RESULTS: Serpine2 mRNA was the major PA inhibitor in the placenta and uterus during the estrous cycle, pregnancy, and lactation, although Serpine1 mRNA had higher expression levels than Serpine2 mRNA in the placenta. Plat seemed to be the major PA in the mouse uterus and placenta. Antiserum against the SERPINE2 protein specifically recognized two forms of SERPINE2 and an extra 75-kDa protein, which was probably a complex of SERPINE2 with a certain protease, from among thousands of protein components in the tissue extract as demonstrated by Western blotting. In the uterus, SERPINE2 was primarily localized in luminal and glandular epithelial cells but it also was detected in circular and longitudinal smooth muscle cells during the estrous cycle and lactation. It was prominently expressed in decidual stroma cells, the metrial gland, and endometrial epithelium of the pregnant uterus. In the placenta, SERPINE2 was expressed in trophoblasts of the labyrinth and spongiotrophoblasts. However, its expression was remarkably reduced in giant cells which existed in the giant cell-decidual junction zone. In contrast, prominent expression of SERPINE2 seemed to be detected on clusters of glycogen cells near the junction zone. In addition, yolk sac membranes also showed high expression of SERPINE2. CONCLUSIONS: These findings indicate that SERPINE2 is a major PA inhibitor in the placenta and uterus during the estrous cycle, pregnancy, and lactation. It may participate in the PA-modulated tissue remodeling process in the mouse placenta and uterus.